Proceedings Book of 42nd Conference For Students PDF

42nd Conference For Students Of
Agriculture And Veterinary Medicine

With International Participation
Proceedings
roceedings book
University of Novi Sad, Faculty of Agriculture

November 16, 2018
UNIV
UNIVERZITET U NOVOM SADU
POLJOPRIVREDNI FAKULTET
ZBORNIK RADOVA
SA 42. SMOTRE NAUČ
ČNIH
NIH RADOVA STUDENATA POLJOPRIVREDE I
VETERINARSKE MEDICINE SA MEĐUNARODNIM UUČEŠĆEM
EŠĆEM
PROCEEDINGS
OF THE 42nd CONFERENCE FOR STUDENTS OF AGRICULTURE
AGRICULTUR AND
VETERINARY MEDICINE
WITH INTERNATIONAL PARTICIPATION
16 Novembar 2018
2
ORGANIZERS OF THE CONFERENCE
UNIVERSITY OF NOVI SAD

FACULTY OF AGRICULTURE
NOVI SAD
FACULTY OF AGRICULTURE
Trg Dositeja Obradovića 8, 21000 Novi Sad SERBIA
Phone: +381 (0)21 4853500
Fax: +381 (0)21 450761
UNIVERSITY OF AGRICULTURAL SCIENCES AND

VETERINARY MEDICINE OF THE BANAT
TIMIŞOARA
Calea Aradului 119, 300645 Timişoara, ROMANIA

Phone: 00 40 (0)256/277-126
Fax: 00 40 (0)256/200-296
3
42nd CONFERENCE FOR STUDENTS OF AGRICULTURE AND VETERINARY
MEDICINE WITH INTERNATIONAL PARTICIPATION
ORGANIZING COMMITTEE:
Dr Branko Ćupina, Vice-dean for science and international cooperation
Dr Dragana Budakov, president of the organizing committee
Dr Mila Grahovac
MSc Mladen Petreš
PROGRAM COMITTEE:
Dr Ljubica Spasojević Kosić, full professor
Dr Emina Mladenović, assistant professor
Dr Mirko Ivković, assistant professor
Dr Dejan Prvulović, assistant professor
Dr Ksenija Mačkić, assistant professor
Dr Mirjana Ljubojević¸ assistant professor
Dr Milena Popov, assistant professor
Dr Ondrej Ponjičan, associate professor
Dr Jasna Grabić, assistant professor
Dr Nataša Vukelić, assistant professor
4
Council of the University of Novi Sad, Faculty of Agriculture, at its fourth meeting on 18 th of
February 1981 made a decision that the Conference for students of agriculture and veterinary
medicine with international participation, held each year at the Faculty of Agriculture will be
held in the memory of academician Dr Petar Drezgić, professor, and the prizes awarded at the
Conference will bear his name.
5
PREFACE
The first Conference for students of agriculture and veterinary medicine with international
participation was held in Novi Sad 42 years ago. To the present date, many papers from national
and international authors were presented (Hungary – Gödölö and Debrecen, Germany – Kassel,
Slovakia – Nitra, Poland – Wroclaw and Warsaw, Romania – Timisoara, Cluj and Bucharest,
Bulgaria – Plovdiv, Russia – Moscow and Kemerovo, Macedonia – Skopje and Bitola, Bosnia
and Herzegovina – Sarajevo, Banja Luka, Croatia – Zagreb). Since 2009, papers presented at the
Conference are published in a special publication named “PROCEEDINGS OF THE 42 nd
CONFERENCE FOR STUDENTS OF AGRICULTURE AND VETERINARY MEDICINE
WITH INTERNATIONAL PARTICIPATION” where all published papers have all elements of
the original scientific paper. Starting this year, we enabled students and mentors who are
interested in benefitting more from this Conference to publish their papers in the journal
Contemporary Agriculture published by the University of Novi Sad, Faculty of Agriculture.
Also, besides the Faculty of Agriculture from Novi Sad, co-organizer of the Conference is also
Faculty of Agriculture from Timisoara and there is a prospect of joining another University
center from the region. This Conference is strongly supported by the Student Parliament of the
Faculty of Agriculture, Novi Sad.
As the host and the principal organizer of the Conference University of Novi Sad, Faculty of
Agriculture has a special honor and a pleasure to wish a warm welcome to all participants,
successful work and amusing socializing in Novi Sad.
Vice-dean for science and international cooperation

Dr Branko Ćupina, full professor
President of the organizing committee

Dr Dragana Budakov, assistant professor
6
INDOOR ECOSYSTEM SERVICES: PLANTS IMPACT ON AIR QUALITY
Martina Zorić
Mentor: doc. dr Emina Mladenović
University of Novi Sad, Faculty of Agriculture, Department of Fruit Growing, Viticulture,

Horticulture and Landscape Architecture, Serbia
Summary: Ecosystem services have been in the focus of most recent research considering
ecology, biodiversity but also its impact on people. Most of the research on this subject is
about the natural ecosystems in the wilderness, on the other hand, small amount of research
part has dealt with indoor and urban ecosystems and its correspondence and influence on
people - our health and quality of life. One of the most common problems in urban and indoor
spaces considering human health is the quality of air. Considering the fact that we spent most
of our time indoor with lack of fresh air due to industry growth and degradation of
environment there is an obvious need for designing a non-invasive and non-obtrusive system
for air purification. This paper presents the results of indoor air quality monitoring: its
changes with the presence of one of the common indoor ornamental plants- Sansevieria
trifasciata ‘Laurentii’. The aim of this research is to build a basis for a creation of biological
purifying system as a low-cost, environmentally friendly method for monitoring and
purification of indoor air which should have the positive impact on user’s health as well on
the quality of life. During this research different air quality parameters were observed:
relative humidity and temperature, the concentrations of carbon-dioxide, methane and overall
VOCs which have previously defined as indoor pollutants. These parameters were measured
with the commercial CO2 data logger Extech SD800 and Arduino Uno based measuring
device with different sensors. Presented results show the change of indoor air quality
measured in the single bedroom, in uncontrolled conditions within the presence or absence of
plants. Monitoring results showed a sudden increment of overall VOC (NH 3, NOx, benzene
and smoke), methane and carbon-dioxide after the plants were removed from the indoor
space. Considering the fact that satisfactory methods for air pollutants monitoring and
removal are not fully developed and implemented, this research has been conducted in order
to define a preliminary and low-cost method for unobtrusive, natural improvement and
monitoring of indoor air quality with the use of ornamental plants and microcomputer's
measurement devices.
Key words: Indoor Ecosystems Services, Sansevieria trifasciata, Air quality, Biological
Purifying System, Quality of Life.
6
INTRODUCTION
Ever since the industrial revolution has happened it seems that people are losing that special
connection with nature which was constant in earlier stages of our development on personal and
society level. As a result, the so-called western style of living has cost us a lot considering the
decrement of the overall quality of life and health (Husti et al., 2016). Considering the number of
health issues and conditions of the human population, its effect on society and economy, the role
of nature in the term of ecosystem services, as an unobtrusive method for improvement of health
and quality of life should be defined and implemented as soon as possible. In recent years the
high number of research on the subject of ecosystem services has been published, but most of it
puts focus on natural environments in the wilderness (Martin et al. 2010), ignoring the fact that
people spend most of their time indoors (Shwartz, A. 2018). Urban and indoor ecosystems and
data in connection with its services are still not adequately described, nor there are clearly
defined methods for its valuation or practical implementation for people (Lyytimäki, J. 2012).
Quality of indoor air is an important factor for determining a level of well-being and has
enormous influence on our health. Indoor air contamination is a complex problem, that according
to World Health Organization (2010) involves the negative effect of particles (such as dust and
smoke), biological agents (molds, spores), radon, asbestos, and gaseous contaminants such as
CO, CO2, NOx, SOx, aldehydes and VOCs (Volatile Organic Compounds). Different volatile
organic compounds found as common components in indoor air are defined as air pollutants, and
can be the triggers or causes for different types of health conditions. Long-term exposure to these
harmful particles such are: formaldehyde, xylene, toluene, benzene, methane, NOx, can lead to
deterioration of chronic diseases, can cause the occurrence of the symptoms similar to flu or
allergies, and some of these compounds (as formaldehyde and benzene) are carcinogenic
(Jones,1999; Kostiainen, 1995; Xu, et al., 2013). According to Guieysse, et al., (2008), based on
data from the conducted study for comparison of present methods for indoor air purification,
devices that included any biological component (plants, microorganisms) were described as the
only method that significantly removed VOCs. Considering the high amount of different
pollutants, including VOCs that we are exposed to in our every-day life, there is a need for
designing air purification systems that could reduce the concentration of pollutants and will not
further harm our environment or health. Certain ornamental plants, commonly used in indoor
spaces such as Mother-in-laws tongue (Sansevieria trifasciata), can improve the quality of air
(Kim et al., 2008; Yoo et al., 2006; Begum, & Gopinath, 2017; Wolverton & Wolverton, 1993;
Kays, 2011). Sansevieria trifasciata as the common indoor ornamental plant is an ornamental
plant that has low maintenance requirements of light, the size, shape and has good general
aesthetic qualities. This plant is commonly called the ‘bedroom plant’due to its characteristic for
the releasement of higher concentrations of oxygen during the night, opposite to most of the
plants. The goal of our research is to promote the indoor ecosystem services, the role of plants in
7
our lives, but also to develop a system for monitoring, valuation and possible improvement of air
that we breathe as one of the most important factors in our everyday surroundings.
MATERIAL AND METHODS
As the goal of this research was to investigate the possible influence of unobtrusive and botanical
purifying system on the indoor air quality in real life conditions, eight pots with Sansevieria
trifasciata ‘Laurentii’ were placed in a small single bedroom. Plant selection was based on the
literature overview. Sansevieria trifasciata 'Laurentii' (Picture 1) was chosen for this research for
its possibility to remove certain indoor air pollutants, but also to release a greater amount of
oxygen during the night, considering that most people spend night time in the bedroom, sleeping.
In this paper, we proposed a low-cost approach to monitor different air quality parameters:
temperature (°C), relative humidity (%RH), as well the concentrations (ppm) of carbon dioxide
(CO2), VOCs and methane. The created biological purifying system has two components: plants -
Sansevieria trifasciata ‘Laurentii’ and monitoring devices. The plants with an average height of
45 cm were potted in standard substrate mix for ornamental plants and placed in two groups of 3
and 5 plants.
Picture 1. Sansevieria Picture 2: Extech Picture 3: Arduino Uno

trifasciata ‘Laurentii’ SD800 based measuring device
with the commercial
sensors
Air quality parameters were continuously measured for 23 days with commercial CO 2 data logger
Extech SD800 (Picture 2) and Arduino Uno based measuring device with the commercial
sensors: DHT11 (temperature and relative humidity sensor) and MQ-135 (Poisonous VOCs
sensor) and methane (MQ-4) (Picture 3). According to the manufacturer, MQ-135 reacts to the
presence of NH3, NOx, benzene and smoke. The research has been conducted in Banjaluka,
Bosnia and Herzegovina region, during December, 2017. The experiment has been divided into
three stages. In the first stage, monitoring devices were placed in the bedroom and data
considering air quality has been measured for 3 days. During the second phase, on the fourth day
8
from the beginning of the research, plants were placed in the room. During the final, third phase
of the research, plants were taken out of the room. It is important to emphasize that there was no
change in behavior of the person who was still using the room regularly. The monitoring of air
quality was constant during all three stages of the research.
RESULTS AND DISCUSSION
Indoor air quality is one of the major factors that contribute to the health and overall well-being
of each individual (Duflo, et al., 2008). The majority of people spend most of their lifetime in
indoor spaces which have different sources of pollution. Even indoor air in our homes is polluted
from building materials, human activities (Hänninen at al., 1999), as well from outdoor air
pollutants. This paper proposes a possible basis for a highly successful, low-cost and natural air
purifying method based on microcomputer's measurement devices and plants. Changes of
temperature (°C), relative humidity (%RH), carbon-dioxide (ppm), VOC (NH 3, NOx, benzene
and smoke) (ppm) and methane (ppm) in the bedroom are shown on Fig. 1. Each graph is divided
into the three stages of research. Starting from left to right: Stage I (black lines) before the plants
were introduced in the bedroom, Stage II (green lines) with the presence of the plants, Stage III
(black lines) after the plants were taken out of the bedroom. Presented data shows the change of
measured parameters in different stages of the monitoring process. Presented temperature data
showed no connection between the presence of plants on these air quality parameters.
Considering the fact that the experiment was conducted during the winter time, and the heating
was on, temperature shows a constant between 20-24°C. Data of measured relative humidity
shows certain decrement during the end of Phase II which is probably the consequence of a
longer period of high temperatures during this period. As for concentrations of carbon dioxide
(CO2), VOC and methane (CH4) there is no clear, measurable evidence that plants do have a
positive impact on these compounds during the Stage II. In stage III, after the plants were taken
out of the room, a sudden increment in concentrations of these compounds is evident and it can
be attributed to the lack of presence of the plants, since no other change has been made in the
bedroom compared to the other days during monitoring.
9
Fig.1 Three stages of the experiment. Starting from left to right: Stage I
(black lines), Stage II (green lines), Stage III (black lines).
These changes are highly noticeable at the graph of monitoring of methane (CH 4). There is an
evident drop in its concentrations during the end of Stage II, and its sudden increment from the
beginning of Stage II, after the plants were taken out of the room. A higher number of peaks can
also be observed in Stage III of monitoring CO2 and VOCs in comparison to measurements
observed during Stage II. The authors are aware that presented data does not prove in concise
shape that Sansevieria trifasciata ‘Laurentii’ has a positive impact on carbon-dioxide, methane
and VOCs concentrations regarding air quality, but shows the sudden increment after the plants
were taken out of the room. This research has shown that inexpensive, custom designed botanical
purifying system for indoor air quality monitoring and improvement can provide data on indoor
air quality and possible positive impact of plants in indoor air pollutants. Although some authors
claim that previously conducted studies have failed to confirm the positive impact on air quality
in indoor spaces (Apte & Apte 2010), others sources claim that there is evident positive
influence on indoor air pollutants of certain ornamental plants such as Ficus elastica, Dracaena
deremensis and Sansevieria trifasciata (Husti et al., 2016). These contrary research reports
confirm that there is a constant need for finding the functional, economically affordable botanical
purifying system with the use of plants.
CONCLUSION
Considering the fact that satisfactory methods for air pollutants monitoring and removal are
not fully developed and implemented, this research has been conducted in order to define a
preliminary and low-cost method for unobtrusive, natural improvement and monitoring of indoor
air quality with the use of ornamental plants and microcomputer's measurement devices.
10
Moreover, components of this biological purifying system can easily be adjusted according to
specific indoor space or users preferences in the terms of expansions of the devices with other
sensors and use of different plant species. This research opens a series of multidisciplinary
questions and subjects for future research, such as the adaptation and creation of calibrated
sensors for air quality monitoring and in the terms of indoor ecosystem services, the plant species
and genotypes used for the improvement of indoor air. Further research in the terms of plant
species and its number in certain space with defined air pollutants and its concentrations would
definitely increase the efficiency of ornamental plants in indoor air quality improvement.
REFERENCES
APTE, M. G., & APTE, J. S. (2010): A pilot study of the effectiveness of indoor plants for
removal of volatile organic compounds in indoor air in a seven-story office building.
BEGUM, K. A. J., & GOPINATH, R. (2017): Development of Step-Wise Ranking for Indoor
Plants as Indoor Air Pollutant Purifiers. Austin Environ Sci, 2(1), 1018.
DUFLO, E., GREENSTONE, M., & HANNA, R. (2008): Indoor air pollution, health and
economic well-being. SAPI EN. S. Surveys and Perspectives Integrating Environment and
Society, (1.1).
GUIEYSSE, B., HORT, C., PLATEL, V., MUNOZ, R., ONDARTS, M., & REVAH, S. (2008):
Biological treatment of indoor air for VOC removal: Potential and challenges. Biotechnology
Advances, 26(5), 398-410.
HÄNNINEN, O., ECONOMOPOULOS, A., & ÖZKAYNAK, H. (1999): Information on air
quality required for health impact assessment. Monitoring ambient air quality for health impact
assessment. WHO Regional Publications, European Series, (85), 9-36.
HUSTI, A., CANTOR, M., STEFAN, R., MICLEAN, M., ROMAN, M., NEACSU, I.,
CONTIUA,I., MAGYARID, K., & BAIA, M. (2016): Assessing the Indoor Pollutants Effect on
Ornamental Plants Leaves by FT-IR Spectroscopy. Acta Physica Polonica, A., 129(1).
JONES, A.P. 1999: Indoor air quality and health. Atmos. Environ. 33:4535–4564.
KAYS, S. J. (2011): Phytoremediation of indoor air—Current state of the art. The value creation
of plants for future urban agriculture. Nat. Inst. Hort. Herbal Science, RDA, Suwon, Korea, 3-21.
KIM, K.J., M.J. KIL, J.S. SONG, E.H. YOO, K.C. SON AND S.J. KAYS. (2008): Efficiency of
volatile formaldehyde removal by indoor plants: Contribution of aerial plant parts versus the root
zone. J. Amer. Soc. Hort. Sci. 133:1–6.
KOSTIAINEN, R. (1995): Volatile organic compounds in the indoor air of normal and sick
houses. Atmos. Environ. 29:693–702.
LYYTIMÄKI, J. (2012): Indoor ecosystem services: bringing ecology and people together.
Human Ecology Review, 70-76.
MARTIN, L.J., ELLIS, E., & BLOSSEY, B. (2010): Ecology: Not in our backyards.Oral
presentation at 95th Ecological Society of America (ESA) Annual Meeting, Pittsburgh, PA.
http://eco.confex.com/eco/2010/ techprogram/P25201.HTM
11
SHWARTZ, A. (2018): Designing Nature in Cities to Safeguard Meaningful Experiences of
Biodiversity in an Urbanizing World. In Urban Biodiversity (Vol. 200, No. 215, pp. 200-215).
ROUTLEDGE in association with GSE Research.
WOLVERTON, B. C., & WOLVERTON, J. D. (1993): Plants and soil microorganisms: removal
of formaldehyde, xylene, and ammonia from the indoor environment. Journal of the Mississippi
Academy of Sciences, 38(2), 11-15.
XU, Q., ZHANG, Y., MO, J., & LI, X. (2013): How to select adsorption material for removing
gas phase indoor air pollutants: a new parameter and approach. Indoor and Built Environment,
22(1), 30-38.)
YOO, M.H., Y.J. KWON, K.C. SON, AND S.J. KAYS. (2006): Efficacy of indoor plants for the
removal of single and mixed volatile organic pollutants and physiological effects of the volatiles
on the plants. J. Amer. Soc. Hort. Sci. 131:452–458.
WORLD HEALTH ORGANIZATION, Denmark: Regional Office for Europe, 2010
12
ASPECTS OF MORPHO-PHENOLOGICAL DEVELOPMENT AT TRITICUM
AESTIVUM L.
Ardelean ABEL
Mentor: Petrescu IRINA

irinuta@hotmail.com
Banat University of Agriculture Sciences and Veterinary Medicine `KING MICHAEL I

of Romania` Timisoara, Faculty of Agriculture, Romania.
Summary: To realize this project, it took the general accumulation of knowledge through
information and documentation in the literature regarding cereal straw: importance of the
chemical composition of the grain, systematic, germination and biological material.
The research were conducted to achieve thematic studies on phenotypic wheat that have been
conducted in the laboratory of Plant Genetics, Faculty of Agriculture U.S.A.M.V.B Timisoara
and aimed to establish differences on phenological phases.
Key word: wheat , morpho-phenological, phases,
INTRODUCTION
Wheat is one of the most important food plants, cultivated in more than 45 countries
today, feeding 35 -40% of the world population . Its main use is in the manufacture of
bread and various products made from flour. In many countries today it’s also used in the feeding
of animals.
Wheat bran is a particularly valuable feed for dairy cows because of their rich protein
(14-15%) and carbohydrates (40-45%) content. Wheat straws are used in animal feed, cellulose
manufacture and animal bedding to produce manure. The stubble after harvest enriches the soil in
organic matter.
Wheat is considered to be the agricultural crop with the largest area cultivated
worldwide, which is over 220 million hectares. In our country the area cultivated with wheat is
approx. 25% of the arable land area and 40% of the cereal cultivated area. The importance to this
culture is the following:
- for almost half of the world's population, bread made from wheat flour is basic food;
- in terms of quality, wheat grains have a balanced ratio between carbohydrates and
proteins, corresponding to the requirements of the human body;
- wheat grains are the raw material for the production or extraction of useful substances
that process a wide variety of agri-food products;
- wheat grains have a long shelf life and can be transported over long distances without
the risk of quality degradation;
13
- can be used directly in animal feed, especially the slurry resulting from the milling
industry, as it has high protein, fat and mineral content.
The purpose of the research was to analyze the phenological differences of one of the
main agricultural cultures in Romania. They were selected as the subject of research due to the
desire to expand the information regarding the production and quality.
The overall objectives of the proposed research are: quantities related to the plant height;
quantitative characters related to the leaves; quantitative characters related to spice.
The study of the phenotypic differences between varieties was carried out between 2017
and 2018. The preparation of the experiment was performed according to the testing
requirements of seed germination. The experiments were carried out during the growth of plants,
in different periods of evolution, starting from winter to the time of their harvesting. Observations
were made 20-25 days from the sprout, 150-190 days from the sprout, 225-250 days from the
sprout and before the harvest.
The variety Alex was chosen as vegetable material for the research.
RESULTS AND DISCUSSIONS
1. Quantitative characters related to the height of the plant
After the winter outbreak, there was a pronounced increase in vegetation due to climatic
conditions during the observation period, there was a good increase in height of 46.13 ± 1.71
cm compared to the values of 18.5 ± 5.1 cm observed at 20-25 days after sprout. Before
harvesting, the height of the plant reached almost normal values by reaching the height of
82.82 value close to 225-250 days from sprout that was 73.67 cm.
Before entering winter (20-25 days from sprout) and after the winter’s end (150-190 days
from the sprout) the average number of internodes was between 1.20 and 3.44. The number
of internodes before harvesting is close to the 225-250 days from the sprout, averaging between 5
and 5.22. At the start of winter, as the plots are in an early stage of development, it can be seen
that the length of the internodes is small 0,10 cm. The length of the internodes for the Alex
variety after leaving the winter (150-190 days from the sprout) is 1.4 cm and it reaches, before
the harvest, normal values of 3.10 cm compared to 2.33 cm at 225-250 days from the
sprout (Table 1).
14
Table 1 Quantities related to the height of the plant in the Alex variety
Phenotypic aspects of the height of the plants number of internodes length of internodes
Triticum aestivum at: (cm) (cm) (cm)
x ± sx s% x ± sx s% x ± sx s%
20-25 days from sprout 18.50 ± 5.10 22.98 1.20 ± 0.13 33.33 0.10 ± 0.01 16.33
150-190 days from sprout 46.13 ± 1.71 11.69 3.44 ± 0.24 12.08 14.95 ± 0.77 8.92
225-250 days from sprout 73.67 ± 1.74 4.08 5.00 ± 0.16 5.47 22.33 ± 0.11 15.23
before harvesting 82.82 ± 1.13 2.37 5.22 ± 0.09 2.98 31.10 ± 0.21 1 0.3 0
2. Quantitative characters related to the leaf

Before entering winter, the number of leaves is reduced, forming only
two leaves, but, after the end of winter, plants in the growing season begin to grow reaching the
values of 4.38 in average 150-190 days from sprout. At 225 days from the sprout the number of
leaves formed was higher on average 5.33 the number that is the same and before the
harvest (Table 2 ) . L the leaf at the entrance of the winter was an average of 29.11 cm and
one225 days from the east the length of the leaves was reaching before harvesting to Alex variety
value is 21.77 cm. Before entering winter, the leaf width is small, its value grows from the
different phenological stage before harvest reaching an average of 0.9 to 3 c m.
Table 2 Quantitative font -related characters in the Alex variety

Phenotypic aspects of Number of leaves leaf length (cm) It's time leaf (cm)
Triticum aestivum at: x ± sx s% x ± sx s% x ± sx s%
20-25 days from sprout 2.00 ± 0.00 0.00 14.96 ± 0.72 8.36 0.30 ± 0.08 4.26
150-190 days from sprout 4.38 ± 0.12 3.56 19,10 ± 1.27 7.05 0.93 ± 0.33 10.87
225-250 days from sprout 5.22 ± 0.08 2.62 21.77 ± 0.24 10.45 1,24 ± 0.82 4.57
before harvesting 5.22 ± 0.09 2.98 25.93 ± 1.06 1.91 1,36 ± 0.27 3.45
3. Quantitative characters related to the spice

The lance of the spice is revealed as soon as it begins to form and reaches the characteristic
values of the Alex variety before the harvest reaches an average of 7.18 cm.
Table 3 Quantitative characters related to spice in the Alex variety

Phenotypic aspects of Triticum aestivum at: Number of bristles spice length (cm)
x ± sx s% x ± sx s%
225-250 days from sprout 31.22 ± 0.73 4.03 7.18 ± 0.45 11.03
before harvesting 31.22 ± 0.73 4.03 7.18 ± 0.45 11.03
At 225 days from the sprout the number of bristles formed was higher, having a value of 32.22 in
average, value maintained before harvesting.
15
CONCLUSIONS
1. Before entering winter, the height had an average of 20.49 cm, because seedlings are
in a very small stage and the length of the internodes is 0.10 cm. Between 225-250 days from
sprout and before sprout the plants had a normal growth reaching a maximum value at plant
height up to 113.73 cm.
2. From the point of view of leaves, the differences are visible. Prior to wintering, the
number of leaves is small. In 225-250 days, the maximum number of leaves was 29,11 cm. The
width of the leaves begins to rise compared to the winter outbreak, the maximum being recorded
averaging 13.04 to 225-250 days from the sprout.
3. 225-250 days from sprout, the difference between the number of spikelets in the three
genotypes has a maximum value of 32.55% for triticale and before the harvest the maximum
value remains the same. The length of the spikes remains unchanged in both periods, and 225-
250 days, and before harvesting.
REFERENCES:
1 . BILETEANU GH., AL. SALONTAI, C. VASILICA, V. BIRNAURE, I. BORCEAN , 1991 -

Fitotehnie . Didactic and Pedagogical Publishing House, Bucharest.
2. BHOWMIK, A., MUREŞAN, T., BURLOI, GH., GHEORHE, V., 1982 - Heredity of protein
content in autumn wheat in a diaphragm crossing system. Problem. genet. theory. Applied, XIV,
5, pp. 391-400.
3 . BRONT, ILONA, CIOBANU, GH., 1995 - Need to know the state of soil fertility in order to
apply rational agricultural technologies. How to cultivate the land in the central area of the west
of the country. Oradea, pp. 24-30.
4 . BUNTA, GH., 1994 - Preliminary studies on the trait of wheat varieties and
lines. Year. Univ. Oradea, I, pp. 46-60.
5 . KABELEA, I., 1983 - Some statistical aspects of genetic analysis of production
capacity. Problem. gender. theory. Applied, XV, 1, pp. 31-49.
6 . CIOBANU, GH., 1995 - How to use chemical and organic fertilizers. How to cultivate the
land in the central area of the West. Oradea, pp. 31-39.
7 . GOMEZ, C., A., GOMEZ, A., A./1984 - Statistical Procedures for Agricultural Research. Ed.
J. Wiley and sons, Inc. Somerset, NY, USA, pp. 130-187.
8 . ITTU, GH., SÂULESCU, N., N., 1988 / c - Improvement of tolerance in triticale to toxicity of
aluminum. Int. Lived. Symp. Gulzow, pp. 95-102.
9 . ICAUFFMAN, M., D., GARDNER, E., H, 1978 / a - Segmental liming of soil and its effect
on the growth of wheat. Agronomy J., 70, pp. 331-336.
10 . KAUFFMAN, M., D., GARDNER, E., H., 1978 / b - A method for so small monoliths for
plant root studies. Agronomy J., 70, pp. 514-516.
11 . KOHLI, M., M., RAJARAM, S., 1988 - Wheat breeding for acid soils: review of brazilian /
CIMMYT collaboration, 1974-1986. Mexico, D., F., CIMMVT, Ed. Kohli, M., M., and Rajaram,
S., p. 50-65.
16
12 . LITTLE, R., 1988 - Plant soil interactions at low pH. Problem solving the genetic
approach. Soil Sc. Pl. Analysis, 19, pp. 7-12.
13 . PETR., F., C., FREY, K., J., 1996. Genotypic correlations, dominance and heritability of
quantitative characters in oats. Crop. Sc., Pp. 259-263.
14. RIEDE, C., R., CAMPOS, L., A., C., 1988 - Development of wheat cultivars with higher
yield and adaptation to different agroclimatic conditions of Parana. Wheat breeding for acid soils:
review of brazilian / CIMMVT collaboration, 1974-
15. SĂRAC I., PETRESCU IRINA , MADOSA E. , ANTONIE I. F. Copper effects at seed
germination of Secale cereale; JOURNAL of Horticulture, Forestry and Biotechnology; Volume
19(4), 84- 88, 2015
16 . TSVETKOV, K., S., 1994 - Tolerance of triticale cv. Vihren (2n = 6x = 42) to the toxicity of
aluminum. Heaven. Res. Comm., 22, pp. 257-263.
17 . VIDA, G., 1984 - Problems of genetic variability. The Possibilities of increasing genetic
variability in the plant kingdom. Martonvsar, pp. 201-203
17
OPEN SPACES AND ELEMENTS ADAPTED TO THE LANDSCAPE OF HORTOBÁGY
NATIONAL PARK
Fernando Arturo Mendez Garzón
Mentor: István Valánszki, PhD

Szent István University, Faculty of Landscape Architecture and Urbanism, Department of
Landscape Planning and Regional Development, Budapest - Hungary
Summary: The paper seeks to show a unique system of outdoor elements and open spaces,
designed specifically for the Hortobágy National Park in Hungary materialized in a group of
preset areas, outdoor furniture and signaling elements that fit harmoniously into the landscape.
The project arises from problems found them in the poor dissemination of information, low
identity with the surrounding, low resistance and quality of materials, impossibility of non-
Hungarian speakers to access the information, the difficulty of recognition and identification of
the places by the visitors and the inexistence of a common morphologic language in the elements
for all National Parks and also for the Hortobágy case. The proposal is framed in the project of
renewal and enhancement of the network of National Parks of Hungary led by the Ministry of
Agriculture.
The project's methodological framework is composed of a nonlinear axis, which covers different
levels of the process; Beginning with the data collection and literature review of worldwide cases
as well as of the Hungarian National Parks, then analysis of collected data and identify the
landscape character, establishment of design parameters, experimentation in the place,
formulate design alternatives, finally assess and qualify the proposal, design in detail and
validate the results.
Thus, integrating concepts such as identity, sustainability, technology, resistant materials, and
universality result in a proposal of high-quality design for the improvement of the
competitiveness and to enhance the people experience in the Hortobágy National Park and the
enjoyment of its landscape through the formulation of a viable and comprehensive design
proposal of open spaces and elements.
Key Words:Natural Parks, Open Space Furniture, Landscape Character, Tourist Services.
INTRODUCTION
Despite the relatively well-developed infrastructure and the great tourist potential of the network
of Hungarian Natural Parks including Hortobágy National Park, nowadays there is a weak system
of furniture, signaling, and lighting that does not fulfill the necessary requirements to satisfy a
growing, international and diversified tourist activity.
18
A comprehensive
ehensive and standardized system of these elements is fundamental for the proper use of
the tourist experience within the parks and that is why today, is easy to find as a trend around
several world parks; the high efforts, and investment in the field of ffurniture urniture and signaling
design, as an indispensable part in the development of landscape design because it directly
influences the experience of the tourist and locals within the national parks. These types of
elements beyond the function can become symbols symbols,, landscape marks and even part of the identity
of a place, hence the great importance of an accurate design and based on the particularities of
each site, likewise with the inclusion of concepts such as universality, usability, readability,
sustainability,, feasibility, etc. that create a sense of identity and belonging in a place. (Salcedo,
2008) In any case, the presence of these elements has been gradually promoting the development
of anomic socialization forms in which is easy to find a de de-structuring off the identity and
collective representations, segregation, and social hierarchy of the population, deficit of
information and difficulty of access to local cultures and permanent obsolescence of traditions.
(Miller, 1994)
The interactions of tourists and locals (users) with

each other and with the artificial elements placed in
those spaces and the type of meanings of this process
and the type of landscape signs (such as furnishing,
equipment, norms, signs, their spatial disposition,
their prioritizationn over time, their contextual
relevance, their ability to produce collective identity,
their quality, etc) become fundamental factors in the
regulation of citizen behavior and defines its
experience. (Salcedo, 2008) (Fig. 1) Fig. 1. Interactions
This shows that the rhythm of construction of the equipment, its location, functional and identity
characteristics and the operation and maintenance processes have not responded to the dynamism
experienced by the tourist boom and to the green development of the regions. (Rosling, Hans,
Gapminder, 2006) The equipment destined to represent the government is poor and deficient,
increasing the lack of ownership of the national and local entities in the National Parks.
Promoting statal presence depends on the correct supply of functional and cultural furnishings
and infrastructure. (Rosling et al., 2006)
The project seeks to enhance the experience of people in the Hortobágy National Park through
formulating a proposal of viable and integral design of artificial elements in and for the landscape
landsc
that allows a better interaction with the natural landscape.
19
Hortobágy is an 800 square kilometers national park located in eastern
Hungary filled with folklore and cultural history. The park, which forms
part of the Great Plain of Hungary, has been listed as a World Heritage
Site. (Junaite, 2017) It is situated in an area where a unique shepherding
culture has evolved over many centuries, manifesting itself in distinctive
traditional styles of clothing and architecture, and the tendingt of
particular types of animals that are not found anywhere else. (Hungary
Guide, 2009) Hortobágy is the largest continuous natural grassland in
Europe, it was established in 1973 as the country’s largest protected
area. Hortobágy has outstanding nat natural
ural features, maintaining great
biological diversity in respect of species and habitats. It is a unique
example of the harmonious coexistence of people and nature based on
the careful use of the land. (Nemzetipark
(Nemzetipark-Igazgatóság,
Igazgatóság, 2017) (Fig. 2)
The proje
project's methodological
framework is composed of a
nonlinear axis, which covers
different levels of the process;
Beginning with the data collection
and literature review of worldwide
cases as well as of the Hungarian
national parks, then analysis of
collected information and identify
the landscape character, establishment of Fig. 3. Methodology.
design parameters, experimentation in the
place with real materials and resources, formulate design alternatives,
Fig. 2. Hortobágy NP. finally assess and qualify the proposal, design in detail and validate the
Location. results. (Fig. 3)
 The data collection: grouping, collecting and gathering the information from different kinds of
media (digital media, brochures, papers and visits to the natural parks). The analysis of collected
information: at this level, the informati
information
on was analyzed and the relevance of this was verified. In
addition, within this analysis, the existing site (Hortobágy NP) was studied, looking at the
existing typologies of artificial elements and the study of primary and secondary sources. The
analysis of case studies abroad (United States, Spain, Colombia, and Thailand) was also made
and the analysis of the implementation site (structure, functions, materials, hierarchy, etc.) began.
 The establishment of design parameters: at this level, the design pa parameters
rameters of both the objects
and the space between those that stood out were proposed; production processes, evaluation
methods, levels of resistance and security, levels of adaptability, identity with the site.
20
 The formulat design alternatives: at this llevel, evel, design alternatives were proposed through
various methods such as; brainstorming, establishing design concepts and methodologies or
adapting existing ones.
 The verification of proposals was made
through fulfilling requirements (must
comply); Resistance,ance, usability, costs,
identity, mainteance and stetic value. The
table shows how the requirement can be
accomplished. (Fig. 4)
 The validation of the results, comparing
and weighing them: at this level all the
checks are validated by comparing them
with each other and with other successful
Fig. 4. Table of Requirements.
cases abroad, measuring the degree of user
satisfaction, finally, all the steps are fed back to make the integral evaluation of the project.
The implementation spaces are the fundamental

axes off the project. These are five patterns that can
be found in most of the Hungary's National Parks.
These spaces have particular characteristics of;
uses, users, functions, elements, that can be
differentiated easily but at the same time can be
improved and replicated along all the National Park
Network through the proposal of landscape design.
(Fig. 5)
Fig. 5. Hortobágy NP. Visitor Centre.
21
Within the implementation site, it is
possible to find some public
buildings and some private ones, to
the north the Visitor Center is
placed where the Museum eum of
Hortobágy Steppe is located, the
administrative offices of the park,
the store of souvenirs and
handicrafts, the parking for official
use. Crossing the avenue can be
found the entrance to the park and Fig. 6. Master Plan.
inside it; the parking lot, a small shrubs garden, an area for temporary
market kiosks within the same parking lot, public rrestrooms, estrooms, the exhibition room of typical
handicrafts, the museum of grazing and a wider area of parking. Taking direction to the
southwest and close to the river there is a pedestrian path that leads to a relatively new open-air
open
theater and beyond is a pedestrian
edestrian bridge that crosses the river towards the park and the wildlife
refuge. (Fig. 6)
The cultural landscape of the Puszta represents

the highest scenic quality, with pleasing and
dramatic patterns and combinations of
landscape features which give it a distinctive
character, including aesthetic qualities and
topographic and visual unity. The unbroken
horizon is only occasionally disrupted by trees,
groves, settlements or linear establishments.
made elements fit harmoniously into this Fig. 7.landscape
Human-made Hortobágy NP. Contemplation Space.
and sustainable land-useland
practices have contributed to the conservation of a diversity of species and biotopes and the
maintenance of the landscape. (UNESCO, 2018) (Fig. 7)
The proposal was designed based on the

landscape character of the National Park
Park,
using a table of parameters and requirements
to fulfill including, morphology,
topography, flora, local materials,
production, usability, and environmental
conditions. Advantages: Optimizes and Fig. 8. Hortobágy NP. Contemplation
ntemplation Space,
transform the perception of the landscape within the Nationa Cross Section.
Nationall Parks through a unique
morphological concept, creates a unified and standardized identity between the parks and the
elements, likewise enhances the identity of each park with the design of elements with specific
22
and unique features of the site. Generates an economically, socially and environmentally
sustainable system of elements achieving a harmonious and integrated balance between site and
users. Finally, takes advantage economically the tourism potential of the National Parks. (Fig. 8)
CONCLUSIONS
The natural landscape in the National Parks is almost already defined and that means that rural
areas need a different approach than urban areas, this different approach makes that there is not
necessarily a strong physical modification of the landscape to preserve the natural landscape of
the parks as much as possible and enhance the perception of the landscape through the open
space design in key sites. (Elements + Spaces + Functions = Open Space Design)
The public space must be understood as a potentiality of life in a community that allows the
appropriation and collective consumption of the spaces. It works as a support for economic,
social and cultural activities and permits mobilization and social interactions. (Salcedo, 2008)
The public space, the quality, and quantity of furniture and equipment define and determine the
citizen interactions, the level of identification and the forms of appropriation that are established
with the landscape and the environment. This means defining and establishing geographical,
rural, architectural, functional, historical, symbolic elements that allow building an image of
public, accessible and enjoyable National Parks, capable of becoming the object of people´s
perceptions, the imagery of the landscape and source of country identity.
REFERENCES
HUNGARY GUIDE. (2009). National Parks Hungary - Pure Nature. Retrieved from:
http://national-park.hungaryguide.info/
INADA, J.I. (2018). Between Landscapes and Gardens. A Collection of Jun-Ichi Inada´s
Landscape Architecture Work´s. Singapore.
JUNAITE, M. (2017). Culture Trip - The Most Beautiful National Parks in Hungary. Retrieved
from: https://theculturetrip.com/europe/hungary/articles/the-most-beautiful-national-parks-in-
hungarythe-most-beautiful-national-parks-in-hungary/
MILLER, J.A. (1994) Signs & Spaces. Rockport Allworth Editions.
NEMZETIPARK-IGAZGATÓSÁG. - Ministry of Rural Development (2014). Magyar Nemzeti
Parkok. Heritage Interpretation Sites.
ROSLING, HANS Y GAPMINDER. (2006) Diseño de información y desarrollo humano.
Retrieved from: http://terremoto.net
SALCEDO, J.P. (2008) Theoretical Designer Specialist in Creativity.
UNESCO. (2018). World Heritage List - Hortobágy National Park - the Puszta. Retrieved from:
https://whc.unesco.org/en/list/474 http://national-park.hungaryguide.info/hortobagy-puszta.html
23
MORPHOLOGICAL DIFFERENCES OF FOREIGN GENOTYPES OF
Corylus avellana L.
COSTEA Marinela, ARDELEAN Abel
Mentor: SARAC Ioan

ionutsarac@yahoo.com
University of agriculture and veterinary medicine `KING MICHAEL I` of Romania,

Faculty of Horticulture and Silviculture, Romania.
Summary: Hazelnut is a species that is suitable for the conditions of culture offered by
our country (west of the country-hill area).
Growth in culture is due to the growing demand for nuts, nutritional value and use in the
food industry (sweets).
The study aims to assess the vegetative growth of imported hazelnut varieties in the first
three years after planting to highlight the degree of adaptation to climate conditions in the
western part of the country.
Key words: hazelnut, genotypes, variability, cantitativ caracters.
INTRODUCTION
World demand for hazelnuts is on the rise . Over the last 10 years, the value of shelled
peanuts on the world market was 7.8 euros / kg. Romania imports 90% of nuts, which makes the
peanut market an attraction for any grocer.
Average annual take for f peanut in shell world is about one million tons (FAO 2014),
an increase of 35% et growth compared to 2000.
Hazel is native to Asia Minor and the Black Sea, where it then spread Peninsula
Bal Canica, Turkey, Greece, Italy, France, Spain, North America and others.(Gürcan et al., 2010)
The world production of hazelnuts for the period 1989-1991 was 917 thousand tons,
in 1996 - 1,064 thousand tons, in 1997-1,120 thousand tons, and in 1998 - 1,108 thousand tons
(of which Asia 559 thousand tons, Europe 290 thousand tons). The main cultivating countries are
Turkey (120 thousand tonnes); Italy, Japan, Iran, USA, Greece, France etc.
In our country, the hazelnut is present as a spontaneous species in the slopes and ridges of
hardwoods in the hilly and hilly area of the country. Hazelnut orchards are very limited in
number (Fruit Growing Research Station Ramnicu Valcea, Falticeni Ştefăneşti Arges), the
hazelnut being cultivated more in family gardens. The recent date
(2010) began to appear commercial plantations in counties Timis ,Sibiu, Alba, Mures and Cluj.
(Merce R. 1997)
24
MATERIAL AND METHOD
Trend current is to cultivate fruited spherical , more advantageous for mechanization extra
ction the core , how much and for industrialization by Dating with different glazes .At us into
the country is being cultivated preponderant Red Lambert variety ,which has force middle,
leaves and involucre colors into the red-green , has fruits small , ovoid , caught 5-
10 in involucre , 53-59% core . It maturesat the end of August .
MATERIAL. OTHER VARIETIES IN THE STUDY

Tonda Gentile delle Langhe . Variety of middle-high force, very productive, with small
spherical fruits, grouped 2-4, with 42-47% core. It matures at the end of August
and early September.
Tonda Gentile delle Langhe is the main cultivar grown in the Hazel area of Piedmont in
northern Italy.
Butler . Strong, resistant to bacteriosis, with a large fruit that contains 47-49% core
and matures at the end of September.
Butler was cultivated as early as 1957 in the United States. It seems to be a hybrid
between Barcelona and Daviana (USDA, 2010).
Tonda Gentile Romana . It is an Italian variety. It is baking at the end of August. Fruits
grouped by 2-4, less often up to 6, suitably large (2,2-2,7g), spherically-rolled with three angular,
prominent ribs, have a pronounced and irregularly convex base, sometimes flat, slightly curly.(
Paolo Boccacci , Roberto Botta; 2008)
Tonda di Giffoni It is a kind of right-wing, middle- strength crown. The core of
the medium, half-round (1.2g), the nut has a weight of 2.5 grams and the peel high yield (46%).
The productivity is high and constant of 3-3,5 t / ha. Baking takes place
in early September. Pollenizers: Tonda Gentile Romana, Camponica , San Giovanni.( Milena
Petriccionea Loredana F. Ciarmielloa Paolo Boccaccib Antonio De Lucaa Pasquale Piccirillo,
2010).
BARCELONA . Bake in late August and early September. Fruits, grouped 2-5, large and
very large (2.8-4.5g), spherical, with slightly convex base . Involucer is
a little longer than honey. The bark of the thick, chestnut color isdarker , thinner , thin and has a
fine pubescent in the upper half .
WORKING METHOD
Making observations in culture.
In research on genetic determinism, genes and their actions cannot be emphasized as such but by
studying their effects, phenomena appearing outwardly. If the genotype shows all
the hereditary possibilities , whether they are manifest or latent, the phenotype includes all the
characters and the attributes of an organism, externalized at a given time under certain concrete
environmental conditions . As such, the phenotype is the result of the interaction of thegenotype
25
(hereditary dowry) with the environment (Sarac, 2005) May many years
of study to characters and qualities of hazelnut varieties for of highlight variability expressed in e
nvironmental conditions exchangeable (Hernea C. 2016).
In the climatic conditions of the Timisoara Teaching and Experimental Center

( Plant House), five varieties of foreign origin were studied, the variability of the quantitative
characters in the period 2016-2018 was analyzed.
VARIABILITY OF THE HEIGHT GROWTH OF Corylus avellana L.

Within the sap height character (cm) it is found that 3 varieties of the studied
ones, Tonda Gentile Delle Langue , Butler and Tonda Di Giffoni , are over the average of
experience, x + sx = 89.82 ± 9.06 , and the Tonda Romana and Barcelona varieties are below the
average in the 2016 study year.
Tonda Gentile Delle Speaker deviates dinstinctiv semnificatv positive than the average ±
SD indicated with d = + 20.98 ± 21.69, and the variety dinstinctivBarcelona deviates significantly
negative d = -35.93 ± 15.32 ± sd.
In 2017, the same varieties are found to be above the x + xs = 119.42 ± 7.08 average, and
the Tonda Romana and Barcelona varieties are below the average. The largest increase over the
mean value is represented by the Butler variety with d ± sd = + 16.48 ± 22.20 and the lowest
increases in the Barcelona variety with d ± sd = -23.64 ± 11.76
In the year 2018 the varieties with values above the average x + xs = 153,63 ± 3,68 are
those mentioned in the previous years, Butler variety showing positive differences d ± sd = +
13,90 ± 13,15 and the variety Barcelona showing a negative deviation with d ± sd = -17.48 ±
7.95(Fig. 1).
26
Height increase
180 164.7 167.53
160 146.27153.48
135.9 136.1 [VALOARE] 136.15
140
110.8 107.1 107.44121.89
120 [VALOARE]
92.22 95.78
100 85.1
[VALOARE]
80
53.89
60
40
20
0
2016 2017 2018
V1.Tonda Gentile V2.Butler V3.Tonda Romana

V4.Tonda di Giffoni V5.Barcelona Media
Figure 1. Variability of height increase (cm) of Corylus seedlings avellana L
VARIABILITY OF THE INCREASE OF THE BUSH DIAMETER (CM) OF

Corylus avellana L.
The character of the diameter of the bush (cm) shows that during the study years the
varieties behave differently from the average. In 2016, the variety with the highest value of the
bush diameter is Tonda Gentile Delle Languewith 46,60 ± 5,92cm compared to the mean
varieties s ± sx = 39,30 ± 4,14 with a positive difference of d ± sd = + 7,3 ± 10,06 and the
Barcelona variety presents the lowest value of this character with x ± sx = 28,22 ± 3,14 ie d ±
sd = -11,08 ± 7,28 from the said average.
In 2017, the ice-varieties under study shows higher values than the average acestra x +
sx = 78,11 ± 3,09 and the second has lower values. The variety with the largest value of the bush
diameter is Butler with x ± sx = 88.00 ± 5.06 with d ± sd = + 9.89 ± 8.15. The smallest wilder is
recorded in the variety Tonda Romana with x ± sx = 68,33 ± 5,88 with a difference d ± sd = -9,78
± 8,97 compared to the varieties average.
Under the conditions of 2018, three varieties have higher values than the average x ± sx =
99,07 ± 2,50 cm and two values lower than this average. The Butler variety shows the highest
mean value x ± sx = 105.73 ± 6.32 and with a difference d ± sd = + 6.73 ± 8.82cm. (Fig.2)
27
Diameter of the bushes
120 105.73 102.76
96.53 91.25 98.53
100 88 [VALOARE]
83.33
77 68.33 73.89
80 [VALOARE]
60 53.3
46.6
35.8 32.56
40 39.296 28.22
20
0
2016 2017 2018

Figure 2. The variability of the diameter of the bushes (cm) of Corylus seedlings avellana L.
VARIABILITY NUMBER OF BRANCHES / BUSHES JUVENILES FROM

Corylus avellana L.
At the number of branches / plant number ( superior buccaneers vs. average) is registered
during the 3 years of study in the varieties Butler, Tonda Romana and Barcelona. The highest
value compared to the averages of the study years is recorded in the Barcelona variety with d ±
sd = + 0.67 ± 0.47 / 2016, d ± sd = + 1.10 ± 0.85 / 2017 and d ± sd = + 1.28 ± 1.04 / 2018 versus
the averages of the crop years that.
The varieties with the lower values of the averages of the growing years
are: Tonda Gentile Delle Langue , Tonda di Giffoni . The smallest values of the number of bush
branches were recorded in the Tonda di Giffoni varietywith mean values of 1.43 ± 0.20 / 2016
and with a difference of δ ± sd = 0.00 ± 0.37 / 2016, with an average of 1.67 ± 0.30 and with
a score of d ± sd = 0.86 ± 0.79 / 2017 and an average value of 3.13 ± 0.61 and with a difference
of - 1.06 ± 1.01.
VARIABILITY OF CHLOROPHYLL CONTENT OF Corylus avellana L.
The chlorophyll content of hazelnut varieties studied during the observation period
was Clorofit Minolta SPAD 502.
As a result of the analyzes carried out, there is a great variability in the chlorophyll
content of the varieties studied during the research. The variety with the lowest value of
chlorophyll is represented by Tonda Romana, and the one with the highest value of
Barcelona. There is a variation in colorophilic content over the years of research.
(Fig.3). (Ludger et al., 2013)
28
Chlorophyll
60
50.5 50.53 49 46.67
46.17 46.2 43.6 47.97
50 44.4 44.17
47.4 42.1738.47 40.53
[VALOARE] [VALOARE] 38.77 38.1
40
30
20
10
0
2016 2017 2018

Fig. 3. The variability of the chlorophyll content of Corylus varieties avellana L.
CONCLUSION
The varieties studied are from Vitro Plant company in Italy, the majority
of Italian origin (or the USA) for multiplying the in vitro culture and expansion, in order
to obtain the peanuts used in the fine candy industry (FererroRocher).
Following the in vitro multiplication, the biological material obtained was used 80% in
Europe and 20% for the establishment of hazelnuts in our country (in the West).
Following experimentation and the results obtained to find out that the variety that shows
the best plasticity in culture, is the variety Tonda di Giffoni, the next variety
is Tonda Romana. The least plasticity in the case of in vitromultiplication was manifested by the
Butler variety (USA).
The greatest adaptability to the transition from in vitro culture to greenhouse cultivation is
represented by the same varieties, Tonda di Giffoni and Tonda Romana, and the smallest is the
Butler variety.
The great productivity and once again the best economy, among the varieties studied, is
the Tonda di Giffoni variety .
REFERENCES
Sarac I., 2005, Genetics and the improvement of forests

pecies, Mirton Publishing House Timisoara
Hernea C., Corneanu M., Sarac I., Turcu E., 2016 ;The potential of Romanian and Swedish
willow for short rotation coppice in the 2specific condition of Banat plain A comparative analyze,
29
Petrescu I., Coradini Cristina, 2010 , Morphological aspects of Calendula officinalis L. flowers
collected from different localities of Timis district. No.7 pp.74-75
Paolo Boccacci , Roberto Botta; 2008, Genetic Diversity of Hazelnut (Corylus avellana L.)
Germplasm in Northeastern Spain.
Ludger L.,Wilfried S. ,Bernhard H. ,Oleksandra K. ,Wolfgang A., Barbara F., Bolko H., Ralf K.,
Martin R., Reiner F., 2013; Genetic variation of chloroplast and nuclear markers in natural
populations of hazelnut (Corylus avellana L.) in Germany;, February 2013, Volume 299 , pp
369–378
Merce R. 1997. Genetic; Variability among hazelnut (Corylus avellana L.) cultivars
Tufan Gökirmak Shawn A. Mehlenbacher Nahla V. Bassil, 2009, Characterization of European
hazelnut (Corylus avellana) cultivars using SSR markers, Volume 56, pp 147–172
K. Gürcan S. A. Mehlenbacher V. Erdoğan ,2010, Genetic diversity in hazelnut (Corylus
avellana L.) cultivars from Black Sea countries assessed using SSR markers,
Milena Petriccionea Loredana F. Ciarmielloa Paolo Boccaccib Antonio De Lucaa Pasquale
Piccirillo, 2010, Evaluation of ‘Tonda di Giffoni’ hazelnut (Corylus avellana L.) clones.
30
MICROBIOME AND IMPORTANCE OF PGPR IN RHIZOSPHERE OF MAIZE (ZEA
MAYS L.)
Nikola HRICÁKOVÁ1, Renata CINKOCKI2
Mentors: 1doc. Ing. Jana Maková, PhD., 2prof. Ing. Soňa Javoreková, PhD.
Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Sciences,

Department of Microbiology, Tr. A. Hlinku 2, 949 76, Nitra, Slovak republic
Summary: Rhizosphere is an environment in the soil, where the most significant interactions
occur between the plant and the microorganisms, which significantly affect its growth and the
overall crop. Metagenomic analysis revealed the dominant presence of Proteobacteria and
Actinobacteria in all samples. The difference was observed in the area of roots (soil, rhizosphere
and rhizoplane) compared to samples taken from the endophytic region of the plant (root, stem
and leaf). The most striking changes were observed in the percentage of bacteria from
Proteobacteria, Actinobacteria and Acidobacteria strains. In maize rhizosphere, we have
selected the three most active strains of Bacillus, Enterobacter and Pseudomonas by following
the biochemical features (IAA, phosphate dissolution, siderophore production, antifungal
activity).
Key words: bacteria, rhizosphere, metagenomic analysis, biochemical features
INTRODUCTION
Analysis of bacterial communities based on nucleic acid isolation allows us to monitor

differences in diversity of rhizosphere microorganisms but also in soils and plants (Gelsomino et
al., 1999). The identification of microorganisms is based on the sequencing of 16S rRNA
fragments by PCR and subsequent second generation sequencing (Engelhard et al., 2010). The
resulting sequences can be applied when classifying microbial populations and determining the
percentage of microorganisms within taxonomic groups.
Microorganisms play an irreplaceable role in every component of the environment. Their activity
is mediated by 80 – 90 % of soil reactions (Coleman and Crossley, 1996). Their functions and
processes affect not only themselves, but also other organisms, especially plants. Interactions
between microorganisms and plants play a key role in the mobilization, transformation and
solubilisation of nutrients (Franco-Correa et al., 2010). Microorganisms are most frequently
present in the places around the root system (rhizosphere) of the plants and decrease their number
from the rhizosphere (Hu et al., 1999). The community of microorganisms in the rhizosphere
significantly influences the nutrient acquisition, the development of phytopathogens and the
resistance to heavy metals. The source of energy for rhizosphere bacteria is mainly in form of
31
root exudates. Their presence is the reason for a different microbial settlement in the area of the
rhizosphere and root surface compared to microbial settlement of the soil (Balík et al., 2008).
The aim of our work was to determine the microbiome of maize in the area of roots (soil,
rhizosphere, rhizoplane) as well as in the plant itself in the endophytic region. In the rhizosphere
area we also observed a significant group of plants growth promoting bacteria and their
bioactivity.
Samples were taken in Kolíňany (40 ° 26'46''N, 79 ° 58'56''W), Slovakia, from 6 randomly
selected Zea mays L. plants in the phenological growth phase of BBCH 15.
The samples were labelled after sampling as follows:
a) soil samples
- soil outside the root system (Soil),
- soil from the rhizosphere (Rhizosphere),
- plant root rinsing (Rhizoplane),
b) plant samples (endophytic sphere)
- Root, Stem, Leaf.
We obtained rhizoplane samples (plant root rinsing) by shaking 10 g of roots in 90 ml of saline
for 20 minutes. Samples of root, stem and leaf were cut and subjected to three-stage sterilization
according to Suna et al. (2008). Samples were sterilized for 60 seconds in 99% ethanol, 6 minutes
in 3,125 % NaOCl (SAVO), 30 seconds in 99 % ethanol, and the last rinse was rinsed in sterile
osmotically treated water, which was repeated 3 times. We used the third rinse as a control
sample. The samples were tested in three replications (labelled as a, b, c).
The environmental DNA was isolated from the MOBIO PowerSoil DNA Kit as instructed by the
supplier. To amplify the V4-V5 region of the 16S rRNA gene, the 515F and 806R primers were
used (Caporaso et al., 2011). The PCR reaction was run in a Surecycler 8800 thermocycler in 96-
well plates. Using a purification kit (Jena Bioscience), PCR products were purified and quantified
by Qubit using fluorimetric measurement. PCR products diluted to the same concentration were
mixed and subjected to PCR with another Illumina XT index kit. The indexed products were
requantified, purified, diluted and loaded onto the Illumina MiSeq cartridges. Sequences were
automatically stored on an Internet repository. Subsequently, they were processed in the SEED 2
program (Vetrovsky et al., 2018), where we removed the poor sequences.
Bacteria from the field of the rhizosphere of the maize (Zea mays L.) were still isolated on TSA
agar, purified and tested for their ability:
1. produce indole-3-acetic acid (IAA) by the method of Gordon and Weber (1951),
2. phosphate solubilizing on the Pikovskayas agar by the method of Gupta et al. (1994),
3. produce siderophores using a CAS agar according to Schwyn nad Neilands (1987),
4. antifungal activity against phytopathogenic fungi strain Rizoctonia sp. and Sclerotinia sp.
according to Sgroy et al., 2009. Inhibition of growth of mycelial phytopathogenic fungi was
evaluated by Quiroga et al. (2001).
32
Bacterial isolates were identified by the MALDI TOF method according to the manufacturer's
protocol (MALDI Biotyper 3.0 Bruker Daltonics GmbH, 2011).

From soil and plant samples, we were generating 100 051 16S rRNA sequences using second
generation sequencing. In soil samples (soil, rhizosphere, and rhizoplane), we found that diversity
was different in percentages of the individual strains compared to plant samples (root, stem and
leaf). The diversity of the bacteria of the individual samples was determined at the strain level
(Fig.1). The presence of individual strains within the soil samples was as follows:
Proteobacteria (18.43 – 31.27 %), Actinobacteria (6.78 – 7.96 %), Verrucomicrobia (4.19 – 7.95
%), Thaumarchaeota (5.06 – 16.95 %), Acidobacteria (17.72 – 28.80 %), Firmicutes (1.59 – 3.58
%). The highest representation of the bacterial strains was in the rhizoplane, while the lowest was
recorded in samples outside the root system (soil). Pereira et al. (2011) analysed bacterial
diversity in soil of maize and found that bacteria from the strain of Proteobacteria prevailed
within the microbiome. Significant differences were also found among the numerous endophytic
bacteria in plant samples. The representation of the individual strains of bacteria was as follows:
Proteobacteria (56.43 – 65.74 %), Actinobacteria (8.85 – 15.78 %), Verrucomicrobia (0.027 –
5.48 %), Thaumarchaeota (0.14 – 7.87 %), Acidobacteria (0.14 – 2.25 %), Firmicutes (9.74 –
20.35 %). The highest number was recorded in stem, while the lowest was in leaf. The most
significant changes between soil and plant samples were observed in the percentages of bacteria
from Proteobacteria, Actinobacteria and Acidobacteria strains as well as the Kniefa et al. (2012),
who studied the bacterial diversity of rice (Oryza sativa) in the area of the rhizosphere and leafs.
We have also focused more on analyzing the diversity of bacteria from the rhizosphere. Bruto et
al. (2012) reported that the most prominent representatives of PGPR bacteria belong to the
Proteobacteria strain. In our rhizosphere samples, the following classes were most frequently
found in the strain: Actinobacteria (7.79 %), Betaproteobacteria (7.06 %), Alphaprotebacteria
(4.86 %), Gammaproteobacteria (4.57 %) and Bacilli (1.08 %). Unlike our results, Roesch et al.
(2007) recorded the highest rate of bacteria in the Gammaproteobacteria (8.43 %) and the lowest
of Actinobacteria (0.95%) in rhizosphere of the maize. In the rhizosphere of the observed maize,
the most common bacteria of the genus were: Actinomyces, Azotobacter, Bacillus, Brevibacillus,
Bradyrhizobium, Enterobacter, Pseudomonas, Rhizoboacter and Serratia.
33
Microgenomates
100% Woesearchaeota
Deinococcus-Thermus
90% Spirochaetes
Hydrogenedentes
80% Parcubacteria
Crenarchaeota
70% BRC1
Cyanobacteria/Chloroplast
60% Ignavibacteriae
Latescibacteria
50% Chlamydiae
Armatimonadetes
40% Chloroflexi
Nitrospirae
30% Euryarchaeota
Fusobacteria
20% Gemmatimonadetes
candidate division WPS-1
10% Planctomycetes
Candidatus Saccharibacteria
0% Bacteroidetes
Firmicutes
Acidobacteria
Thaumarchaeota
Verrucomicrobia
Sample groups Actinobacteria
Proteobacteria
Fig.1 Graph showing the microbial diversity of soil, rhizosphere, rhizoplane, root, stem and leaf at the
strain level
Metagenomic analysis revealed the microbial diversity of the rhizosphere, but our aim was also to
investigate the biochemical activity of these bacteria by culture methods. Of the total number of
isolates (11) we selected the most active three by the biochemical assay, which we identified by
the MALDI TOF method at the genus level, such as Enterobacter sp., Pseudomonas sp. and
Bacillus sp. The same biochemical traits were tested in Enterobacter sp. by Kumar et al. (2014),
Pseudomonas sp. by Jošić et al. (2011) and Bacillus sp. by Došen (2015).
Values of the observed biochemical features are shown in Table 1. As shown, the most active
bacterial isolate identified as Pseudomonas sp., which had a low index of dissolution of
phosphate (1.65 mm), shown is a positive production of siderophores and IAA produced at a
10.56 μg.ml-1. Growth of phytopathogenic fungi of the genus Sclerotinia sp. and Rhizoctonia sp.
inhibited to over 80%.
34
Table 1: Bioactivity of selected isolates of PGPR
Antifungal
Bacterial isolation
activity %
IAA µg.ml-1
Identification
Siderophores
SI mm
(genus)
Classes
Rhizoctonia
Sclerotinia
sp.
sp.
KmiJP17 Enterobacter
Gamma- 1.29 15.74 + - +++
B089 sp.
proteo-
bacteria KmiJP17 Pseudomonas
1.65 10.56 + +++ +++
B090 sp.
KmiJP17
Bacilli Bacillus sp. 2.96 3.13 - +++ ++
B091
SI – Solubilization index; IAA - Indole-3-acetic acid; Production of siderophores – (negative), + (positive);
Antifungal Activity - ( 10%), ++ (40 – 80 %), +++ (> 80 %)
CONCLUSION
Our research, we found that the highest proportion of bacteria of the strain Proteobacteria was
found in soil samples in rhizoplane and plant samples in stem. In terms of biochemical activity in
the area of the rhizosphere we were isolated 11 bacteria including 3 isolates showed high
bioactivity. Especially the isolate KmiJP17B090 (Pseudomonas sp.) appears to be emerging
(high IAA production, positive production of siderophores and antifungal activity against
phytopathogen) for further research on its use as a component of biofertilizers and biopesticides.
ACKNOWLEDGEMENTS
This work was supported by the National Institutes of Ministry of Education, Science, Research
and Sport of the Slovak republic, by the Slovak Research Grant Agency from projects VEGA No.
1/0305/17, KEGA No. 014SPU4/2017 and APVV No. 150543.
REFERENCES
Balik, J. (2008) Relations between polyphenols content and antioxidant activity in vine grape and
leaves. Czech Journal Food Science, 26, 25 – 32.
Caporaso, J.G. - Lauber CH.L. et al. (2011) Global patterns of 16S rRNA diversity at a depth of
milions of sequences per sample. PNAS, 108 (1). DOI:10.1002/jobm.201000171
Coleman, D. C. – Crossley, D. A. (1996) Soil ecology.
35
Došen, Nataša. (2014) Proizvodnja i primena mikrobiološkog preparata „Bacillomix specijal“ :
master rad. Novi Sad : Pojoprivredni fakultet. 63 s.
Franco – Correa, M. et al. (2010) Evaluation of actinomycete strains for key traits related with
plant growth promotion and myccorrhiza helping activities. Apllied Soil Ecology, 45 (3), 209 –
217.
Gordon, S. A. – Weber, R. P. (1951) Colorimetric estimation of indole acetic acid. Plant
Physiology, 26, (1) 192 - 195.
Gupta, R. et al. (1994) A modified plate assay for screening phosphate solubilizing
microorganisms. Journal of General and Applied Microbiology, 40 (3), 255 - 260. DOI:
https://doi.org/10.2323/jgam.40.255
Hu, S. J. et al. (1999) Dynamics of bacterial populations in relation to carbon availability in a
residue–amended soil. Applied Soil Ecology, 13 (1) 21 – 30.
Jošić, D. et al. (2011) Selection of indigenous fluorescent pseudomonad isolates from maize
rhizospheric soil in Vojvodina as possible PGPR. Romanian Biotechnological Letters, 16 (5),
6580 – 6590. ISSN 1224-5984.
Knief, C. et al. (2012). Metaproteogenomic analysis of microbial communities in the
phyllosphere and rhizosphere of rice. The ISME Journal, 6, 1378 – 1390.
Kumar, A. – Maurya, B. R. – Raguwanshi, R. (2014) Isolation and characterization of PGPR and
their effect on growth yield and nutrient content in wheat (Triticumaestivum L.). Biocatalysis and
Agricultural Biotechnology. 3, 121 – 128. DOI: http://dx.doi.org/10.1016/j.bcab.2014.08.003.
Periera, et al. (2011). Analysis of the Bacterial Diversity Associated with the Roots of Maize
(Zea mays L.) through Culture – Dependent and Culture – Independent Methods. ISRN Ecology,
2011, 1 – 10. DOI: 10.5402/2011/938546.
Quiroga, E. N. – Sampietro A. R. – Vattuone, M. A. (2001) Screening antifungal activities of
selected medicinal plants. Journal of Ethnopharmacology 74, 89 – 96.
Roesch, L. F. W. – Passaglia, L. M. P. – Bento, F. M. – Triplett, E. W. – Amargo, F. A. O.
(2007). Diversity of diazotrophic endophytic bacteria associated with maize plants. Revista
Brasileira de Ciencia do Solo, 31 (6). DOI: dx.doi.org/10.1590/S0100-06832007000600015.
Sgroy, V. – Cassán, F. – Masciarelli, O. – Del Papa, M.F. – Lagares, A. – Luna, V. (2009)
Isolation and characterization of endophytic plant growth promoting (PGPB) or stress
homeostasis-regulating (PSHB) bacteria associated to the halophyte Prosopis stombulifera.
Applied Microbiology and Biotechnology, 85 (2) 371 - 381. DOI:
https://doi.org/10.1007/s00253-009-2116-3.
Sun, Y. – Lin, L. et al. (2008) Structural changes of bamboo cellulose in formic acid.
BioResources, 3(2). DOI: http://ncsu.edu/bioresources
Vetrovsky T., Baldrian P. et al. (2018) Effects of land use on the level, variation and spatial
structure of soil enzyme activities and bacterial communities. Soil Biology and Biochemistry, 34
(13) DOI:https://doi.org/10.1016/j.soilbio.2011.03.018
36
GERMINATION AND PROTOCORM FORMATION OF OPHRYS SPHEGODES MILL.
– IN VITRO PROTOCOL FOR AN ENDANGERED ORCHID SPECIES
Ines Prlainović
Mentor: Mirjana Ljubojević, assistant professor

University of Novi Sad, Faculty of Agriculture, Department for Fruit Science, Viticulture,
Horticulture and Landscape Architecture, Serbia
Summary: Ophrys sphegodes Mill. is protected and threatened wild species, due to its
pollination biology, small seed and habitat destruction. The aim of this study was to establish
asymbiotic germination protocol for the purpose of ex situ conservation. Two basal media KC
and MM, with organic additives (peptone, L-glutamine, folic acid, casein hydrolysate) and
control media KC-K and MM-K. were used in the present research. Seeds were examined under
two illumination conditions, 0/24 light/dark and 16/8 light/dark. According to the obtained
results, the best results were achieved on MM-PE media, cultured in dark.
Keywords: conservation, terrestrial, ex-situ, orchids
INTRODUCTION
Ophrys sphegodes Mill., also known as the early spider-orchid, is a species of terrestrial
orchids (family Orchidaceae) found on alkaline meadows, chalk or limestone. Although O.
sphegodes is widespread across Europe and the Middle East, it is considered rare and vulnerable.
In Serbia, O. sphegodes is strictly protected wild species by CITES convention. On the other
hand, many of the remaining populations on the British Isles consist of very few plants
(Hutchings, 1989). According to the mentioned author, if the rate of decline observed over the
last fifty years continues, the species would disappear from the British Isles by the end of this
century. Hutchings (1989) stated that the main reasons why these orchids are threatened are
climate changes and, related to that, their pollination biology. O. sphegodes reproduce through
sexual deception of male Andrena nigroaenea bees (pseudocopulation). Slightly warmer springs
are causing the males to emerge much earlier, meaning they are less synchronized with the
orchids’ flowering (Davy et al., 2014). Furthermore, other extrinsic factors involved in
population reduction are habitat extermination, often for real estate land, and plant exploitation
for ornamental and edible purposes. The basic problem with terrestrial orchid propagation, in
general, is a small probability of seed germination due to its extremely small size. The orchid
seed has an undifferentiated embryo and lack of nutrients for growth. Hence, they form a
symbiotic relationship with fungi known as orchid mycorrhiza, which provides nutrients for
protocorm formation. To overcome these obstacles, and the rapid decrease of O. sphegodes
population, ex situ conservation and in vitro asymbiotic propagation are estimated as the best
solutions for conservation. Knudson (1922) pointed out that germination is possible on a
37
substrate which contains minerals and carbohydrates, in absence of fungi. Germination depends
on a large number of factors, such as temperature, light, minerals, carbohydrates, pH, vitamins,
hormones (Rasmussen, 1995; Arditti, 1967; Stewart and Kane, 2006; Godo et al., 2010; Chen et
al., 2015; Ponert et al., 2013; Gupta, 2016). Seed usually germinate at 20-25°C and pH 4.8-5.8
and often demand Fe and Mn for germination; also coconut water, pineapple juice, peptone,
tryptone, yeast and salep are used as supplements (Marić, 1995).
Thus, the aim of this study was to establish optimal asymbiotic germination protocol for
the purpose of ex situ conservation of endangered orchid species O. sphegodes.
Plant material
Maturate capsules were sampled in the late June of 2017 (Figure 1). Extracted seeds were
dried at 26˚C for 48h to eliminate excess moisture, placed in Petri dishes over silica gel and
stored at 4˚C in continuous darkness until use.
The seed surface was sterilized with 7% calcium hypochlorite containing 0.1% Tween 20 for
20 minutes. Seeds were further rinsed with distilled water three times. Sterilization was
accomplished in laminar flow hood with 70% ethanol for 2 minutes, followed by triple rinsing in
sterile distilled water.
Figure 1. Plant material: A) Mother plant; B) Viable seeds - red colored
Cultivation media composition and culture conditions

Two basal media ‒ KC (Knudson C, 1946) and MM (Malmgren, 1996), supplemented with
different organic additives except for the control media (KC-C and MM-C), were applied. Basal
medium Knudson C was enriched with casein hydrolysate 400 mg l -1 with folic acid 0,5 mg l-1
(KC-CA) and L-glutamine 100 mg l-1 with folic acid 0,5 mg l-1 (KC-A) while Malmgren medium
was enriched with peptone 2 g l-1 (MM-PE) and L-glutamine 100 mg l-1 (MM-A). All nutrition
media contained 2% sucrose, 7% agar and 1% activated carbon, while their pH was adjusted to
5.8 ± 0.02 before autoclaving at 121 ° C for 20 minutes. After successful sterilization seeds were
spread on the medium followed by wrapping of Petri dishes with a single layer of parafilm and
38
stored at a temperature of 23 ± 2 ° C. To examine the effects of light conditions on seed
germination and protocorm formation, Petri dishes were placed under two illumination conditions
- 0/ 24 light/dark (L/D) and 16/8 (L/D). Counting of germinated seeds was carried out eight and
twelve weeks after sowing.
Data processing and statistical analysis

Germination percentage and seedling development were calculated by dividing the number of
germinated seeds with the total number of seeds observed. Data were subjected to analysis of
variance (ANOVA) and the mean values of protocorm size were separated using Duncan’s
Multiple Range Test (DMTR). The collected data were analyzed in Statistica 13 (StatSoft,
DELL) software.
Presented results indicate a huge influence of illumination and nutrition media on seed
germination and protocorm formation. Seed germination was overall significantly more
successful in dark conditions (0/24 L/D) than with lighting (16/8 L/D). This result coincides with
Godo et al. (2010) who found that light conditions have an inhibitory effect on terrestrial orchid
Calanthe tricarinata Lindl. germination. Results showed that MM medium has a considerably
better impact on germination and protocorm formation, while on KC medium seeds stay in
swelled condition and stopped with further development. This result can be explained by the
composition of nutrition media. While MM medium contains an only organic form of nitrogen
(glycine and casein hydrolysate) KC medium contains an inorganic form of nitrogen which in
some cases has been shown to have an inhibitory effect on seed germination and protocorm
formation (Ponert et al., 2013, Sgarby et al. 2009).
After the first measurement, seeds cultured in continual darkness on KC-CA medium had the
highest full germination percentage (62%) (Figure 2.). However, the appearance of protocorm
was notable on seeds sown on MM-PE, MM-A, and MM-K, while shoots and rhizoids appeared
only on MM-PE and MM-A mediums and cultured in continual darkness (Figure 3). Results
presented in this paper show that KC medium promotes only swelling of the seeds without
protocorm, shoots and rhizoids formation.
39
Figure 2. Measurements 8 Figure 3. Influence of Illumination and nutrition media 8 weeks after
sowing: A) Swelled
elled embryo; B) Rupture of testa; C) Appearance of
weeks after sowing – full protocorm; D) Appearance of shoot and rhizoids
germination
Figure 4. Measurements Figure 5. Influence of Illumination and nutrition media 12 weeks

after sowing: A) Swelled embryo; B) Rupture of testa; C)
12 weeks after sowing –
Appearance of protocorm; D)Appearance nce of shoot and rhizoids
full germination
40
The second measurement showed that on KC mediums seeds remained swelled. Protocorm
formation, shoots and rhizoids appearances were achieved only on MM-PE and MM-A mediums,
in continual darkness (Figure 4, 5, 6.). This result indicates a positive influence of organic
supplements on germination rate. With light, no growth was achieved at all.
The measurements of the embryo size confirmed that darkness and MM medium
supplemented with additives promote germination and protocorm formation of O. sphegodes
(Table 1.). The highest embryo size during the first measurement was achieved on MM – PE
(height 0.70 mm; with 0.62 mm; shoot height 0.16mm) and on MM – A (height 0.64 mm; width
0.56 mm; shoot height 0.14mm). During the second measurement increased embryo size was
achieved only on MM-PE (height 0.81 mm; with 0.75 mm; shoot height 0.32mm), MM-A (height
0.77 mm; with 0.65 mm; shoot height 0.24mm) and MM-C (height 0.55 mm; with 0.50 mm;
shoot height 0.06 mm) which indicates a positive influence of MM medium and darkness on
germination. During the second measurement, there were no changes in embryo size on seeds
cultured on 16L/8D illumination.
This study presents a procedure for the in vitro asymbiotic germination and protocorm
formation of O. sphegodes that can be used for conservation programs and mass production
protocol. Furthermore, because of the robust and attractive character of O. sphegodes as well as
long-living flowers, presented research could provide an impulse to the use of this species as a
garden or potted plants. In nature, germination of terrestrial orchid seeds is conditioned by the
establishment of symbiosis with mycorrhizal fungi and this process can take up to several years
(Rasmussen, 1995). Presented procedure accelerates the germination period and can provide a
large number of plants in a relatively short period of time.
41
Table 1. Mean values of embryo size during the first and second measurement
8 weeks 12 weeks
Nutrition
Illumination Height Width Shoot height Height Width Shoot height
media
(mm) (mm) (mm) (mm) (mm) (mm)
a a a a a
MM-PE 0.70±0.02 0.62±0.06 0.16±0.10 0.81±0.19a 0.75±0.18 0.32±0.08a
MM-A 0.64±0.10b 0.56±0.05b 0.14±0.10a 0.77±0.2a 0.65±0.16b 0.24±0.22a
0L/24D MM-C 0.58±0.06c 0.51±0.05c 0.06±0.07b 0.55±0.09b 0.50±0.08c 0.06±0.06b

KC-CA 0.17±0.03d 0.10±0.01d 0.00±0.00c 0.17±0.02c 0.11±0.01d 0.00±0.00b
KC-A 0.16±0.01d 0.10±0.02d 0.00±0.00c 0.16±0.02c 0.10±0.02d 0.00±0.00b
KC-C 0.15±0.02d 0.10±0.02d 0.00±0.00c 0.16±0.01c 0.10±0.02d 0.00±0.00b
MM-PE 0.14±0.02d 0.11±0.02d 0.00±0.00c 0.14±0.02c 0.11±0.02d 0.00±0.00c
MM-A 0.16±0.02d 0.13±0.01d 0.00±0.00c 0.16±0.02c 0.13±0.01d 0.00±0.00c
MM-C 0.14±0.02d 0.10±0.02d 0.00±0.00c 0.14±0.02c 0.10±0.02d 0.00±0.00c
16L/8D
KC-CA 0.17±0.02d 0.11±0.03d 0.00±0.00c 0.17±0.02c 0.11±0.03d 0.00±0.00c
KC-A 0.15±0.01d 0.10±.02d 0.00±0.00c 0.15±0.01c 0.10±.02d 0.00±0.00c
KC-C 0.15±0.01d 0.10±0.02d 0.00±0.00c 0.15±0.01c 0.10±0.02d 0.00±0.00c
*Means ± standard deviation within a column followed by the same letters are not significantly different using DMTR at p ≤ 0.05
A) B)
Figure 6. Seed germination: A) Swelled embryo; B) Protocorm with shoot and rhizoids
42
CONCLUSION
This paper presents successful in vitro germination of mature seeds of terrestrial orchid
species. Both MM and KC media caused swelling of the embryo, but protocorm and shoots as
well as rhizoids were formed only on MM media. Also, during this research, a positive influence
of organic additives on germination rate was achieved. Remarkably better results were obtained
in dark conditions (0/24 L/D), which was also previously confirmed by several other authors.
After 12 weeks, significant results were achieved on MM-PE and MM-A media, especially on
MM-PE media. The best protocol for Ophrys sphegodes, therefore, is culturing seeds in the dark
conditions, on MM-PE media.
REFERENCES
ARDITTI, J., (1967): Factors affecting the germination of orchid seed. The Botanical Review
33: 1-97.
CHEN, Y., GOODALE, U., FAN, H.L., GAO, J.Y. (2015): Asymbiotic seed germination and
in vitro seedling development of Paphiopedilum spicerianum: An orchid with an extremely small
population in China. Global Ecology and Conservation 3:367-378.
DAVY, A.J., HUTCHINGS, M.J., ROBERTS, D.L, ROBBIRT, K.M., (2014): Potential
Disruption of Pollination in a Sexually Deceptive Orchid by Climatic Change. Current Biology
24: 2845–2849.
GODO, T., KOMORI, M., NAKAOKI, YUKAWA, T., MIYOSHY, K., (2010): Germination
of mature seeds of Calanthe tricarinata Lindl., an endangered terrestrial orchid, by asymbiotic
culture in vitro. In vitro Cellular and Developmental Biology - Plant 46: 323-328.
GUPTA, A., (2016): Asymbiotic Seed Germination in Orchids: Role of Organic Additives.
Interantional Advanced Research in Science. Engineering and Technology 3(5): 143-147.
HUTCHINGS, M.J., (1989): Population biology and conservation of Ophrys sphegodes,
Modern methods in orchid conservation: the role of physiology, ecology and management.
Cambridge University press, 101-115.
KNUDSON, L., (1922): Non-symbiotic germination of orchid seeds. Bot. Gaz. 73: 1–25.
KNUDSON, L., (1946): A new nutrient solution for the germination of orchid seed. American
Orchid Society Bulletin 15, 214-217.
MALMGREN, C., (1996): Orchid propagation. Theory and practice. In: Allen C (Eds.) North
American native terrestrial orchids. Propagation and production. North Am Native Terrestrial
Orchid Conf Proc. C. Allen, Germantown, Md. pp. 63–71.
MARIĆ, M.M., (1995): Plant tissue culture. Publishing house Draganić, 77-82.
PONERT, J., FIGURA, T., VOSOLSOBE, S., LIPAVSKA, H., VOHNIK, M., JERSAKOVA
J. (2013): Asymbiotic germination of mature seeds and protocorm development of Pseudorchis
albida (Orchidaceae) are inhabited by nitrates even at extremely low concentrations, Botany 91,
662-670.
43
PONERT, J., VOSOLSOBE, S., KMECOVA, K., LIPAVSKA, H., (2011): European orchid
cultivation – From seed to mature plant. European Journal of Environmental Sciences 1(2): 95-
107.
RASMUSSEN, H.N., (1995): Terrestrial orchids, from seed to mycotrophic plant. Cambridge
University Press, Great Britain.
SGARBI, E., GRIMAUDO, M., DEL PRETE, C., (2009): In vitro asymbiotic germination and
seedling development of Limodorum abortivum (Orchidaceae). Plant Biosystems 143, 114–119.
STEWART, S., KANE, M., (2006): Asymbiotic seed germination and in vitro seedling
development of Habanaria macroceratitis (Orchidaceae), a rare Florida terrestrial orchid. Plant
Cell, Tissue and Organ Culture 86: 147-158.
44
ANALYSIS OF THE DEVELOPMENT OPPORTUNITIES OF AGRICULTURAL
LANDSCAPE WITH GREEN INFRASTRUCTURE NETWORK IN A HUNGARIAN
RURAL AREA
Gabriella Balha
Mentor: István Valánszki, Kinga Mezősné Szilágyi

Szent István University, Faculty of Landscape Architecture and Urbanism, Dept. of
Landscape Planning and Regional Development Hungary
Summary: The aim of the research is to analyze the possibilities of development of a

hungarian rural area with green infrastructure network. Researching for GI elements that
reflect typical local conditions, and analyzing the GI network of elements based on different
aspects. The analysis of the analytical aspects is based on the ecosystem services,
sustainability aspects, and the increase of biodiversity. The analysis is recorded by a created
GIS that assigns qualitative and quantitative metrics to geographic units based on a specific
indicator system. The resulting database can serve as a basis for developing a monitoring
system, sustainability, and analysis of other areas.
Key words: green infrastructure network, rural, GIS, indicator, monitoring
INTRODUCTION
The aim of the GI network is to preserve the population, to improve the quality of life, to support
climate change to mitigate climatic extremities, to address environmental risks through efficient
resource management, money resources, water, land, air, etc. Responsible and resourceful
exploitation, emphasizing the protection of living organisms and their habitats, increasing
diversity and continuity, thus increasing environmental values and stability (Vaszócsik V.et al
2014). The Green Infrastructure Approach is the key to good and sustainable land use, especially
in compact and rapidly growing European cities with particularly strong pressures on the
landscape (EEA 2006, Poelmans And Van Rompaey 2009). The European Commission has also
integrated it into the second objective of the European Biodiversity Strategy 2020 document:
"Building green infrastructure and restoring at least 15% of degraded ecosystems will stay and
improve ecosystems and services by 2020" (COM/2011/0244) In the present study, I will present
an indicator system that can be a tool to assist the implementation of the green infrastructure
concept in the national planning practice (NCCS 2008) Hungary is one of the most vulnerable
countries in Europe with regard to the impact of climate change on biodiversity, is on the species
richness of the wildlife. Hungary's National Climate Change Strategy addresses a number of
aspects (fields, forests, water management, nature conservation, etc.) affecting the elements of the
green infrastructure network. To stop the loss of green space, to decline of the outer areas,
increasing forest areas are the main goals which are connected with GI network. Land use needs
45
to be re-regulated in the light of climate protection considerations. This study is intended to
research the above strategies in rural areas. The development of the ZI network is focused on the
management of the urban green space system. Several studies have been carried out in order to
preserve biodiversity, not only the green areas of the city, but also the green space of the
outskirts, of the same importance in several aspects (land protection, climate protection, water
management. A large part of Hungary is rural area, where the development of the GI network is
not only directed towards the green space system in the settlement area, but mainly the
surrounding of the settlement. The main goal of the study is to provide a system that will provide
sustainable development opportunities for rural areas using the green infrastructure system.
During the literature research, I determined that the selection of the elements may vary as a use of
the landscape and function, so I have designated a sample area for examining the development
possibilities of the GI network of Hungarian agrarian regions. I chose a sample area, which is a
tourist-oriented but inherently agricultural area. Hajdúszoboszló's spa is famous for as a
traditionally agricultural town. I set the system of green infrastructure elements in line with local
characteristic, based on the mapping of the green infrastructure elements of the region. I
identified all the elements that are typical of the particular rural, agrarian region.
The research is based on the definition of the typical GI elements of the region. The physical
components of the Green Infrastructure are green and blue areas through the natural functions
and processes are maintained (forest areas, line of tries, fields, protected areas, blue elements,
cultivated areas, etc...)
These elements form the green infrastructure network as a service system. By upgrading the
cadaster, we can keep track the green infrastructure elements and network development. The
physical system of the green infrastructure was analyzed on four levels: elements, network,
settlement, and region. The green infrastructure cadaster based on the definition of the elements
contains the "green" and "blue" elements of the network and their systems. In the map depicted, I
have grouped the analysis of existing and potential green infrastructure elements on the basis of
an on-site survey on the outskirts of the settlement and the available historical maps and
development plans.
After the elements were determined, a GIS database was created, I connected the
geographical units with quality (protection, confusion) and quantitative indicators (area, length,
ratio) that provides assistance for cadastral analysis (ZHF 2017). The value of the elements and
the network of the green infrastructure can be evaluated through the quantity and quality and
efficiency of the services. Therefore, an assessment of the ecosystem services is an important
element of the analysis of green infrastructure. Depending on the ecosystem that can be assigned
to each item (for a single item, several services can be ordered) the network continuity of each
ecosystem service can be mapped. The thematic levels of each service collectively outline the
46
territorial color of the network, outlining the part of the network, how strong / weak it is
(Effenberg, 2011).
In addition to the ecosystem services of the individual elements, I described the supply,
regulation, maintenance and cultural services assigned to the territorial units, analyzing the GI
supply, balancing surfaces, the intensity of the green area, analyzing the protection of GI
elements, network connectivity (CICES 2015). For the analyzes, it was necessary to determine
indicators to be objectively measurable and monitorable:
Green mass (balancing surface). The amount of biomass in urban green spaces is a characteristic
measure of the viability of an urban ecosystem, one of the fundamental features of sustainability.
Indicator: Based on NDVI vegetation index.
Fig.1.Green space intensity
The utilization of rainwater on site is important both ecologically and economically. The drainage
of rainwater may be justified by the local exploitation potential. It is desirable to describe them
and to include them on the map in the relevant item. Indicator: The percentage of rainwater in the
area (%). The value may be greater than 100% when the water is drawn or stored elsewhere.
Indicator: Proportion of areas not suitable for local use of rainwater (%).
The degree of protection of the elements gives valuable information about the overall social value
and importance of the green infrastructure element. Indicator: The number and extent of affected
areas of the relevant green infrastructure element (local, national, international), multiple
protection ratings)
The development of the monitoring system serves the continuous maintenance of the cadastre and
the monitoring of the state of the green infrastructure changes. The monitoring system identified
different indicators for tracking development.

47
During the research, a GI cadaster was created based on the sample area and the elements
specified in the study can be adapted to analyze the GI network of any major agrarian area.
Defining elements first sets item groups that can contain different elements per region.
Table.1.
ITEM GROUP ITEM
Forest economic forest
public welfare forest
protective forest
Cultivated area general agricultural land
gardened farmland
restricted agricultural land
lawn
“Blue” infrastructure watercourse
still water
sewage system
Recreational areas sports and recreation area
tourist area
airport
camping
Line infrastructure bicycle path
alle
baulk
vineyard
transport areas
Potential green biologically inactive surfaces
infrastructure elements brownfield Sites
wetland
suggested area for protection
Cadastre was recorded in a GIS system that allowed geographical qualifications to be provided
with qualitative and quantitative indicators. This is important for sustainable development, it is
easy to trace the qualitative and quantitative changes in each element.
element type: line element,
element: tree,
quality indicator: tree status, tree species, protection,
quantitative indicator: length, number of individuals
The method of capturing the data assigned to the cadastral elements in a GIS database can easily
be adapted to analyze the GI network of another area that allows monitoring and perpetuation.
48
It reveals the system's shortcomings by objective factors, resulting in an analysis of the role of
each element in the GI network and the system's shortcomings. The indicator system defined in
the analysis provides further opportunities to define an area GI network with objective indicators.
On the summary map you can see the weakness of the system, the element system to be
developed and the network of GI. In order to keep the system sustainable the monitoring system
set up serves the development, the development horizon and the responsible persons with the
established indicators.
Fig.2. Analysis of GI elements after protection indicator
I grouped the ecosystem serccies in tree main groups, and folloving eight sub-groups:
A:supply services: tangible goods provided by ecosystems and which can be sold directly or
indirectly can be consumed, for example: vegetable and animal food, raw materials and fibers;
biomass-based and energy source of animal origin;
B: Regulatory and Maintenance Services: Includes all the ways in which ecosystems regulate or
modify biotic and abiotic environmental factors. They can not be consumed or usable directly, for
example: bio-remediation, flood protection, erosion protection, pollination, pest and disease
control; soil formation and soil structure; climate control;
C: cultural services: intangible (non-material) goods that have a symbolic, cultural or intellectual
significance, such as: tradition, heritage, aesthetics, recreation, recreational facilities; educational
and research activities.
Following the analysis of ecosystem services, one of the main results of the research was the
creation of an indicator system that is also suitable for the examination of other areas.
Elements of the green infrastructure can work well if a single, are assembled. To this end, the
connectivity between the elements must be ensured both in the internal and the internal areas, and
in addition to the administrative boundaries, the development of the region and the landscape.
In the GIS system, the following map shows the ecosystem services groups:
49
Fig.3. Map of Ecosystem services
CONCLUSION
The cadastre-geospatial-analysis-monitoring system run through the designated pilot area

demonstrates that it provides a sufficient amount of data for the analysis of the given field type.
Further research requires that the so-called cadastral system, the methodology and indicator
system of analysis, the GIS database, and the monitoring system can be applied to the regional
and landscape development of the GI network of other regions.
REFERENCES
MAGYARORSZÁGI területi tervezést támogató térképes indikátor rendszer kialakításának

lehetséges lépései a zöld infrastruktúra koncepció megvalósításáért Vaszócsik Vilja , Göncz
Annamária1 , Schneller Krisztián , Tóth Péter , Prokai Réka Tájökológiai Lapok 12 (2): 411–428.
(2014)
LANDSCAPE AND URBAN PLANNING Detecting And Modelling Spatial Patterns Of Urban
Sprawl In Highly Fragmented Areas: A Case Study In The Flanders–Brussels Region EEA 2006,
Poelmans And Van Rompaey 2009
EUROPEAN Biodiversity Strategy to 2020 (COM/2011/0244)
NATIONAL Climate Change Strategy –Hungary (2008)
ZÖLDINFRASTRUKTÚRA-HÁLÓZAT FEJLESZTÉSE MTA Ökológiai Kutatóközpont
Ormos Imre Alapítvány Budapest, 2017
Spatial Cadastral Information Systems Wolfgang Effenberg 2011
Common International Classification of Ecosystem Services (CICES classification version 4.3,
2015)
50
IPARD – CHALLENGES AND PERSPECTIVES OF AGRICULTURAL HOLDINGS IN
SERBIA
Aleksandar Anđelković
Mentor: Marija Nikolić, Ph.D., Assistant Professor

University of Belgrade, Faculty of Agriculture, Institute of Agriculture Economics, Serbia
Summary: The implementation of the IPARD - Instrument for Pre-Accession Assistance for
Rural Development as the EU pre-accession program in Republic of Serbia represents a
significant opportunity for the agricultural sector. The most important challenges are the
improvement of the situation for farmers in the primary sector and for enterprises in processing
and marketing. There are public calls for Investment into agricultural products and Investments
in the processing and marketing of agriculture and fishery products. The objective of this paper
is to emphasize most importance challenges of the holders of agricultural holding related to
IPARD and compare with experience in other countries. Considering the experiences of the
countries in the region, where most of the requests submitted were related to the purchase of
tractors and new machinery and equipment. The interview was conducted on 30 agriculture
holdings. The biggest unknown is how to fulfill the requirements of animal welfare, food safety,
production of environment, veterinary and phytosanitary requirements. Funding is one of the
most important issues of agriculture and rural development of the Republic of Serbia. Also,
significant problem for agricultural producers is unresolved ownership relations and age
structure of agricultural holding. Of the total number of interviewed holders of agricultural
holdings, only one applied for funds from IPARD. The biggest problem for this holding was the
change of the Rulebook after the public call. By calculating the coefficient of correlation,
hypotheses have been confirmed that: the size of UAA has an impact of the holders knowledge
about IPARD, the size of UAA has an impact on financial resources for the purchase of new
tractors and machinery. The perspective of using the IPARD fund certainly exists. A large
number of holdings are interested in IPARD in the future.
Key words: IPARD, rural development, agricultural holdings, Republic of Serbia
INTRODUCTION
Agriculture is one of the most important sectors in the economy of the Republic of Serbia.
According to the 2012 Agriculture Census, the total number of holdings in Serbia is 631.552. On
family agriculture holding are employed 1.414.564 persons (Bogdanov and Babović, 2014).
Serbia's farm structure is complex, consisting of small subsistence agricultural holdings, small
semi-subsistence farms, large family farms, as well as large enterprises with a mixed ownership
51
structure. The average plot of utilized agricultural area (UAA) per farm in Serbia is 5.4 ha, which
is about one third of the EU-27 average (14.5 ha). The average standard output (SO) per holding
in Serbia is EUR 5.939 which is 4 times less than the EU-27 average (Cvijanović et al., 2012).
The future of agriculture holdings and their competitive position on the liberal market largely
depend on the creators of agriculture policy. The integration of Serbia's agricultural policy with
the European Union Common Agricultural Policy (CAP) is one of the central issues of the
Republic of Serbia's accession to the European Union (EU). By receiving the status of candidate
on March 1st, 2013, the Republic of Serbia won the right to use funds from the Instrument for
Pre-Accession Assistance for Rural Development - IPARD. IPARD is the most complex IPA
component and it comprises measures that can be grouped into three axes. The procedure is very
complex and strict monitoring of project activities is implemented. The availability of IPARD
funds should strengthen rural communities and agriculture. “It is important that the funding
provided by IPARD II programme should be focused at: competitiveness of the agri-food sector;
alignment with the veterinary, phytosanitary, environmental and food safety standards of the EU;
restructuring and modernization of the sector; development of sustainable resource management
practices; strengthening of the LEADER approach” (Radović et al., 2018). The most important
challenges are the improvement of the situation for farmers in the primary sector and for
enterprises in processing and marketing. Therefore from the overall budget for the period 2014 –
2020 about 80 % are planned for the measure “Investments in physical assets of agricultural
holdings“ and “Investments in physical assets concerning processing and marketing of
agriculture and fishery products” (Ministry of Agriculture and Environmental
Protection,Republic of Serbia, 2015).
The first public call for applications within IPARD for investments in physical assets of
agricultural holdings – purchase of new machinery and equipment – was launched on 25
December 2017. This invitation covers the sectors of milk, meat, fruits and vegetables and other
crops. The amount of payments that users can make can range from EUR 5.000 to 1.000.000,
depending on the sector. So far, three public calls have been announced, and the last one on
October 22 2018, for the construction of storing facilities and equipment and procurement of
mechanization funds. According to the data of the Directorate for Agrarian Payments (DAP),
over 500 applications have been received. In Macedonia, 195 applications were received for the
stated measure in the three-year period, of which only 38 % were accepted (Stevanovski and
Ziberovski, 2012). The Paying Agency for Agriculture, Fisheries and Rural Development in
Croatia received 1.143 applications in total. The most project contracts within the Measure 1
were signed – 426 (Kralik and Zmaić, 2014). In the period from December 2012 to May 2014,
255 applications for investments in physical assets were received in Albania (Bezhani, 2015).The
first public call for applications for the approval of the IPARD project “Investments in physical
assets concerning processing and marketing of agriculture and fishery products” in the
procurement of new equipment in Serbia was called on 27 March 2017 with deadline 27 April
2017.
52
Serbia has a big advantage in using IPARD funds, because it has experiences, analyses and
recommendations from other countries (Šestović et al., 2017). Will use their advantage, will
show results. The aim of this paper is to emphasize and identify the most significant problems
and challenges that the holders of agricultural holdings meet on IPARD and point to the
prospects in the future. Authors performed research in which they wanted to confirm the
following hypothesis: H1) The age of holders of agricultural holdings has an impact on the
holders knowledge about IPARD; H2) The size of UAA has an impact of the fholders knowledge
about IPARD; H3) The size of UAA has an impact on financial resources for the purchase of new
tractors and machinery.
For the purpose of this study, they interview was conducted, by which the data on the main
challenges and problem of holders of agricultural holding in IPARD were collected. The
interview was conducted on 30 agriculture holdings. The holders of agricultural holdings
answered questions about the main reasons for not applying for funds from IPARD funds, or
central problems when they applied. Data was collected from the respondents by using structured
interview schedule. After the evaluation of the data, the key problems faced by agricultural
holdings are highlighted. Secondary data sources were used, such as data from the Statistical
Office of the Republic of Serbia (SORS), the Ministry of Agriculture Forestry and Water
Management of Serbia (MAFWM), the Directorate for Agrarian Payments of Serbia (DAP). We
have also brought out the experiences of those countries that have gone through IPARD.
On the basis of available literature and empirical research we set 3 hypotheses to test factor that
have impact on the interest of holders of agricultural holdings for IPARD. Measures of
dependence that were used in this paper are Pearson’s coefficient of correlation. The values of the
correlation coefficient r range from -1 to 1. If the values obtained are closer to -1, then this
represents a strong negative relation. The values closer to 1 mean that a strong positive relation
between the variables is strong. Correlation coefficient equal to zero indicates that there is no
correlation.
Calculating of the Pearson’s coefficient of correlation:
∑ (𝑥𝑖 − 𝑥̅ )(𝑦𝑖 − y)
𝑟=
∑ (𝑥𝑖 − 𝑥̅ ) ∑ (𝑦𝑖 − y)
53
RESULTS AND DISCUSSI
DISCUSSION
Of the 30 interviewed holders of agricultural holdings, 96.7 % are men. Although the amount of
returns for women is higher, according to IPARD ru rules,
les, the number of such family holdings in
Serbia is extremely low. Only 15.9 % of family holding managers are women. According to the
Rulebook on the entry in the Register of agricultural holdings ("Official Gazette of RS", No.
17/13), spouses cannot have separately registered agricultural holdings.
One of the most significant demographic characteristics of the rural areas of the Republic of
Serbia is the unfavorable age structure of the population. According to the Statistical Office of
the Republic of Serbia,
rbia, only 4.8 % of holders of agricultural holdings are under 35 years of age.
A number of European countries are also facing this problem. For example, in Holland, the share
of the said group is only 3.1 %, in Romania it is 4.7 %, while the EU28 average is 5.8 %
(Eurostat). The largest number of interviewed holders of agricultural holdings (32 %) belongs to
the age group 18-35
35 years. Participation of respondents from 36 36-45
45 years is 25 %, while the share
of holders of agricultural holdings from 46 46-55, or 56-65 65 years, to the level of about 20 %. The
lowest percentage of respondents is over 65 years of age. Although this age group is significant at
the level of the Republic of Serbia, it is difficult to talk about their interest in IPARD.
Agricultural holdingss with 51 ha and more ha of UAA have the largest share in this survey – 33
%. Then there are holding with 66-1515 ha with share of 22 %. The share of holdings with 16-30 16 ha
and 31-50
50 ha UAA is 19% (Figure 1). The smallest share is owned by holdings with 5 or less
hectares of UAA. In Republic of Serbia the share of holding with more than 50 ha is only 9.1 %
(SORS).
Figure 1.. Agriculture holding of respondents by utilized agriculture area (UAA)
Professional competencies of the users rrepresent

epresent an important factor in the application for
IPARD. Applicants should prove sufficient agriculture experience and competences in one of the
following categories: agricultural secondary school education or at least three years of
agricultural experience
nce (proved by a professional service record from the employer or registered
for that time in the Register of Agricultural Holdings) or university degree or secondary school
education and commitment in writing that they will follow a training course with a minimum
duration of at least 50 teaching hours in the relevant sector before applying for the final payment.
54
In the case of legal entities, the above requirements apply to managers (MAFWM, 2017). More
than half of the respondents have completed secondary school, 27 % are basic, and only 17 %
have a high level of education. In Serbia, only 1.4 % of holders of farms completed a higher
agricultural school or college. 60 % of interviewed holders own a registered holding for more
than 3 years, and this rule does not represent a significant challenge for potential users. Research
in Bosnia and Herzegovina shows that over 85 % of farmers in this country meet at least one of
the professional competence (ACED, 2012). Experience from Turkey shows that the beneficiaries
of funds with a higher degree of education used more funds from IPARD (Yardimci et al., 2018).
Of the total number of interviewed holders of agricultural holdings, only one applied for funds
from IPARD. Considering the experiences of the countries in the region, where most of the
requests submitted were related to the purchase of tractors and new machinery and equipment,
the information and limitations related to this measure were examined. Out of the total number of
interviewed owners of agricultural holdings, only 24 % did not know that they could apply for a
refund for purchasing a new tractor or new machinery and equipment. Regarding IPA programs
and funds the level of awareness among farmers in Croatia is high: 91% of the surveyed farmers
are familiar with IPA, however, only 15% of interviewed is actually planning to apply for some
form of pre-accession aid (Tankosić Vapa and Stojsavljević, 2014).
However, 59 % of the respondents point out that it is not sufficiently informed about the
application procedure, the necessary documentation and the procedure of control (Figure 2). The
biggest unknown is how to fulfill the requirements of animal welfare, food safety, production of
environment, veterinary and phytosanitary requirements. Namely, holders of agricultural holding
who apply for IPARD in sector of other crops must fulfill the animal welfare rules for domestic
animals for their own needs. That can be one pig or three hens. As the European Commission’s
Progress Report on Serbia from 2014 noted, in the areas of food safety, veterinary and
phytosanitary policy, Serbia remains moderately advanced.
Similar to the experiences of farmers in Bosnia and Herzegovina (ACED, 2012), the majority of
respondents in the IPARD fund were informed on the Internet - 63 %. Through television and
radio, 22 % of respondents, from neighbors and other farmers, accounted for 10 %, and only 5 %
of respondents on the IPARD fund were informed by the advisory and expert services.
55
Figure 2.. Informing the holders of agricultural holdings about IPARD Measure for purchase new tractor s or
machinery and mechanization
The holders of agricultural holdings point out that they are not satisfied with the number of
organized trainings or courses about IPARD program. Namely, only 5 % of interviewed holders
of agricultural holdings attended some kind of training. This pr problem
oblem is not only related to the
IPARD program, but the whole agricultural sector of Serbia. According to the 2012 Agricultural
Census, only 7 % of farm managers attended some kind of training in the previous 12 months
from the moment of the Census.
Fundingg is one of the most important issues of agriculture and rural development of the Republic
of Serbia, because insufficient financial resources allocated to this sector of the country’s
economy are considered the key limitation for better valorization of ag agricultural
ricultural resources and the
revitalization of rural areas (Ristić et al., 2018). In this survey
survey,, 57 % of respondents point out that
there are not enough financial resources for the purchase of new tractors, machinery or equipment
(Figure 3). All investments are made on the principle of pre pre-financing,
financing, that is, it is essential that
the approved project is completed with its own funds (or credit), and the payoff follows after the
completion of the entire investment and determination of compliance with the standards of the
investment itself. A recipient can claim the support, irrespective of the total value of the
investment, for eligible expenditure within the following ceilings: minimum EUR 10,000,
maximum EUR 1,000,000. 45.9 % of holdings have SO less than EUR 2,000. Respectively,
about 80 % of holding have SO less than EUR 8,000. It is evident that a large number of
agricultural holdings will not be able to use those funds.
Figure 3.. Availability of financial resources of respondents
56
A significant problem for agricultural producers is unresolved ownership relations. According to
the Rulebook on IPARD incentives for investments in the physical property of agricultural
holdings ("Official Gazette of RS", No. 84/17), land and buildings must be owned or long-term
lease by the Applicant. A common case in our households is that on the same estate, more
holders of registered agricultural holdings live and work. For example, the land is owned by one
member of the household, an economic yard owned by another and the machinery owned by a
third. The slow process of changing ownership structure suggests that the farm is seen as a family
asset, rather than a workplace and economic system (Kotevska, et al., 2015).Which is contrary to
the rules. The faster transfer of property rights to young farmers could contribute to their
inclusion in agriculture activities and better use of financial funds.
Among the respondents, only one agricultural holding applied for the use of funds from the
IPARD. The subject of the application was the purchase of new tractor within the scope of
Measure 1 - investments in the physical assets of agricultural holdings. The biggest problem for
this farm was the change of the Rulebook after the public call, ie after submitting the request. In
accordance with the Rulebook on IPARD for Investing in the Physical Properties of Agricultural
Holdings ("Official Gazette of RS" No. 84/17 and 112/17), the second public call for applications
for the approval for investments in the physical properties of agricultural holdings for purchase of
tractors was launched on January 4 2018, with deadline 26 February 2018 (DAP,2018).
Complement number 1 of the second public call was published on January 8, 2018. The basic
change relates to respecting emission standards of exhaust gases. According to this
Complementary, fund are only granted for tractors that meet the Stage III A emissions standards,
ie Tier 3, Stage IIIB, Tier 4, as well as Stage IV. As the ownership of agricultural holding points
out, the problem arose when it was necessary to re-purchase 3 bids for tractors that meet the
stated requirements provided for in the Complement.
The impact of age holders of agricultural holdings on their IPARD information was tested by
Pearson's correlation coefficient. The value of R is -0.0054. Although technically a negative
correlation, the relationship between variables is only weak. This hypothesis has been confirmed,
but the impact of increasing age on information is less pronounced.
By testing the dependence between the size of the UAA and the information of the holders of the
holding, the hypothesis was confirmed. Although it is technically a positive correlation, the value
of R is 03582. The relationship between variables is not high, but enough to confirm the
hypothesis that there is better information and interest of farms with a larger UAA.
By researching the relationship between the UAA and the answer to the available financial
resources, the hypothesis confirmed. The value of R is 0.7117. This is a moderate positive
correlation, which means there is a tendency for high X variable scores go with high Y variable
scores (Table 1).
57
Table 1. Calculate coefficients of correlation for hypotheses
The name of hypothesis The value of R

H1: The age of holders of agricultural holdings has an impact on
-0.0054
the holders knowledge about IPARD
H2: The size of UAA has an impact of the holders knowledge
0.3582
about IPARD
H3: The size of UAA has an impact on financial resources for
0.7117
the purchase of new tractors and machinery
It is necessary to point out that all holdings that are not similar to IPARD can apply for funds
from the National Rural Development Measures. An important document that defines all other
documents related to rural development and agriculture is a “National Program for Agriculture
and Rural Development of the Republic of Serbia from 2018 to 2020 - NPRD (Goverment of
Republic of Serbia, 2017). This is a document that is complementary to IPARD program and
defines measures to support rural development in accordance with the current national legal
regulations and requirements, and defining the criteria and elements of financial support. So,
when you meet the requirements and can apply for NPRD, you cannot be registered to a
competition for IPARD. (Gluščević et al., 2017). If unresolved ownership relations were resolved
immediately for IPRAD application, artificially created conditions for achieving the advantage in
applying could be determined, which is contrary to the rules.
CONCLUSION
The implementation of the IPARD program provided significant opportunities for the
development of agriculture in the Republic of Serbia. Serbia has a big advantage in using IPARD
funds, because it has experiences, analyses and recommendations from other countries. The
experiences of countries in the region show that the utilization of the IPARD fund is less in the
initial stages of program implementation. The number of IPARD applications in Serbia is at a
satisfactory level, but it remains to be seen how much the application will be accepted, ie how
much funds will be approved and paid out.
Using these benefits does not depend exclusively on farmers, but also from the Ministry of
Agriculture Forestry and Water Management and Directorate of Agrarian Payments. The
utilization of IPARD funds will largely depend on professional competence and intercession of
employees in these institutions. Only with the mutual cooperation of farmers and institutions can
they make maximum use of resources.
Informing the holders of agricultural holdings is not satisfactory. More attention must be paid to
education of farmers and their inclusion in IPARD. The organization of seminars and courses
would increase the awareness and interest of agricultural producers. It is for farmers to resolve
58
unresolved ownership relations. The transfer of property rights to young farmers could contribute
to their inclusion in agricultural activities at an earlier age. Funding is one of the most important
issues of agricultural holding. The agricultural holdings most likely to benefit from IPARD are
entrepreneurial, have a willingness to communicate with other farmers, and have financial
resources and the knowledge. Agricultural holdings who have little UAA, who have fewer assets,
it will be more difficult to use the benefits of the IPARD. Other holdings that are not ready of
IPARD will use fund from national measures of the rural development. The perspective of using
the IPARD fund certainly exists. More than 60% of the respondents said they might use IPARD
in the future, while only 13% said they did not plan at all, and 11% said they planned to use
IPARD funds.
REFERENCES
AGENCIJA ZA SARADNJU, EDUKACIJU I RAZVOJ - ACED. (2012). IPARD u Bosni i

Hercegovini kroz Hrvatska istustva. ACED.
BEZHANI, E. (2015). The Result and the Impact of Project Ipard for the Rural and Agricultural
Development. Mediterranean Journal of Social Sciences, 602-604.
BOGDANOV, N., & BABOVIĆ, M. (2014). Radna snaga i aktivnosti poljoprivrednih
gazdinstava. Beograd: Republičli zavod za statistiku.
EUROPEAN COMISSION. (2014). Progress report on Serbia. Retrieved November 1, 2018,
from https://ec.europa.eu/neighbourhood-
enlargement/sites/near/files/pdf/key_documents/2014/20140108-serbia-progress-
report_en.pdf
GLUŠČEVIĆ, S., MAKSIMOVIĆ, S., PEJANOVIĆ, R., & SIMEUNOVIĆ, T. (2017).
Possibility of rural sector development in serbia using iprad program. Economics of
Agriculture, 753-767.
GOVERMENT OF REPUBLIC OF SERBIA. (2017). Retrieved November 3, 2018, from
National Program for Agriculture and Rural Development of the Republic of Serbia from
2018 to 2020: https://goo.gl/9fxoQ8
KOTEVSKA, A., BOGDANOV, N., NIKOLIĆ, A., DIMITREVSKI, D., STOJCHEVSKA
MARTINOVSKA, A., TUNA, E., et al. (2015). The impact of socio-economic structure
of rural population on success of rural development policy - Macedonia, Serbia and Bosna
and Herzegovina. Skopje: Association of Agricultural Economists of Republic of
Macedonia.
KRALIK, I., & ZMAIĆ, K. (2014). Owerview of the IPARD program fund usage in the Republic
of Croatia. International Symposium on Animal Science 2014 (pp. 502-509). Belgrade:
University of Belgrade Faculty of Agriculture.
59
MINISTRY OF AGRICULTURE AND ENVIRONMENTAL PROTECTION, REPUBLIC OF
SERBIA. (2015). IPARD programme for 2014-2020. Retrieved November 1, 2018, from
http://www.minpolj.gov.rs/wp-
content/uploads/datoteke/korisna_dokumenta/IPARD_II_Program_ENG_05072017.pdf
MINISTRY OF AGRICULTURE FORESTRY AND WATER MANAGMENT -
DIRECTORATE FOR AGRARIAN PAYMENTS. (2017). Retrieved November 3, 2018,
from First public call for submitting approval for the approval of the IPARD project
subsides for investment in physical property agricultural holdings in purchase of new
equipment, machine and mechanization: https://goo.gl/mZgVBb
MINISTRY OF AGRICULTURE FORESTRY AND WATER MANAGMENT -
DIRECTORATE FOR AGRARIAN PAYMENTS. (2018). Retrieved November 3, 2018,
from The second public call for submitting approval fir the approval of the IPARD project
subsidies for investments in physical property agricultural holdings in purshing new
tractors: https://goo.gl/VgSiBF
RADOVIĆ, G., KOŠIĆ, K., & DEMIROVIĆ, D. (2018). Financing as a key factor of the strategy
of sustainable rural tourism development in the Republic of Serbia. Economics of
Agriculture, 413-426.
RISTIĆ, L., TODOROVIĆ, V., & JAKŠIĆ, M. (2018). Limitations and opportunities for funding
agriculture and rural development in the Republic of Serbia. Economics of Agriculture,
1123-1138.
ŠESTOVIĆ, M., RADOSAVLJEVIĆ, K., & CHRONEOS KRASAVAC, B. (2017). The
importance of EU pre-accession fund for agriculture and their influence on country's
competitiveness. Ekonomika preduzeća, 506-517.
STATISTICAL OFFICE OF REPUBLIC OF SERBIA. (2013). Census of Agriculture 2012. -
Agriculture in the Republic of Serbia. Belgrade: Statistical Office of Republic of Serbia.
STEVANOVSKI, M., & ZIBEROVSKI, J. (2012). Structure of foreign investments and their
impact on agribusiness in Republic of Macedonia. Journal of Hygienic Engineering and
Design, 23,29.
TANKOSIĆ VAPA, J., & STOJSAVLJEVIĆ, M. (2014). EU common agricultural policy and
pre-accession assistance measures for rural development. Economics of Agriculture, 195-
210.
YARDIMCI, M., HIKMET, A., & ASLAN, R. (2018). The impact of IPARD supports on
structural and managerial features of dairy enterprises in Afyonkarahisar province. Indian
Journal of Animal Research, 151-156.
ZEKIĆ, S., MATKOVSKI, B., & KLEUT, Z. (2016). IPARD funds in the funtction of the
development of the rural areas of the Republic of Serbia. Economic Horizonts, 165-175.
60
HISTOMORPHOMETRIC AND HISTOLOGICAL STUDY OF THE EFFECT OF FREE
OMENTAL PEDICLE GRAFT ON NON UNION MODEL IN RABBIT RADIUS
Ena Oster
Mentor: Ozren Smolec, assistant professor, PHD. dr. med. vet.

Veterinary faculty, University of Zagreb, Clinic for the surgery, ortopaedics and ophtalmology,
Croatia
Summary: The study was carried out on 16 male adult New Zealand rabbits divided in 2
groups. A large segmental defect was created in the radius. In the group B the defect was filled
with a piece of omental tissue. After euthanasia the bones were removed for histomorphometric
analysis. Significant differences between the mean values for histological-grading score in the
control and experimental groups were detected (p < 0,05). We showed that free graft of
autogenous greater omentum could stimulate the formation of competent bone in an enviroment
deprived of its normal vascularization.
Key words: rabbit, omentum, bone, healing
INTRODUCTION
Bone healing is a complex, regenerative process initiated in response to injury, resulting in

optimal skeletal repair and restoration of skeletal function. The repair of large bone defects
remains a major clinical orthopaedic challenge. Bone is a highly vascularised tissue reliant on the
close spatial and temporal connection between blood vessels and bone cells to maintain skeletal
integrity. Angiogenesis thus plays a pivotal role in skeletal development and bone fracture repair.
The development of a microvasculature and microcirculation is critical for the homeostasis and
regeneration of living bone (Schmid et al., 1997). There are a number of factors involved in
neoangiogenesis, the main protagonists are: Vascular Endothelial Growth Factor (VEGF), basic
Fibroblast Growth Factor (bFGF), various members of the Transforming Growth factor beta
(TGFα) family. Omental grafting may affect bone healing positively not only through release of
VEGF, but also as a source of stem cells, basic fibroblast growth factor and transforming growth
factor-β; all of which are reported to have positive effects on bone repair (Matoba et al., 1996;
Rosier et al., 1998). Omental stromal cells express both adult stem cell and pluripotent embryonic
cell markers and may play a role in bone healing. Omentum as a free tissue graft to stimulate
healing of severely damaged tissue has been used successfully in humans and experimentally in
dogs ( Bigham-Sadegh et al., 2012)
The omentum is a serous membrane made up of a lattice of blood vessels and fat (Valat and
Moisonnier, 2001). Basically, it is highly vasculated organ with a rich source of angiogenic
factors that promote the growth of blood vessels into whatever tissue it is placed close to. Recent
studies have revealed that the omentum, apart from being a great source of various growth factors
61
and also contains omnipotent stem cells that can differentiate into a variety of cell types. On the
other hand, the role of greater omentum in augmentation of tissue repair and its great ability in
revascularization of tissues have been well documented. It’s use has been reported in
reconstruction of osteoradionecrosis of mandibulae (Moran and Panje, 1987), in experimental
tibial defects in rabbits (Oloumi et al., 2006), in healing of critical size defect in rabbit radius
(Kos et al., 2008), in nonunion radial defects in dogs (Saifzadeh et al., 2009), and in diaphyseal
fractures of the radius and ulna in dogs weighing less than 6 kg (Baltzer et al., 2015; Ree et al.,
2018).
The objective of our investigation was to estimate possibilities for application of free
transplant of the greater omentum in improving the bone defect healing process.
Eighteen male New Zealand rabbits, 2.50 +/- 0.30 kg, were kept in the same living conditions
(room temperature 20+/- 1 °C, relative humidity 55+/-5%). The animals were anaesthetized by
injecting with the injection of 5mg/kg xylazine and 50 mg/kg ketamine intramuscularly.
Eighteen rabbits were randomly divided into two groups. A 6 mm critical size-defect was
created in the right radial diaphysis with an oscillating saw under saline irrigation (leaving the
ulna intact) of each animal B group and in group A. A critical size bone defect (CSD) is defined
as the smallest intra-osseous wound characterized by an absence of spontaneous healing, which
would not heal by bone formation during the life time (Parfitt et al., 1987).
In group B the abdominal cavity was approached through 2 cm ventral midline incision
between the umbilicus and pelvic inlet. A 1x1 cm piece of greater omentum was isolated and
placed as a free graft in the osteotomy gap. Ketofen® was administered intramuscularly 3 days to
control pain. At 8 weeks post surgery the animals were euthanized and the bones removed for
histomorphometric evaluation. The operated radii were removed, cleaned of soft tissue and tissue
from grafted bone defects were fixed in 4% neutral buffered formalin. The bone samples assigned
for histomorphometry were embedded undecalcified in methylmetacrylate. Sections of 5 μm
thick were cut with a 2040 outcut microtome (Reichert-Jung, Heildelberg; Germany) and stained
with Goldner-Mason. Sections were analyzed with a Opton microscope. Nomenclature follows
the recommendations of the American Society for Bone and Mineral Research (Parfitt et al.,
1987).
Eight weeks after operation, the histological findings showed that the greater omentum graft
provoked the significant bone activity with predominant bone forming, and the bone turnover rate
with osteoblast domination (Figure 1), whereas in control group showed low bone activity, no
62
bone turnover rate is observed (Figure 2). Levence test for equal variance has shown that in the
case of osteoblast interface (Ob.S/BS) the samples had equal variance. Therefore, analysis has
been performed on these samples (Analysis of Variance, ANOVA). Osteoblast interface has
proved to be statistically significant parameter (p
(p=0.005).
=0.005). Osteoblast interface was found in the
treated group with respect to controlled one (Figure 3). The surface of trabecula covered with
osteiod and osteoblast interface have shown a high degree of positive linear correlation both in
the controlled and
nd the treated group.
Figure 4 The specimen without free omental graft, showing low bone activity and fibroplastic reparation( arrow),
Goldner-Mason staining , (x10)
Figure 5 Specimen with free omental

tal graft, the significant bone activity with predominational bone forming present
(arrows), Golden Mason staining,(x10)
Figure 6 Levence test has proved that osteoblast interface (Ob.S/BS) is statistically significant parameter (p=0.005)
63
Our presumption is that free transplant of the greater omentum implanted at the ectopic site
into critical-size bone defect will maintain its vitality and results by the forming new bone tissue.
Omentum contains stem cell, that can differentiated into a variety of cell type and good source of
angiogenic factor like vascular growth factor that provide oxygen at site of fracture and stimulate
mesenchymal cell to differentiate into osteoblast cell to form trabecular bone tissue when
compare with control cases. Moreover, omentum contains a potent mixture of growth factors,
progenitor cells (such as positive for CXCR-4 and WT-1), and chemotactic factors (such as SDF-
1α), which further helps in the recruitment of progenitor cells from the bone marrow and local
tissue to accelerate tissue repair (Litbarg et al., 2007). With the rapid explosion in the field of
tissue engineering and regenerative medicine, a greater understanding of the omentum may result
in the use of the omentum or its constituents to repair, replace, and possibly regenerate damaged
or defective tissues (Naveed et al., 2010).
CONCLUSION
In conclusion, our study shows that statistically significant osteoblast interface wakes to
conclude that autogenous free omental graft has influenced on bone turnover, especially on the
formation of newly-created bone. This result refers to the affect of omentum on the fracture site
by stimulation of undifferentiation mesenchymal cells at the site of fracture by local growth
factor metaplastically altered in the osteoblast cells that increase the osteogenic cells number.
Hence it could be recommended to enhance healing when the fractures are at risk of nonunion.
REFERENCES
BALTZER, W.I., COOLEY, S., WARNOCK, J.J., NEMANIC, S., STIEGER-VANEGAS, S.M.
(2015): Augmentation of diaphyseal fractures of the radius and ulna in toy breed dogs using a
free autogenous omental graft and bone plating., Vet Comp Orthop Traumatol., February, 28(2),
131-139.
BIGHAM-SADEGH, A., MIRSHOKRAEI, P., KARIMI, I., ORYAN, A., APARVIZ, A.,
SHAFIEI-SARVESTANI, Z. (2012): Effects of adipose tissue stem cell concurrent with greater
omentum on experimental long-bone healing in dog, Connect Tissue Res., 23 Jan, 53(4), 334-42.
LITBARG, O.N., KRISHNAMURTHY, P.G., SETHUPATHI, P., ARRUDA, A.I., DUNEA, G.,
SINGH, A.K. (2007): Activated omentum becomes rich in factors that promote healing and tissue
regeneration. Cell. Tissue. Res., 328, 487-497.
MATOBA, Y., KATAYAMA, H., OHAMI, H. (1996): Evaluation of omental implantation for
perforated gastric ulcer therapy: findings in a rat model, J. Gastroenterol., December, 31(6), 777-
784.
64
MORAN, W.J., PANJE, W.R. (1987): The free greater omental flap for treatment of mandibular
osteoradionecrosis., Arch Otolaryngol Head Neck Surg., April,113(4), 425-427.
NAVEED, U., SAQIB, M.D., McGUIRE, P.G., HOWDIESHELL, T.R. (2010): The omentum is
a site of stromal cell–derived factor 1α production and reservoir for CXC chemokine receptor 4–
positive cell recruitment. Am.J. Surg. 200, 276–282.
Kos, J., Smolec, O., Krpan, D., Babić, T., Vnuk, D., Kreszinger, M., Pirkic, B., Hock, K. (2008):
Healing of critical size defect on diaphiseal bone in rabbits by using free omental graft, Bone, 43.
OLOUMI, M.M., DERAKHSHANFAR, A., MOLAEI, M.M., TAYYEBI, M. (2006): The
angiogenic potential of autogenous free omental graft in experimental tibial defects in rabbit:
Short-term preliminary histopathological study, Journal of Experimental Animal Science
Volume 43, Issue 3, 12 December, 179-187.
PARFITT, A.M., DREZNER, M.K., GLORIEUX, F.H., KANIS, J.A., MALLUCHE, H.,
MEUNIER, P.J., OTT, S.M., RECKER, R.R. (1987): Bone histomorphometry: Standardization
of nomenclature, symbols and units. J. Bone Miner. Res. 2, 595–610.
REE, J.J., BALTZER, W.I., NEMANIC, S. (2018): Randomized, controlled, prospective clinical
trial of autologous greater omentum free graft versus autogenous cancellous bone graft in radial
and ulnar fractures in miniature breed dogs., Veterinary Surgery, 17 April, 47(3), 392-405.
ROSIER, R.N., O'KEEFE, R.J., HICKS, D.G. (1998): The potential role of transforming growth
factor beta in fracture healing. Clin Orthop Relat Res., October, (355 Suppl), 294-300.
SAIFZADEH, S., POURREZA, B., HOBBENAGHI, R., NAGHADEH, B.D., KAZEMI, S.
(2009): Autogenous greater omentum, as a free nonvascularized graft, enchances bone healing:
an experimental nonunion model., Journal of investigative surgery, 22, 129-137.
SCHMID, J., WALLKAMM, B., HAMMERLE, C.H., GOGOLEWSKI, S., LANG, N.P. (1997):
The significance of angiogenesis in guided bone regeneration, A case report of a rabbit
experiment. Clin. Oral Implants Res. 8, 244-248.
65
SYSTEMIC EFFECTS OF EPIDURAL ANESTHESIA WITH LIDOCAINE-
ADRENALINE IN DOGS
Tara Kostanjšak1
Mentor: Marko Pećin2

1
Student of 6th year at Faculty of Veterinary medicine, University of Zagreb
2
Assistant Professor at Orthopaedics and Ophthalmology Clinic, Faculty of Veterinary
medicine. University of Zagreb.
Summary: Published studies show that lumbosacral epidural anaesthesia can have
systemic effects (Nolte et al., 1983). Cardiovascular and respiratory effects can be noted after
administration of epidural anaesthesia (Veering and Cousins, 2000). This work describes
systemic effects of lumbosacral epidural anaesthesia with 2% lidocaine+0.0125 mg/ml
adrenaline solution in 28 dogs. Effects were compared with control group of also 14 dogs
that had lumbosacral epidural anaesthesia with only 2% lidocaine. In both groups of dogs 1
ml of solution per 6kg of animal body mass was administered. Differences between time of
onset, duration of anaesthesia, and time needed for dogs to start to support their body weight
were measured. During 60-minute period of time parameters like rectal body temperature,
pulse, breathing rate, arterial blood pressure, pulmonary artery blood pressure and cardiac
output were measured also showing cardiovascular and respiratory effect of epidural
anaesthesia. Effects of adrenaline added in anaesthetic solution on cardiovascular system
were more visible than the effects on respiratory system. In addition, there were some
differences between two anaesthesia’s in time of onset, duration and time needed for dogs to
support themselves. Adrenaline added to lidocaine in epidural anaesthesia have no
significant influence on prolonging the duration of anaesthesia with minimal effects on
cardiovascular and respiratory system in dogs.
KEY WORDS: epidural anaesthesia, lidocaine-adrenaline solution, systemic effects, dog
INTRODUCTION
Epidural anaesthesia is one of the most commonly used regional anaesthesia techniques
because of its simplicity, safety and efficiency for surgical procedures caudally from
umbilicus (Bradley et al., 1980). In veterinary medicine, it was first administered in the
1920s. From then on, it was used daily in animals for reducing pain during surgeries, for
reducing the skeletal muscle tone and for reducing the dose of other anaesthetics used in
combination with epidural anaesthesia. Minor surgical procedures on hind legs, perineum,
pelvis and tail of the dog or short-term postoperative analgesia are main indications for using
epidural anaesthesia. Its analgesic activity is most commonly used for orthopaedic surgeries
66
where, in combination with intravenous or inhalation anaesthesia, it provides satisfactory
anaesthesia. It is widely used in emergency C-sections because it does not affect the foetus,
and because of its efficiency and fast activity on reducing pain in abdominal surgeries. With
development of anaesthesia the techniques of epidural anaesthesia are also improved, and
new, more potent anaesthetics are introduced. In addition, local anaesthetic lidocaine is
combined with adrenaline to improve and prolong its efficiency time. Duration of epidural
anaesthesia depends on type of anaesthetic used, its concentration, dosage and presence or
lack of different compounds that have pharmacological activity in anaesthetic solution (Hall
and Clarke, 1991). Selection of local anaesthetic, or its combination with other
pharmacologically active compounds (like as adrenaline), and its concentration and volume
depend on the size of the dog, desirable expansion of anaesthetic and desirable time of onset,
duration and effect. For example, dosage of 1 ml 2% lidocaine on 4,5 kg of body mass will
completely anesthetize caudal part of the abdomen, hind legs and pelvis caudally from first
lumbar vertebra. Good epidural anaesthesia is achieved with dosage of 1 ml of 2% lidocaine
or 0,5% bupivacaine on 5 kg of body mass. Even though there is possibility of combining
anaesthetics with other pharmacologically active compound, because of its cost, local
anaesthetic and combination of local anaesthetic and epinephrine (adrenaline) were used in
this experiment. Local anaesthetics applied in epidural space in dogs can be found in
cerebrospinal liquid, epidural venous plexuses and lymph system or they can be partially
distributed in fat tissue of epidural space (Ward et al., 1985). Also, they can be resorbed and
have systemic influence (Ward et al., 1985). In this article, systemic influences of lidocaine-
adrenaline solutions, which consisted the dosage of 1 ml/6 kg of 2% lidocaine and 0.0125
mg/ml epinephrine will be shown.
MATERIALS AND METHODS
Experiment was done to compare time of onset, duration and influence of lidocaine-
adrenaline lumbosacral epidural anaesthesia on cardiovascular and respiratory system in dogs.
Control group was consisted of dogs on which epidural anaesthesia with 2% lidocaine was
used. Parameters, which were measured, were: body temperature, pulse rate, arterial blood
pressure, pulmonary artery pressure, cardiac output and breathing rate. These parameters were
measured in 28 dogs. First measurement was done right before application of epidural
anaesthesia. Second measurement was done 5 minutes after application of epidural
anaesthesia, third was done 15 minutes after application, and every other was done 15 minutes
after the last one. The last measurement was done 60 minutes after application of epidural
anaesthesia.
Dogs were divided into two exact groups of 14 dogs (group A and group B). Group A
consisted of 14 dogs anesthetized with solution of 2% lidocaine and group B consisted of 14
dogs anesthetized wit solution of 2% lidocaine to which adrenaline, in dose of 0.0125 mg to 1
67
ml of lidocaine, was added. Dosage in both groups was 1 ml of anaesthetics per 6 kilograms
of body mass.
All dogs were 3-4 years of age, smaller breeds, males, which had no cardiovascular or
respiratory problems in their history or clinically. Range of body mass was between 5.25 and
7.70 kilograms. In both groups, dogs were sedated with intramuscularly applied 0.2 mg per
kilogram of Acepromazine half an hour before application of epidural anaesthesia. Applied
dosage does not effect significantly on respiratory or cardiovascular system and therefore is
suitable for this experiment (Hall and Clarke, 1991).
Lumbosacral epidural anaesthesia was applied with disposable 22G hypodermic needles,
3.5 cm length. During application, dogs were in sternal position. Right after application, the
dogs were placed in standing position for establishing time of onset. When onset was
established, they were put back to sternal position in which they stayed until the end of the
measurement.
Number of inhales and exhales was measured for one minute. The pulse was measured
with help of ECG monitor, and body temperature with rectal thermometer. Cardiac output and
pulmonary artery pressure were invasively measured with SWAN-GANZ catheter (figure 1.)
that was put through jugular vein in pulmonary artery while arterial pressure was measured
invasively through femoral artery.
Fig. 1. Swan- Ganz catheter for invasive measuring of blood pressure
1. Differences in time of onset and duration

Time of onset and duration of two different epidural anaesthesia’s were shown in table 1.
Time of onset was measured as time passed from the moment of application in epidural space
until the moment the dogs were not able to stand on their own, and when they did not react on
pressure caused by haemostat between toes on their hind legs. When the pressure was applied
they did not try to move nor, did they show any painful reaction.
68
Duration was measured from time of onset until the dogs started moving their hind legs
and started showing painful reactions on stimuli. The pressure was done every 5 minutes for
total of 25 minutes. After 25 minutes passed the pressure was applied every minute. In
addition, time needed for dogs to support themselves on their hind legs was measured.
Table 1. Differences in time of onset and duration between group A and group B. Values
are in minutes.
Time of onset Time of duration
Average A 1,7 35
Average B 2,215 37,25
The difference in time of onset and duration of epidural anaesthesia, between the two
groups is evident. In dogs that epidural anaesthesia of 2% lidocaine was applied, the onset
began after 1,7 minutes. The shortest period needed was 0,8 minutes (48 seconds), and the
longest period needed was 2,5 minutes. Onset of epidural anaesthesia with added adrenaline
began in average after 2,215 minutes. The shortest time needed and the longest were 1,5
minutes and 3 minutes.
Duration, until the dogs started feeling pain caused by pressure with hemostat, in both
groups was in average 36 minutes. In group where lidocaine was combines with adrenaline
(group B), time when dogs started showing signs of pain was between 35 and 39 minutes after
application of epidural anaesthesia, while in group where only lidocaine was used (group A)
animals started showing signs of pain 30 to 45 minutes after application.
2. Differences in cardiovascular and respiratory affects
The results of experimental measurements in cardiovascular and respiratory effects
between epidural anaesthesia with lidocaine and lidocaine-adrenaline are shown in table 2.
Average values measured in 28 dogs are shown. Average results of group A (where only 2%
of lidocaine was used) are shown in red, while average results of group B (where 2%
lidocaine and 0,0125 mg/ml adrenaline were used) are shown in blue.
69
Table 2. Body temperature, pulse, breathing, sys art – Systolic arterial pressure, dias art –
diastolic arterial pressure, mean art – mean arterial pressure, ap sys – systolic pressure
pulmonary artery, ap dias – diastolic pressure in pulmonary artery, ap mean – mean arterial
pressure in pulmonary artery.
Minutes 0 5 15 30 45 60
Body
38.2 38.7 38.3 38.6 38.2 38.6 38.6 38.7 38.4 38.8 38.4 38.8 °C
temp.
Pulse 86 91 84 118 91 104 82 115 76 106 77 102 n/min
Breathing 16 15 15 16 15 15 14 14 15 14 13 14 n/min
sys art 165 170 167 160 167 161 144 162 151.5 164 154 161 mmHg
dias art 94 88 93 91 84 90 75.5 101 81 83 82 81 mmHg
mean art 118.5 112 113 113.5 114 116 99 119 93 115 106 106 mmHg
ap sys 27 28 28 30 19 31 19 26 21 24 22 28 mmHg
ap dias 11 14 12 15 9 16 7 13 8 13 9 15 mmHg
ap mean 17 20 19 22 14 22 10 18 13 17 14 20 mmHg
Experimental measurement showed some differences between these two epidural

anaesthesia’s. Epidural anaesthesia of 2% lidocaine with added 0,0125 mg/ml adrenaline
resulted with relatively higher average onset time and longer period needed for dogs to
recover and to support themselves on their hind legs. Difference in onset time, between group
A and group B, was 0,425 minutes (25, 5 seconds).
Time needed for dogs to feel pain, by applying pressure in 3 dogs in group A (2%
lidocaine) was shorter than 34 minutes while in group B (lidocaine + adrenaline) that time
was, in all dogs, longer than 35 minutes. From these results, it is evident that there is
influence of adrenaline on duration of anaesthesia but that difference, because it is only few
minutes, is not significant.
Time needed for dogs to support themselves on their hind legs is shorter in group A. It can
be concluded that adrenaline slows removal of lidocaine from spinal cord and prolongs its
activity.
Systemic influences of epidural anaesthesia on cardiovascular system are more
pronounced in group B. All dogs from group B had tachycardia after application of epidural
anaesthesia. During that time, average frequency of heartbeat was never over physiological
boundary of 120 beats per minute (Ettinger et al., 2016).
In addition, cardiac output and pressures in pulmonary artery were higher than in other
dogs anesthetized only with lidocaine.
In dogs from both groups breathing was not significantly changed. It stayed the same (+/-
2 breaths).
By using both anaesthesia’s, in all 28 dogs for 60 minutes, there were no noticeable
changes in breathing and heart work. Lack of these changes imply that there are no harmful
influences of these anaesthesia’s.
70
CONCLUSION
Experimental measurements have confirmed that epidural anaesthesia with solution of 2%

lidocaine, local anaesthetic, in dosage of 1 millilitre per 6 kg of body mass in dogs, sedated
with Acepromazine, did not effect functioning of cardiovascular or respiratory system. It was
confirmed as safe method for shorter surgeries for caudal parts of the body with average
duration time of 35 minutes.
Adrenaline, as addition to solution of local anaesthetic, in lower doses, applied locally,
prolongs time of duration and onset of anaesthesia. Despite this, added adrenaline had no
significant influence on time of duration and onset of anaesthesia. Minor differences were
measured, but they can be considered insignificant.
Influence of adrenaline resorbed from epidural space to cardiovascular system was seen as
higher heart rate with consequent higher cardiac output, and blood pressure. Influence on
respiratory system was not seen in either anaesthesia.
Considering this facts, results of experimental measurements of two different epidural
anaesthesia’s, and price of lidocaine and adrenaline, the advantage should be given to the
anaesthesia using only 2% lidocaine.
REFERENCES
ETTINGER, S.J., FELDMAN, E.C., COTE, E. (2016): Textbook of Veterinary Internal

Medicine, 8th Edition: 733
NOLTE, I., WATNEY, G.C.G., HALL, L.W.(1983): Cardiovascular effects of epidural
blocks in dogs. J. Small Anim. pract. 24: 17-29.
BRADELY, R.L., WITHROW, S.J. AND HEATH, R.B.(1980): Epidural analgesia in the
dog. Vet. Surg. 9: 153-156.
WARD, R.J, ,BONICA, J.J., FREUND, F.G., AKAMATSU, T., DANTZINGER, F.,
ENGLESSON, S. (1985) : Epidural and subarahnoid anesthesia:cardiovascular and
respiratory effects. JAMA. 191-275.
VEERING, B.T., COUSINS, M.J.(2000): Cardiovascular and pulmonary effects of epidural
anaesthesia. Anaest. Int. Care. 28: 620–635.
71
PANTARSAL ARTHRODESIS USING FIXIN HYBRID PLATE IN A FRENCH
BULLDOG
Niko Ivkić1
University of Zagreb, Faculty of Veterinary Medicine, 6 th year student
Mentor: Assistant Professor Marko Pećin 2, PhD, DVM

University of Zagreb, Faculty of Veterinary Medicine, Clinic for Surgery, Orthopedics and
Ophthalmology, Croatia
Summary: Pantarsal arthrodesis is a surgical fusion of a talocrural, intertarsal and

tarsometatarsal joints to form a bony ankylosis (Piermattei et al., 1997). Tibiotarsal joint
disorders are mostly traumatic and due to automobile accidents. Indication for arthrodesis are
numerous, for example irreparable fracture of the joint, chronically unstable joint, chronic
severe degenerative joint disease from any cause or neurological injury causing partial paralysis
of the limb especially of the carpal and tarasal joints (Piermattei et al., 1997). Over the years,
several techniques of arthrodesis have been described, but all procedures follow some basic
principles, such as removal of the entire articular cartilage, rigid fixation at a functional support
angle and application of spongy bone graft (Rahal et al., 2006). Pantarsal arthrodesis is
considered the most appropriate procedure when arthrodesis of the tibiotarsal joint is required.
Recently, pantarsal arthrodesis is performed with the use of a bone plate implanted on the medial
surface of the joint. The aim of this study is to describe the performance of pantarsal arthrodesis
using a stainless steel Fixin hybrid plate with titanium screws. The application of the plate was
on the cranial surface (dorsal) of the joint with the purpose of limb salvage in a French bulldog
after laceration injury caused by a car accident.
Key words: pantarsal arthrodesis, hybrid plate, dorsal plate, bulldog
INTRODUCTION
Pantarsal arthrodesis is a surgical fusion of a talocrural, intertarsal and tarsometatarsal joints

to form a bony ankylosis, and it is considered the best procedure when arthrodesis of the
talocrural joint is necessary (McKee 1994). The advantages of pantarsal arthrodesis over selective
talocrural arthrodesis include the availability of more bone for the placement of implants and
arthrodesis of the tarsal and tarsometatarsal joints which helps to mitigate the development of
osteoarthritis. Plantar bone plate fixation (Klause and others 1989), dorsal bone plate fixation
(Klause et al., 1989, Decamp et al., 1993), linear external skeletal fixation (Klause et.al 1989) and
circular external skeletal fixation (Lewis et al., 1999) have all been described. Dorsal bone plate
fixation is considered to be mechanically unsound because the plate is on the compression
(concave) side of the tarsus (Klause et al., 1989). DeCamp et al., (1993) reported that in five of
72
10 dogs treated with a dorsal plate the implant became loose, but still we have to use this
tehnique because of the specific findings intraoperatively. The aim of this study is to describe the
performance of pantarsal arthrodesis using a Fixin hybrid plate for application to the cranial
surface of the joint with the purpose of limb salvage in a French bulldog after laceration injury
caused by a car accident.
CASE
George, a 2 years old French bulldog weighing 12,6 kg, was attended at the Surgery,
Orthopedics and Ophthalmology Clinic of the Faculty of Veterinary Medicine, University of
Zagreb with history of recent car accident. After physical and radiographic examinations he was
diagnosed with an Gustilo Anderson type IIIB open fracture of the left medial malleolus and
luxation of tarsal joint and the left hip joint with an extensive laceration in right femoral and
gluteal region (Figure 1-2).
Fig. 1-2 Radiographic image in ventrodorsal and laterolateral projection demonstrating luxation of the left
tibiotarsal and hip joint with fracture of the left medial malleolus after traumatic injury.
Under the intravenous anesthesia, surrounding of the wound on a left tibotarsal joint was
clipped and the wound was debrided, cleaned, and copious lavaged with 0,5L of Hartmann
solution. After that Robert- Jones bandage was applied with sulfadiazine silver cream in contact
layer. Extensive laceration of right femoral and gluteal region was managed at the same way and
because of specific location of the laceration the Tie-over bandage with sulfadiazine silver cream
in contact layer was performed. Further, first 9 days bandages was changed every day in
intravenous anesthesia, necrotic parts of the wounds was removed, non-vital parts of wound
73
edges was revitalized and honey, sugar or no- adhesive dressing were applied in contact layer.
George was also under antimicrobial therapy for 7 days. Combination of enrofloxacinum and
amoxicilinum clavulante acid was applied. Analgesia was provided through Fentanyl patch in
duration of 2,5 weeks and (NSAID) for 7 days. For the next 3 weeks George came every 2 nd day
for observation and wound cleaning, bandaging and debridement . Progression in wound healing
was observed (Figures 3-6). At the appearance of the granulation tissue ®Granugel was applied,
when epithelialization started, combination of sulfadiazine silver cream and insulin were putted
in contact layer of bandage. Two months later the surgical procedure occurred.
Fig. 3-4. Wounds in left tarsal region and right femoral and gluteal region at one of the first presentations.
Presence of necrotic and a-vital tissue.
Fig. 5-6. Wounds in left tarsal region and right femoral and gluteal region 2,5 weeks after the first presentation.
Presence of healthy granulation tissue and beginning of the epithelisation.
In general inhalant anesthesia, with the dog in dorsal recumbence the tarsal joint was accessed
by incision in the dorsal region of the limb that extended from the distal third of the tibia to the
middle third of the metatarsal bones. Subcutaneous tissue was dissected and musculature and
tendons were carefully separated for exposure of the distal portion of the tibia, tarsal and
74
metatarsal bones. The curettage and grafting of tarsal calcaneoquartal, centrodistal and
tarsometatarsal joints is done primarily from the dorsal, medial and lateral aspects, leaving the
large plantar ligaments intact. Debridment and grafting of talocrural and talocalcaneocentral was
also made even though the fracture of the Talus was present. The bone plate is pre-contoured to
the desired angle of 135˚ using a table top plate bender. The most common angle for the dog
ranges from 135˚ to 145˚ (Swaim et al., 2015). The Fixin 1,9-2,5 hybrid plate was placed on
dorsal side of tibia and metatarsus and the subsequent order in which the 1.9mm and 2.5mm titan
screws was drilled and tightened was: (1) proximal metatarsal bone, (2) distal metatarsal bone,
(3) remaining metatarsal bone, (4) distal tibia and remaining tibia (Figure 7).
Fig. 7. Intraoperatively placed Fixin 1,9-2,5 hybrid plate at the dorsal side of tibiotarsal joint.
Muscle fascia, subcutaneous tissue and skin sutures were performed in a conventional
manner. Immediately after procedure the control X-ray examination was performed (Figure 8). In
the postoperative period, antimicrobial drug (Cefazolinum) was administered for 6 hours after the
procedure and the Meloxicam (NSAID) for 10 days after the surgery. Surgical wound cleaning
and partial activities restrictions were recommended for 30 days. Robert.-Jones bandage was
applied over the left leg in duration of 8 weeks for providing extra stability.
75
Fig. 8. Control X-ray examination showing that all bones and implants are in position.
On the follow up the patient showed functional support of the limb, even though the hip joint
is still luxated. One month after the procedure the radiographic exams revealed a proliferative
bone reaction in the interarticular regions. The use of dorsal plates for pantarsal arthrodesis, with
their ease of application, is common in dogs. However, dorsal plates are placed on the
compression side of the construct; therefore, they bear large bending moments because of the
talocrural fusion angle and the long moment arms provided by the tibia and tarsometatarsus on
either side of the construct. Such constructs are biomechanically weaker than those involving a
plate placed on the tension side of the construct. Dose constructs are more susceptible to plastic
deformation or breakage early in the postoperative period as a result of acute overload or more
commonly to fatigue failure over an extended period (Klaus et al.1989). Plate breakage with
canine dorsal plate pantarsal arthrodesis is one of the most significant complications reported in
the literature (Dechamp et al.1993). Dorsal plates have been associated with a greater incidence
of implant failure, owing to the suboptimal biomechanical properties of the plate-tibiatarsus-
metatarsus construct (Guilou et al. 2008). According to McKee et al. (1994), customized Plates
applied medially or laterally are a great alternative for pantarsal arthrodesis compared to the
plates used on the dorsal or plantar surface of the limb. Nonetheless, in this case we have to use
dorsal approach because of the absence of normal talus. According to Swaim et al. (2015) a
dynamic compression plate (DCP) or lengthening plate can be applied, but in this case due to its
structure that combines stainless steel and titan alloy the Fixin 1.9-2.5 100mm plate was used.
Titanium bushings allow titanium screws to be applied and locked onto stainless steel body of a
plate (Figure 9).
76
Fig. 9. Fixin 1,9-2,5 hybrid plate with eight holes, long bridge and titanium bushings allowing titanium screws to
be placed and locked.
The Fixin plate was also used because unlike customized plates applied medially or laterally
it allows pre-contouring to the desired angle of 135 ˚. According to Davis (2013) stainless steel
have a higher module of elasticity compared with titanium alloy. Presence of the stainless steel
should improved the elasticity of the plate and reduce the possibility of implant failure. The
bending forces and early postoperative complications are also reduced by placing the Robert-
Jones bandage over the left leg for 8 weeks. Because of the absence of tarsal bones and a great
part of the talus we were not able to place pre-customized titanium plate at the standing angle to
the medial or lateral surface of the joint. For that reason, the plate was placed dorsally. As the
composition is concerned, the titanium alloy plate wouldn’t be satisfactory owing to cyclic
movements and material fatigue. There was no available titanium plate with satisfactory long
bridge and if bending of the plate to the standing angle was performed, there was a great
possibility of plate deformation with screw holes deforming what would disable normal screw
locking. The same case is less possible with plates that have bridge. Therefore taking all this facts
into consideration Fixin stainless steel plate with bridge and titan screws has been chosen.
Furthermore, Fixin hybrid plate enables 2,5mm screws for Tibia and 1,9mm for Metatarsal bones
to ensure that the ones don’t break.
77
CONCLUSION
Even tough the customized prebended plates applied medially or laterally are according to
some authors better alternative for pantarsal arthrodesis compared to the plates used on the dorsal
or plantar surface of the limb, this case shows how fractured Talus can annul all the benefits of
those techniques. Locking plates have become the gold standard for the fixation of complex
fractures, as they combine fixation with anatomic articular reduction. Benefits of locking plates
are numerous. The unique conical screw-plate locking system ensures the distribution of force
throughout the structure, eliminating the risk of implant breakage and screws backing out or
cross-threading. Taking all into consideration it is concluded that the Fixin Hybrid plate for
pantarsal arthrodesis proved to be a satisfactory method, which promoted adequate interarticular
stability and favored bone proliferation and fussion.
REFERENCES
DAVIS, J.R. (2003): Handbook of Materials for Medical devices. ASM International. 265p.
DECAMP, C. (1997): Piermattei Brikner and Flo Handbook of Small Animal Orthopedics and
Fracture Repair, 3rd edition, Elsevier. 636p.
DECAMP, C. E., MARTINEZ, S. A., JOHNSTON, S. A. (1993): Pantarsal arthrodesis in dogs
and a cat: 11 cases (1983-1991). J. of the Am. Vet. Med.Assoc. 203: 1705-1707.
GUILLOU, R. P., FRANK, J. D., SINNOTT, M. T., MEYER, E.G., HAUT, R.C., DÉJARDIN,
L. M. (2008): In vitro evaluation of medial plating for pantarsal arthrodesis in dogs. Am. J. Vet.
Res. 69: 1406-1412.
KLAUSE, S. E., PIERMATTEI, D. L., SCHWARZ, P. D. (1989): Tarso-crural arthrodesis:
complications and recommendations. Veterinary and Comparative Orthopaedics and
Traumatology 3: 119-124.
LEWIS, D. D., RADASCH, R. M., BEALE, B. S., STALLINGS, J. T., LANZ, 0. I., WELCH, R.
D., SAMCHUKOV, M. L. (1999): Initial clinical experience with the IMEX circular external
skeletal fixation system. Vet. Comp. Orthop. Traumat. 12: 108-117.
MCKEE, W. M. (1994) Intractably painful joints. In Manual of Small Animal Arthrology. Eds J.
E. F. Houlton, R. W. Collinson. Cheltenham, BSAVA Publications. 115-134pp.
MCKEE, W.M., MAY, C., MACIAS, C., LAPISH, J. (2004): Pantarsal arthrodesis with a
customized medial or lateral bone plate in 13 dogs. The Veterinary Record. 154: 165-170.
RAHAL, S.C., VOLPI, R.S., HETTE, K., TEIXEIRA, F.J., VULCANO, L.C. (2006):
Arthrodesis tarsocrural or tarsometatarsal in 2 dogs using circular external skeletal fixator: Case
Report. The Can.Vet. J. 47: 894-898.
SWAIM, S.F., WELCH, J., GILLETTE, R.L. (2015): Managment of Small Animal Distal Limb
Injuries. 267-269pp.
78
DIAGNOSIS OF HIP DYSPLASIA IN DOGS ACCORDING TO THE GUIDELINES OF
THE INTERNATIONAL CYNOLOGICAL ASSOCIATION (FCI)
Tijana Kukurić
Mentor: Prof. dr. Bojan Toholj

University of Novi Sad, Faculty of Agriculture, Department of Veterinary Medicine, Serbia
Summary: The detection of hip dysplasia in dogs is a significant diagnostic challenge, which
aims to eliminate this disease from the population. During the research a total of 14 dogs were
examined. We designed work tables to evaluate the parameters of the degree of hip dysplasia.
Evaluation is formed especially for each hip joint, and the final estimate of dysplasia is guided by
that hip joint that has given a poorer score. In this way, we evaluated the presence of hip
dysplasia in dogs and the frequency of individual parameters.
Key words: hip, dysplasia, evaluation, parameters.
INTRODUCTION
Canine hip dysplasia (HD) is significant and relatively frequent disease of dogs. The disease
has a strong hereditary component. However, non-genetic factors such as nutrition, lack of
movement or intensive training at an early age are also important for the development and
manifestation of the disease (Barr, 1987). Heavy dogs, with rounded conical conformation, less
developed muscles and early physical maturity are the most affected (Mikkelsen, 2007). It has
been noted that breeds with a more developed musculature, with a flat foot position, are less
susceptible to disease (Riser, 1975). According to the definition, hip dysplasia is an abnormality
in development. Puppies are therefore born with normal hips, and the instability of coxofemoral
joints occurs during postnatal re-engineering (Kealy, 2005). It is generally accepted that the
initial instability factor is the initial instability of the joint. Then follows a vicious circle, in which
the basic instability of the joint worsens with every movement and weight, stimulating the
femoral head beyond its anatomical position (Richardson, 2010). Clinical signs related to HD can
be manifested as mild or occasional lameness to a clinical picture in which the signs are not
expressed, as a consequence of the development of degenerative changes and pain (Fry, 1992).
Diagnosis of hip dysplasia is performed by clinical examination and radiography. Clinical tests
for early diagnosis of dog HD are based on the determination of increased joint mobility. The
most common is the Ortolani test. Many organizations, such as the Orthopedic Animal
Foundation (OFA), the Federation Cynologique Internationale (FCI) and the British Veterinary
Association/Kennel Club (BVA/KC) have developed diagnostic protocols for HD. In addition,
specific HD estimation programs have been developed: for example, Pennsilvania Hip
Improvement Program (PennHIP) and Dorsolateral Subluxation Score (DSL) (Grundström,
2014). The Orthopedic Foundation for Animals, FCI and BVA/KC use standard ventro-dorsal
79
projections for the radiographic recording of animals in the sedation. The condition of the hip is
assessed based on the described assessment methods. Radiographic assessment of the condition
focuses on signs of incongruence, degenerative joint diseases and joint instability. Differences
between programs are in evaluation methods (Verhoeven, 2012). The recommended age, for most
diagnostic protocols, in which the radiographic assessment is performed is one year old, or over
18 months in large breeds (Grundström, 2014).
The aim of this research was analysis of the protocol for diagnosis of hip dysplasia according
to the FCI system and its application to dogs of the appropriate age, as well as the setting of a
decision algorithm for the existence of hip dysplasia in dogs, using the table.
The investigatoin was carried out at the Veterinary teaching hospital, University of Novi Sad.
A total of 14 dogs, different breeds and both sexes, were brought by their owners, to a diagnosis
for hip dysplasia and/or obtaining the appropriate certificate. In order to enable regular diagnosis,
it is necessary to respect all prerequisites. It establishes a condition for the dog to be involved in
the diagnosis, that he has a minimum of one year of age, and in addition, that he is permanently
marked with a microchip. In order to enable the patient's correct positioning, sedation is
necessary. For sedation we used medetomidine (Domitor, Orion Corporation Orion Pharma,
SRB) at a dose of 10-20 μg/kg. After i.m. applications, effects are seen within 10 to 15 minutes,
in the form of sedation and muscle relaxation. After obtaining a X-ray images, we used
atipamezol (Antisedan, Orion Corporation Orion Pharma, SRB) as reverse drug to increase a
speed recovery of dogs from sedation. We used the X-ray device ZooMax (EU), with the setting
of parameters kV and mAs according to the size of the patient, and Agfa CR 10-X digital
procesing unit. We assessed the degree of hip dysplasia according to FCI guidelines. The
estimate is based on a total of 6 parameters: Norberg angle, coverage of femur head, craniolateral
edge of the acetabulum, presence of subhondral osiification, changes in the femur head and
Morgan lines. Each of these parameters has been assigned a certain numerical value, in order to
obtain their final numerical value by their addition, and accordingly assign appropriate final
estimates. Estimates according to the parameter are shown in Table 1.
80
Table 1. Working table for assessing hip dysplasia according to the FCI guidelines (CFH- center of the femur head,
DEA- dorsal edge of acetabulum).
Points 0 1 2 3 4 5
Parameter 1 >105˚ <105 ˚ 100-105 ˚ 90-100 ˚ 80-90 ˚ <80 ˚
Norberg angle
Parameter 2 CFH CFH CFH CFH CFH CGBK
Coverage of medially medially covers the laterally in laterally in laterally in
femur head in relative to DEA relation to relation to relation to
relation the DEA (>2mm) the DEA (1- the DEA (6- the DRA
to the (1-2mm) 5mm), the 10mm), the (>10mm),
DEA joint space joint space the joint
(>2mm) centrally significantly space in the
extended expanded luxation
Parameter 3 Parallel Horizontal Slightly Moderately Significa- Edge is
Craniolateral with the flattening, flattening, ntly missing,
edge of the head of mild medium flattening, the
acetabulum the exostoses exostoses medium acetabulum
femur exostoses is significa-
bone ntly
deformed
Parameter 4 Narrow, Wide, Lateral Lateral Laterally Connected
Presence of evenly evenly thickening thickening significant with the
subhondral thicke- thickening of the of the thickening, lateral
osiification ning small medium medially not pelvic edge
degree, stage, visible or missing
mild medium
reduction reduction
medial medial
Parameter 5 Head Round Head Head Head Head
Changes in round head and slightly medium medium deformed,
the femur and neck flattening, flattening, flattening, neck with
head neck cylindrical neck with neck with neck with massive
clearly mild mild medium exostoses
expre- exostoses exostoses exostoses
ssed
Parameter 6 Not Small thin Conspicu- Conspicu- Conspicu- Very
Morgan lines visible ridge, up ous ridge ous ridge up ous ridge up expressed
to 1 mm 1-2 mm to 3 mm to 4 mm ridge, like
exostosis
The estimate of dysplasia is formed especially for each hip joint, by the same score system,
and the final grade is guided by that hip joint that has given a poorer score. Therefore, there are 5
categories A, B, C, D and E. Categories A and B represent a normal and approximately normal
finding, and C, D and E state of dysplasia. The final score was based on Table 2.
Table 2. Category of hip dysplasia.

Left hip/Right hip
Sum of points Category Degree of dysplasia
0-2 A Normal hip
3-6 B Approximate normal hip
7-12 C Mild dysplasia
13-18 D Medium heavy dysplasia
>18 E Heavy dysplasia
81
In this study, the diagnosis of hip dysplasia in dogs and assessment of the degree of dysplasia
according to the FCI system was performed. By observing X-ray, correctly positioned patients,
we used the guidelines from Table 1, to evaluate a degree of hip displasia. Each parameter
received a score from 0 to 5, separately for the left and for the right hip. These numerical values
were collected and then according to Table 2 we determined a presence of hip dysplasia and
degree of same, based on the more deformed joint, as shown in Table 3.
Table 3. Results by category ( L- left hip, R- right hip).
Patient Sum Category Patient Sum Category

1 L 12, R 13 D 8 L 4, R 4 B
2 L 2, R 1 A 9 L 2, R 2 A
3 L 0, R 0 A 10 L 3, R 1 B
4 L 2, R 0 A 11 L 3, R 2 B
5 L 25, R 24 E 12 L 19, R 16 E
6 L 6, R 10 C 13 L 0, R 2 A
7 L 11, R 9 C 14 L 4, R 5 B
The final results of this research, which refer to the number of patients who received grades A
to E, while also pointing to the prevalence of hip dysplasia, are given in Table 4. Of the total of
14 examined dogs, 5 dogs are dysplastic.
Table 4. Research results.
Category Number of dogs

A 5
B 4
C 2
D 1
E 2
By analyzing the x-ray, we determined the presence of changes caused by dysplasia. Changes
in the form of a reduction in Norberg's angle and the head of the femur bone were most common.
Out of a total of 14 dogs, changes in Norberg's angle were present in 11 patients, and changes in
the coverage of the femur head in 13 patients. There were some slight departures, even in dogs
that were rated A, that is, Norberg's angle was slightly less than 105˚, or the center of the femoral
head was less than 2 mm in relation to the dorsal margin of the acetabulum. However, we also
had a patient with ideal hips, who did not have any changes to the parameter, and refers to a
patient number with a serial number 3 (Table 3).
On the other hand, the lowest Norberg angle below 80˚ had patients with serial number 5 and
grade E, a patient with a serial number 7 and a grade C and a patient with a serial number 12 and
an estimate of E. As for changes in the coverage of the femur head the most expressed is in a
patient with serial number 5, in which the joints were completely in the luxation, the center of the
femur head compared to the dorsal edge of the acetabulum was over 10 mm. Changes in the
82
femur bone are the next parameter to the frequency, which was present in 9 dogs. Most often, this
was the initial stage in which the head of the femur remained largely unchanged, but the changes
involved the neck of the femur. The neck loses its shape, becomes cylindrical with a milder
degree of exostoses, and in a patient with grade E, serial number 5, the head was completely
deformed with massive exostoses. Due to the inadequate position between the head of the femur
and the acetabulum, its easy to have deformation on the craniolateral edge of the acetabulum,
covered with exostoses, also present in 9 dogs. The occurrence of the subhondral osssification of
the cranial edge of the acetabulum was less commonly observed in 4 patients. Morgan's lines
were the least represented, in only 2 cases, as changes that usually occur in the older age. Patient
with serial number 5 had the worst finding of severe dysplasia.
Comparing estimates in regard to the sum of the left and right hip estimates, we note that the
changes in the majority of cases were present in proportion to both the left and right hip.
Based on this study, hip dysplasia in dogs is often disease that is transmitted genetically,
even though it is not noticeable phenotypically and leaves significant repercussions on the health
of dogs. Therefore, the breeding of dysplastic dogs is not recommended. The influence of this
data on the eradication of hip dysplasia in dogs lies mainly in the breeders hands and their ability
to understand and accept the results, as well as to adhere to the veterinarian's recommendations.
CONCLUSION
Hip dysplasia has a significat incidence, five (which is 35%) of 14 examined dogs, were
dysplastic. The most presented parameter (13 dogs) was a insufficient coverage of femur head
with acetabulum edge, while the least-represented parameter (2 dogs) was the presence of
Morgan lines.
REFERENCES
BARR, A.R.S., DENNY, H.R., GIBBS, C, (1987): Clinical hip dysplasia in growing dogs: the
long-term results of conservative management. Journal of Small Animal Practice 28, 243–252.
FRY, T.R., CLARK, D.M., (1992): Canine hip dysplasia: clinical signs and physical diagnosis.
Veterinary Clinics of North America: Small Animal Practice 22, 551–558.
GRUNDSTRÖM S. (2014): Infuence of nutrition at young age on canine hip dysplasia in german
shepherd dogs. Univeristy od Helsinki, Small animal surgery, Licentiate of Veterinary Medicine
Degree, p 2-11.
KEALY JK, MCALLISTER H. (2005): Bones and joints. In: Winkel AJ, editor. Diagnostic
Radiology Ultrasonography of the Dog and Cat. fourth edition ed. Elsevier Saunders; p. 301-41.
MIKKELSEN J.M. (2007): Dynamic ultrasonography of the hip joint in early diagnosis of canine
hip dysplasia. University of Copenhagen, Copenhagen, Denmark
RICHARDSON DC, NAP RC, ZENTEK J, TOLL PW, HAYEWINKEL HAW, ZICKER SC,
TOWELL TL. (2010): Musculoskeletal disorders. In: Hand MS, Thatcher CD, Remillard RL,
83
Roudebush P, Novotny BJ, eds. Small animal clinical nutrition. 5th ed. Topeka, Kan: Mark
Morris Institute, 666-713.
RISER WH. (1975):The dog as a model for the study of hip dysplasia. Growth, form, and
development of the normal and dysplastic hip joint. Vet Pathol; 12(4):234-334.
VERHOEVEN G, FORTRIE R, VAN RYSSEN B, COOPMAN F. (2012): Worldwide screening
for canine hip dysplasia: Where are we now? Vet Surg, 41: 10-19.
84
SELECTION CRITERIA OF BREEDING BULLS FOR MILK PRODUCTION TRAITS
Igor Obranović
Mentor: Prof. dr Snežana Trivunović

University of Novi Sad, Faculty of Agriculture, Department of Animal Science, Serbia
Summary: The aim of this study is to see what is the most commonly used criteria for the
selection of bulls in dairy cattle breeding. For this study were used results of the progeny tested
Holstein-Friesian bulls. A survey has shown that the most common decision factor for the bull
selection is the price of the semen. For most traits, a high and positive correlation is calculated
between phenotypic values of milk production traits of the first-calf heifers and breeding values
of the progeny tested bulls.
Key words: breeding values, bulls, progeny tested
INTRODUCTION
Dairy cattle breeding is an important segment in modern livestock production where highly-
productive cows are used to produce big amounts of good quality and healthy milk. As a result of
long-term selection and planned animal breeding, high-productive breeds were created with great
genetic potential and their traits are mostly passed on to the next generation. If our goal is to pass
on the good and desirable traits on to the offspring, a strict and precise selection must be applied
where we mate the genetically best animals between each other, and in these situations, the best
tool for this kind of process is the estimation of breeding values of animals. The estimation of
breeding values of animals represents the estimation of the genetic potential of the animal, it is
expressed in relation to the average population and its result depends on the type and amount of
information, as well as from the accuracy of the measurement. Since in cattle breeding, using
artificial insemination, bulls give more offspring than cows, it is necessary to pay more attention
on the breeding values of bulls.
For the estimation of breeding values, most commonly used method is linear mixed models,
like the BLUP animal model. Using linear mixed models, we can estimate the animals genetic
potential with great precision due to the all available information about the animal and its
relatives. Some of the most important biological and economic traits of dairy cows are: a)
producing large amounts of good quality milk, b) reproductive efficiency, c) good conformation,
d) longevity, e) good health status (Berry, 2015). Also, along with these traits, animal welfare is
becoming more and more significant factor in modern livestock breeding. (Oltenacu and Broom,
2010).
On the territory of the Autonomous Province of Vojvodina, implementation of selection
measures are defined by breeding programs and in its realization, alongside the farmers,
85
participate breeding organizations which include: Main breeding organization, regional breeding
organizations and primary breeding organizations. In the controlled population on the territory of
Autonomous province of Vojvodina, the most dominant breed of cattle is the Holstein-Friesian
breed (Trivunović et al. 2018). Based on the information given by the Main breeding
organization, in 2005 this breed accounted for 98% of the controlled population but in the past
decade there had been some changes in the breed structure and increasaed interest among the
farmers to breed breeds of cattle with combined traits (Simmental - SIM and Brown Swiss) and
beef cattle (Hereford, Aberdeen- Angus, Charolais, Limousin), so in 2017. breed structure is 76%
HF, 21% SIM and 3,16 % others breeds (Trivunović et al. 2018). Milk production is one of the
most important traits of breeding goals. Based on the data from the professional report, the
average milk yield of the Holstein-Friesian breed in 2017. in full lactation was 7.722 kg of milk,
294 kg of milk fat and 252 kg of milk protein, and compared to the results in 2016 production is
increased by 125 kg of milk, 5 kg of milk fat and 6 kg of milk protein. Milk yield of the Holstein-
Friesian breed in the standard lactation length (305 days) in 2017. was: 6.984 kg of milk, 264 kg
of milk fat and 224 kg of milk protein. Compared to the production results in 2016, based on the
standard lactation length, the milk production in 2017 was increased by 98 kg of milk, 4 kg of
milk fat and 3 kg of milk protein. The average of milk fat percentage in 2017. was 3,78%, which
is an increase for 0,01% compared to the production in 2016., while the average percentage of
milk protein in 2017. was 3,21% which is the same amount of milk protein like in 2016. The aim
of this study is to see what is the most commonly used criteria for bull selection in dairy cattle
production among the farmers. Alongside that, it will be established is there a connection
between the ranks of the phenotypic values for milk traits of first-calf heifers and breeding values
of the progeny tested bulls and what is the correlation between them. The assumption is that these
values are in a high positive correlation, that is, the genetically advanced bulls will have a more
productive female offspring.
As a material for this study, we used the results of the progeny test of bulls on milk traits,
based on the information gained by the milk production of the first-calf heifers in their first
lactation. The results of the progeny test are announced one per year in the Professional report
and the results of the performed control tasks for the implementation of the breeding programs in
the Autonomous Province of Vojvodina. The base consisted of 168 black Holstein-Friesian bulls
and of 67 red hosltein-friesian bulls where, next to the identify marks of the bulls (name and HB
number), as the results of the progeny test, the following values are shown: number of female
offspring, phenotypic and breeding values for milk yield, amount of milk fat, milk fat percentage,
amount and percentage of milk protein.
Based on the available data, descriptive statistics have been made, where the following
parameters were calculated: average value, standard deviation, minimum and maximum value in
Microsoft Office Excel. With Spearman’s rank correlation coefficient has been calculated what is
the connection between the ranks of the bulls. The bulls are ranked on the basis of two criteria:
86
average phenotypic values of their daughter's production results and the breeding values of the
bulls. According to the breeding program for the Holstein-Friesian breed, the estimation of
breeding values is calculated by using the BLUP animal model.
A survey research had also been conducted on the breeding organizations (primary and
regional) to see how do farmers make their decisions when it comes to selection of the bulls, how
often do they ask the breeding organizations for advices and are they familiar with the terms
breeding values of bulls and results of the progeny test.
During this research, the results are divided into several parts. The first part of the research
refers to the survey who was conducted on the breeding organizations in order to get the
information how do farmers make their decisions when it comes to selecting bulls for
insemination cows of heifers on their farms. Most of the respondents answered that the farmers
rarely contact them for advices about making a mating/insemination plan ( 84%). Tips and
advices are mostly used by bigger farms with a longer tradition in this segment in livestock
production. When the farmers don't seek for advices from the breeding organizations, they
exclusively rely on the advice of the veterinarian (42%), 30 % of the farmers rely on the advice of
the veterinarian or from a person employed in the distribution center. A certain number of
farmers this decision make on their own (21%), and a small number of farmers seek advice from
a person employed in the distribution center (7%). According to the answers given by the
respondents, the main deciding factor for choosing the bulls for insemination cows and heifers is
the price of the semen (43%), next most important factor for the farmrs are the production results
of the bulls related to milk traits (32%). Alongside these, other factors are: availability of the
semen by the veterinarian, fertility of the semen, the origin of the bull, that is, genetical
connection to the animals on their farm (relationship), and the main traits that the farmers want to
improve are: body matter, udder, longevity calving ease. The answer to the question is the
farmers familiar with the term breeding values, 55% answered yes, and 26% said that they are not
familiar with that term. The rest of the answer was that the concept of breeding values was
familiar to the farmers who are more seriously involved in cattle breeding. The highest number of
respondents answered that the information about these values can be found by the help of the
breeding organizations, in distribution centers and catalogs of centers for artificial insemination,
and some of them answered that they could find these informations on the internet and from the
veterinarian who performs the insemination.
The aim of the second part of the research was to study the results of the progeny test through
the average, minimum and maximum values of the phenotypic and breeding values of the tested
bulls. In the tables 1 and 2, we can see the results of the average phenotypic values of the black
and red Holstein-Friesian bulls.
87
Table 1.Descriptive statistics of average values of milk yield traits of the first-calf heifers of progeny tested black
Holstein-Friesian bulls.
Trait n 𝑋±SD Min Max
Milk, kg 168 7135,68±1046,68 3779,71 9735,78
Milk fat, kg 168 263,47±34,86 150,05 381,80
Milk fat, % 168 3,72±0,18 3,28 4,21
Protein, kg 168 227,90±33,30 125,48 304,76
Protein, % 168 3,20±0,06 3,06 3,42
Based on the criteria of average values of milk yield trait the bull RUFFIAN is the first
ranked bull whose daughters in average produced 9735,78 kg of milk in the first lactation. The
worst ranked bull for milk yield trait is the bull who was used for natural mating( V-2030) whose
daughters produced 3779,71 kg of milk in the first lactation. In relation to the average values, the
female offspring of the bull RUFIAN produced 2600,10 kg of milk more than the average
population, while the female offspring of the bull used for natural mating (V-2030) produced
3355,97 kg of milk less than the average population.
Table 2. Descriptive statistics of average values of milk yield traits of the first-calf heifers of progeny tested red
Holstein-Frieasian bulls.
Milk, kg 67 6114,08±859,61 3865,76 8058,19
Milk fat, kg 67 233,34±31,62 162,78 303,01
Milk fat, % 67 3,84±0,15 3,46 4,24
Protein, kg 67 196,40±28,02 129,24 262,25
Protein, % 67 3,22±0,05 3,13 3,38
Based on the criteria of average values of milk yield trait the bullREXFOR RED is the first
ranked whose daughters in average produced 8058,19 kg of milk in the first lactation. The worst
ranked bull for milk yield trait is the bull who was used for natural mating(V-2020)whose
daughters produced3865,76 kg of milk in the first lactation.In relation to the average values, the
female offspring of the bull REXFORD RED produced 1944,11 kg of milk more than the average
population, while the female offspring of the bull used for natural mating(V-2020)produced
2248,32 kg of milk less than the average population
Considering that the genetic potential of the Holstein-Friesian breed for production of large
amounts of high-quality milk, the average milk yield within the population on the territory of
Vojvodina is not so satisfying. The average milk and milk fat yield is lower than the average
values in Croatia where the average production of milk within the breed is 7889 kg of milk and
4,0% of milk fat (CAA, 2018), while in Slovenia, the average milk production is 8042 kg and
3,97% of milk fat. (Kmetijski Institut Slovenije, 2018). Trivunović et al. (2018) points out that
we must understand that the population if the Holstein-Friesian breed in the Autonomous
Province of Vojvodina is very heterogeneous in many parameters(herd management, selection
88
activities, size of the herd, and so on) and that in some former state farms, the average milk yield
is 8500 kg of milk.
Table 3. Descriptive statistics of breeding values progeny tested black Holstein-Friesian bulls on milk production
traits
Milk, kg 168 461,56±1695,20 -4210,54 4571,17
Milk fat, kg 168 12,90±55,84 -143,44 176,76
Milk fat, % 168 -0,05±0,29 -0,71 0,74
Protein, kg 168 14,73±53,96 -136,68 135,98
Protein, % 168 0,004±0,125 -0,41 0,28
Based on the criteria of breeding values we came to the same results. Within the population of
the black Holstein-Friesian, for milk yield trait, the bull RUFFIAN was, again, the best-ranked
bull with a result of 4571, and the lowest ranked was a bull who was used for natural mating (V-
2019) with a result of -4210,54.
Table 4. Descriptive statistics of breeding values progeny tested red Holstein-Friesian bulls on milk production traits
Milk, kg 67 16,44±1374,99 -3528,44 3562,78
Milk fat, kg 67 3,17±49,85 -108,21 125,60
Milk fat, % 67 0,04±0,27 -0,62 0,79
Protein, kg 67 1,73±44,83 -103,93 123,57
Protein, % 67 0,03±0,11 -0,20 0,26
Within the population of the red Holstein-Friesian, based on the estimated breeding values on
milk yield traits, the bull REXFOR RED was, again, ranked first with a result of 3562,78, and the
lowest ranked bull who was used for natural mating (V-2020) had a result of -3528,44.
In the third part of the study, a comparison was made between the ranks of bulls based on the
results of milk production traits who are shown as average phenotypic values with the breeding
values.
Table 5. Correlation ranks between progeny tested bulls

Breed HF RHF
Trait Spearman’s rank correlation coefficient
Mleko, kg 0,995 0,961
Mast, kg 0,995 0,967
Mast, % 0,955 0,921
Protein, kg 0,995 0,956
Protein, % 0,749 0,689
Based on the information from the table 5, we can see that for most traits, ranks of bulls based on
the phenotypic values and ranks based on the breeding values are in a tight and positive
89
correlation. This means that the farmers could use both criteria for bull selection so that they
could improve milk production traits like production large amounts of milk, milk fat and milk
protein. For the trait percentage of milk protein, it was calculated a medium-strong positive
correlation, which means that for that trait the ranks of the bulls vary in a certain degree. This
means that the genetically better-ranked bull is not ranked in the same way when it comes to
phenotypic values of the production results of his female offspring. This can be a consequence of
increased variability of this trait which at the phenotypic level expresses itself in two ways: by
the action of the environmental factor, but also by the influence, above all of the additive effects
of the gene. Next to this, it should be known that this trait is, in our population, the least used
criteria for selection and that the genetical variation is expected.
CONCLUSION
When we decide to choose a bull for insemination of the cows and heifers, we must gather all
available information about the bull so that we could know what offspring could we expect in the
future, and will that offspring satisfy out production goals. If the farmer's interest is to have good
quality cows on his farm who will produce big amounts of high-quality milk, he should,
primarily, rely on the estimated breeding values of the bulls because it is the most precise way to
make genetical improvement and improvement of the dairy cattle production both in Vojvodina
and the rest of Serbia.
The results of the survey had shown that a large percentage of farmers rarely contact the
breeding organizations for advice when making a mating plan, and when they don't seek help
from them, they mostly rely on the advice and decision of the veterinarian. When it comes to
factors that are important for bull selection, based on the survey, the deciding factor was the price
of the semen, which means if the farmer cannot pay more money for a better bull semen, he will
choose the cheaper one even if that semen isn't genetically good enough. After the price, other
significant deciding factors are the production parameters (mostly the milk yield), and a certain
impact when choosing the bull semen is the availability of the semen, and semen offered by a
veterinarian. Still, we must keep in mind that even if the price of the semen is higher than the
average, the farmer will profit more if he chooses the more expensive good-quality semen
because it will pay off through the production of the genetically better female offspring.
Given that, besides the price, milk production traits are very important for defining the
selection criteria, the gained results about the correlation of bull ranks on the examined
population had shown a high and positive connection between the phenotypic values of the
female offspring progeny tested bulls and breeding values of the bulls. This means that the
farmers can use both criteria when choosing a bull for insemination of their cows and heifers on
their farms but for the more precise selection and quicker genetical progress, it is recommended
that they should more often use the breeding values of the bulls.
90
REFERENCES
BERRY DONAGH P. (2015): Breeding the dairy cow of the future: what do we need? Animal
Production Science 55, 823-837.
GLAVNA ODGAJIVAČKA ORGANIZACIJA, POLJOPRIVREDNI FAKULTET,
DEPARTMAN ZA STOČARSTVO, NOVI SAD.(2018): Stručni izveštaj i rezultati obavljenih
poslova kontrole sprovođenja odgajivačkih programa u AP Vojvodini za 2017. godinu.
CROATIAN AGRICULTURAL AGENCY-CAA. (2018): Annual report 2017-cattle breeding.
Križevci.
KMETIJSKI INSTITUT SLOVENIJE. (2018): Results of Dairy and Beef Recording Slovenia
2017. Ljubljana.
OLTENACU, P.A., BROOM, D.M. (2010):The impact of genetic selection for increased milk
yield on the welfare of dairy cows. Animal Welfare, 19(S): 39-49.
TRIVUNOVIĆ, S., ŠTRBAC, LJ., JANKOVIĆ, D., IVANOVIĆ, D., RADOVIĆ, I., MIRKOV,
M., PIHLER, I., BJEDOV, S., ŠARAN, M.,(2018): Livestock production development in AP
Vojvodina, Acta Agrarira Debreceniensis, University of Debrecen, 501-514.
91
EFFECTS OF NONSTEROIDAL ANTI-INFLAMMATORY DROGS ON BONE
HEALING
Elena Maduna1
Mentor: Ozren Smolec2
1
Student of 6th year at Faculty of Veterinary Medicine, University of Zagreb
2
Assistant Professor at Orthopaedics and Ophthalmology Clinic, University of Zagreb Veterinary
medicine, University of Zagreb
Summary: This review provides an unbiased analysis of the current literature regarding
nonsteroidal anti-inflammatory drugs and their effect on bone healing. This review demonstrates
that NSAIDs inhibit or delay fracture healing to a greater or lesser degree, depending on the
specific drug, mode of administration, administration period and ability to inhibit COX-2. Most
animal studies and the few human studies that exist support this conclusion. Since it is
increasingly clear that the function of COX-2 is critical for bone regeneration, defining the risk-
benefit ratio for NSAID use is critical. Patient co-morbid conditions, such as, fracture severity,
advanced age, diabetes, and cardiovascular health, will need to be considered when assessing
the risk-benefit ratio. Future research is needed to define the role of COX-2 in bone regeneration
and whether NSAID therapy will further impair fracture healing or other regenerative processes
in the presence of these co-morbid conditions. Research using rodent and rabbit models provides
strong evidence that NSAID negatively affect bone healing. Whether this effect is clinically
important is controversial in both human and veterinary medicine. Further experimental and
clinical trials involving domestic species and veterinary NSAID are needed to guide
veterinarians as they decide which, when, at what dose, and for how long NSAID should be
prescribed after bone injury.
Key words: NSAID, bone healing, cyclooxygenase
INTRODUCTION
Mechanism of action of nonsteroidal anti-inflammatory drugs was discovered when Ro and

associates noticed their ability to inhibit bone healing process. They discovered significantly
decreased bone healing in rodent treated with indomethacin (RO et al., 1978). This discovery has
started a series of researches and simultaneously cyclooxygenase-2 (COX-2) and biological
healing mechanisms of bone were discovered. Prostaglandins and inflammatory proteins have
affected on development and differentiation of bone structure, and inflammatory proteins can
have affects on development and differentiation of osteoblasts and osteoclasts. Anti-inflammatory
prostaglandins and modulating inflammatory proteins can inhibit forming of osteoclasts.
Therefore, immune system could be a link between bone formation and resorption (
ANANDARAJAH et al. 2009).. Treating bone disease often includes use of anti-inflammatory
92
drugs (NSAID) because of their analgesics and anti-inflammatory characteristic. Non-selective
NSAID affects the metabolism of prostaglandin by inhibiting synthesis of COX 1 and COX 2.
Non-specific and COX-2 selective non-steroidal anti-inflammatory drugs (NSAID) inhibit
cyclooxygenase isoenzymes and effectively reduce pain and inflammation associated with acute
or chronic musculoskeletal pathology. However, it is considered that use of NSAID as analgesics
negatively contributes to bone healing.
MECHANISAM OF ACTION OF NSAID
Nonsteroidal anti-inflammatory drugs (NSAIDs) produce their therapeutic activities through

inhibition of cyclooxygenase (COX), the enzyme that makes prostaglandins (PGs). They share,
to a greater or lesser degree, the same side effects, including gastric and renal toxicity. Recent
research has shown that there are at least two COX isoenzymes. COX-1 is constitutive and
makes PGs that protect the stomach and kidney from damage. COX-2 is induced by
inflammatory stimuli, such as cytokines, and produces PGs that contribute to the pain and
swelling of inflammation. Thus, selective COX-2 inhibitors should be anti-inflammatory
without side effects on the kidney and stomach (BOTTING et al.1998.) With damage of cell
membrane endogen peptides lipocortin’s starts to release and they activate phospholipase A2.
This is stimulated with destruction of membrane by infection, trauma, temperature or platelet
aggregation. Phospholipase A2 i C starts to release arachidonic acid from membrane
phospholipids. Mechanism of action of NSAIDs includes inhibition of several inflammatory
mediators in a cascade of arachidonic acid. Arachidonic acid starts to form eicosanoids with
action of COX and lipoxygenase (LOX). Prostaglandins, prostacyclin’s and thromboxane are
produced by COX action, while LOX produces leukotriene and lipoxin. Prostaglandin synthesis
is catalysed by COX-1 and COX-2. COX-1 is enzymatically active in different tissues
including gastric tissue, intestines, kidneys and platelets. COX-1 activity is primarily
physiological, it includes protection of gastric mucosa, renal perfusion and vascular
homeostasis. In some cases, COX-1 can have inflammatory effect. COX-2 is induced with
mediators as serum growth factor, cytokine and mitogen. Activity of COX-2 is primarily
related with pathological processes. COX-2 also has a physiological effect related with
maintenance of renal perfusion, reproduction etc.
CYCLOOXYGENASE INHIBITORS
NSAIDs are usually used for acute or chronic musculoskeletal disease. Acute disease includes
localized oedema, inflammation, pain, distension and partial rupture of ligaments or tendon and
bone fracture or chronic diseases like osteoarthritis. NSAIDs therapy reduces localized oedema
and pain inhibiting cyclooxygenase. Traditional NSAIDs inhibit COX-1 and COX-2 and can
have side effects especially on gastrointestinal system, kidney function, blood pressure and
coagulation parameters. The use of NSAIDs can cause gastrointestinal bleeding and perforations
that can be lethal. These side effects are caused by COX-1 inhibition. COX-2 was discovered in
93
1989. It has been found that expression of COX-2 is induced with tissue injury and after damage
caused by inflammation (SIMMONS et al., 2004). Soon after that it was assumed that NSAID
therapy reduces pain and inflammation by inhibiting COX-2 rather than COX-1. Therefore, new
medicine was developed which selectively inhibit COX-2. The assumption was that COX-2
selective inhibitors decrease side effects because homeostatic functions of COX-1 remain
preserved. The research has shown that the use of NSAID and COX-2 selective NSAID can
prolong bone healing and decrease mechanical integrity of healing bone. (DIMMEN et al., 2009;
O'CONNOR et al., 2009).
INFLUENCE OF COX-2 SELECTIVE INHIBITORS ON BONE HEALING

With discovery of COX-2 selective NSAID doctors started to use them for acute and chronic pain
management. As a result, scientists began researching the influence of COX-2 selective NSAID
on bone healing. The effects of COX-2 inhibitors is still debatable. Simon et al. have shown that
healing of femur is significantly decreased in rodents treated with celeocoxib or rofecoxib. X-ray
and histological examination of femur healing in COX-1 and COX-2 rodents has shown
significant bone callus that is subjected to endochondral ossification. Bone healing in rodents that
were given rofecoxib (3 mg/kg QD) has also shown significant deterioration based on x-ray and
histological examination. On the other hand, treating rodents with celeocoxib (4 mg/kg , QD) was
less harmful for femur healing based on the analysis of torsional mechanical examination. Results
of other research are confirming these results proving that COX-2 inhibitors like celeocoxib,
rofecoxib or meloxicam are unfavourable on bone healing in rodents (KARACHALIOS et al.,
2007; MURNAGHAN et al., 2006). Gerstenfeld et al 2003. has shown that oral dose of parecoxib
(1,5 mg/kg) caused a delay in bone healing in male rodents. This research has also shown that
delaying application of celeocoxib seven days or more after fracture improves healing. Other
studies confirm these results which indicates that avoiding the application of NSAID in early
stages of fracture treatment can reduce negative effects (GERSTENFELD et al., 2003;
VIRCHENKO et al., 2004). Negative effects of celeoxib depends on dosage of medicine,
duration of treatment and starting point of therapy.
COX-2 AND ITS ROLE IN BONE HEALNIG

COX-2 has a positive role in bone healing. Although, mechanisms in which COX-2 benefits or
complicates bone recovery are unknown. Is probable that loss of COX-2 function changes
cellular signal pathways necessary for bone healing because of NSAIDs. One theory explains that
COX-2 activity is essential for converting mesenchymal cells in osteoclast. Therefore, inhibition
of COX-2 activity has negative effect on osteogenesis. Other theory explains that analgesic effect
of NSAIDs enables experimental animals to lean on the injured leg too early, which can lead to
repeated injury and prolonged healing. This theory was tested on rodents with fractured femur
using celecoxib (3 mg/kg SID or 6 mg/kg SID) I acetaminophen (60 mg/kg SID OR 300 mg/kg
SID) for pain management in duration of 10 days after fracture (COTTREL et al., 2009). Results
have shown that celecoxib inhibits bone healing while acetaminophen didn’t affect the healing
94
process. All medication provided significant pain management, although only celecoxib affected
bone healing. That indicates that analgesic effect isn’t adequate cause of negative effects of
celecoxib or NSAID therapy in bone healing.
DISCUSSION
Although NSAIDs have negative influence on bone healing, there are not enough researches to
stop the usage of NSAIDs during orthopaedics procedures or fractures in domestic animals. In
one hand fractures and osteotomy can heal with NSAID therapy and they play an important role
as anti-inflammatory and analgesic components. On the other hand, when it is important to
reduce time of healing because of delayed healing, inability to heal or patients that are expected
to have problems with healing we need to consider if it is advised to use NSAIDs (BURD et
al.,2003).. When deciding whether to use COX-2 selective or nonselective NSAIDs other criteria
such as side effects, price and availability should be considered. High dosage and long-term
application of NSAIDs have a negative effect on bone healing. Research also shows that NSAID
induced inhibition of bone healing can be reversed when NSAID application is stopped. Research
that shows inhibition of bone healing on humans and dogs includes long-term NSAID
application, therefore NSAID therapy should be stopped when there is no more need for its
analgesic effects (LEONELLI et al., 2006).. The complexity of the fracture, used force, soft
tissue injuries and fracture stability can affect the outcome. It is possible, for example, that
NSPUL interferes only in the endochondral oscillation, therefore, if the fracture is
multifragmented and very unstable, the NSPULs can have a detrimental effect on the
consolidation process, while in a more stable fracture NSPULs may be completely ineffective
(MBUGUA et al., 1989).
CONCLUSION
NSPUL, due to its ability to inhibit prostaglandin production, alleviates the local inflammatory
response, desensitizing peripheral pain receptors. Although, they are good painkillers, some
studies have shown that they can inhibit bone healing, others disagree. It is recommended to use
NSAIDs with caution, and to avoid them when it is important that the bone heals faster. In lack of
strong scientific evidence, it is impossible to determine whether NSAIDs should be used. In
authors opinion there are no strong evidence that could completely contraindicate use of NSAIDs
for bone fracture treatment. Medical workers should use them in low risk patients and use it no
longer than a week after the fracture. Additional well-controlled clinical trials are required.
95
REFERENCES
ANANDARAJAH, A. P., E. M. SCHWARZ (2009): Bone loss in the spondyloarthropathies: role
of osteoclast, RANKL, RANK and OPG in the spondyloarthropathies. Adv Exp Med Biol.
649:85–99.
BOTTING R.M., VANE J.R. (1998) : Anti-inflammatory drugs and their mechanism of action.
Inflammation research, vol 47, suppl 2, pp 78-87.
BURD, T. A., M. S. HUGHES, J. O. ANGLEN (2003): Heterotopic ossification prophylaxis with
indomethacin increases the risk of long-bone nonunion. J. Bone Joint Surg. Br. 2003, 85B, 700–
705.
COTTRELL, J.A., J. P. O'CONNOR (2009): Pharmacological inhibition of 5-lipoxygenase
accelerates and enhances fracture-healing. J. Bone Joint Surg. Am. 91, 2653–2665.
DIMMEN, S., L. ENGEBRETSEN, L. NORDSLETTEN, J. E. MADSEN (2009): Negative
effects of parecoxib and indomethacin on tendon healing: An experimental study in rats. Knee
Surg. Sports Traumatol. Arthrosc. 17, 835–839.
GERSTENFELD, L.C., M. THIEDE, K. SEIBERT, C. MIELKE, D. PHIPPARD, B. SVAGR, D.
CULLINANE, T. A. EINHORN (2003): Differential inhibition of fracture healing by non-
selective and cyclooxygenase-2 selective non-steroidal anti-inflammatory drugs. J. Orthop. Res.
21, 670–675.
KARACHALIOS, T., L. BOURSINOS, L. POULTSIDES, L. KHALDI, K.N. MALIZOS
(2007): The effects of the shortterm administration of low therapeutic doses of anti-COX-2
agents on the healing of fractures. An experimental study in rabbits. J. Bone Joint Surg. Br. 89,
1253–1260.
LEONELLI, S. M., B. A. GOLDBERG, J. SAFANDA, M. R. BAGWE, S. SETHURATNAM, S.
J. KING (2006): Effects of a cyclooxygenase-2 inhibitor (rofecoxib) on bone healing. American
Journal of Orthopedics, vol.35, no.2, pp.79–84.
MBUGUA, S. W., L. A. SKOGLUND, AND P. LØKKEN (1989): Effects of phenylbutazone
and indomethacin on the post-operative course following experimental orthopaedic surgery in
dogs. Acta veterinaria Scandinavica, vol. 30, no. 1, pp. 27–35.
MURNAGHAN, M., G. LI, D. R. MARSH (2006): Nonsteroidal anti-inflammatory drug-induced
fracture nonunion: An inhibition of angiogenesis? J. Bone Joint Surg. Am. 88 (Suppl. 3), 140–
147.
O'CONNOR, J.P., J. T. CAPO, V. TAN, J. A. COTTRELL, M. B. MANIGRASSO, N.
BONTEMPO, J. R. PARSONS (2009): A comparison of the effects of ibuprofen and rofecoxib
on rabbit fibula osteotomy healing. Acta Orthop. 80, 597–605.
RO, J., N. LANGELAND, J. SANDER (1978): Effect of indomethacin on collagen metabolism
of rat fracture callus in vitro. Acta Orthop. 49, 323–328.
SIMMONS, D.L., R. M. BOTTING, T. HLA (2004): Cyclooxygenase isozymes: the biology of
prostaglandin synthesis and inhibition. Pharmacol. Rev.56, 387–437.
VIRCHENKO, O., B. SKOGLUND, P. ASPENBERG (2004): Parecoxib impairs early tendon
repair but improves later remodeling. Am. J. Sports Med. 32, 1743–1747
96
THE EFFECT OF CALCIUM CHLORIDE APPLICATION ON STORAGE CAPACITY
AND QUALITY OF KIWIFRUITS DURING COLD STORAGE
LAMA ISMAIEL*
* Institute of Food Technology, Faculty of Agricultural and Food Sciences and Environmental
Management, University of Debrecen, H-4032 DEBRECEN, Böszörményi út 138. Hungary,
Summary: This research was carried out during two seasons (2013-2014) on the Kiwifruits (type
Hayward), where the fruits were treated in the post-harvest period by calcium chloride Cacl 2
with the following concentrations (2, 3 and 4%) at 20 °C for 2 minutes while the fruits of the
control samples were not treated. And then all fruits were stored at 1 °C and a relative humidity
of 90-95%, in order to study the effect of the treatment on the quality of the fruits and their
storage capacity. The firmness of the kiwi fruits, their total acidity and the content of vitamin C
were decreased during the storage period, while the weight and absolute loss were increased.
Treatment resulted in a significant increase in the storage period of "Heyward" kiwi compared to
the control samples. The results showed that the firmness of the control kiwi fruit was decreased
after 4 months of storage from (7.37) kg/cm2 to (0.55) kg/cm2 in average, while the firmness of
the treatments (T1, T2, T3) (2, 3, 4%) Cacl2 were decreased from (7.36-7.35-7.39) kg/cm2 to
(1.52-2-2.09) kg/cm2 respectively. This treatment significantly decreased the loss of vitamin C.
The vitamin C content of the control samples after 4 months of storage was decreased from 121.7
mg / 100g to 37.06 with / 100g on average, while the content of the treatment samples (T1, T2,
T3) was decreased from (121.3-120.88-120.05) mg/100 g to (48.69-51.27-54.18) mg/100g in
average, respectively. The loss of weight was 10.07% for the control sample, and (5.24, 6.96,
9.22) % for (T3, T2, T1) respectively after 4 months of storage. The percentage of absolute loss
during the study period was 100% for the control sample, (24, 34, 76) % for the treatment
samples (T1, T2, T3) respectively after 5 months of storage. There was not a significant effect of
calcium chloride treatments on the total acidity and the total dissolved solids during refrigerated
storage on 1 °C. It was found that the calcium chloride treatment with 3% concentration was
more appropriate than 2% concentration for the effectiveness in maintaining the firmness of the
fruits and in reducing the percentage of weight loss, total losses and vitamin C. The results in the
two seasons were identical.
Keywords: Kiwi - Calcium Chloride - Storage - firmness.
INTRODUCTION
Food and agriculture organization (FAO) estimates that around 20-30% of global
agricultural production is destroyed between harvest and consumption period depending on the
type of the product and the technology in which it is used in the production, transport and storage
operations. Reducing the loss of agricultural crops is no less important than increasing the
97
production quantity and it is the main task of the workers in the section of vegetables and fruits
storage.
Kiwi cultivation has developed rapidly in the global level since the beginning of the eighties of
the last century, after identifying the nutritional value of its fruits, especially that it is considered
as a fruit rich in vitamin C, as well as the economic importance of some of its varieties such as
the "Hayward" type, which is characterized by high productivity in the unit area and gradual
maturity, thus they can be stored for long periods up to six months, and they can tolerate loads
and transport (Antunes et al., 2005). Despite the characteristic of gradience in the maturity of the
kiwifruits, it does not eliminate the need for refrigerated storage to preserve the quality and
nutritional value of them.
It is known that calcium ions Ca+2 delay maturity of fruit, so calcium treatment has been
successfully used in many fresh fruits in stages before and after harvesting fruits, in order to
reduce tissue firmness loss and slow the maturation (Souty et al., 1995). Preserving the quality of
the fruits through the contribution of Ca+2 ions by linking the pectin substances inside the cellular
walls, which increases their cohesion and coherence, thus Ca +2 contribute to reducing the
deterioration rate and maintaining the quality longer (Franco et al., 2008).
1. Plant material: Kiwi fruits were harvested randomly and carefully during the seasons of 2013
and 2014 from Actinidia Deliciosa trees (var. Hayward), after the completion of physiological
maturity with a firmness ranging between (7.33 - 7.42) kg/cm 2.
2. Treatments have been used:
First treatment (T1): fruits covered with Cacl2 (2%) at 20 °C for 2 minutes.
Second treatment (T2): fruits covered with cacl2 (3%) at 20 °C for 2 minutes.
Third treatment (T3): fruits covered with Cacl2 (4%) at 20 °C for 2 minutes.
3. Storage of fruits: All fruits were stored at 1 °C and a relative humidity of 90-95%.
4. Tests have been conducted: (Abdullah and Ali, 2010)
4.1: Measuring the firmness of the fruit: using Penetrometer measuring method.
4.2: Determination of content of ascorbic acid (vitamin C): By calibration the fruit's squeezer
with 2,6-Dichlorophenolindophenol dye (Tillmans dye) by reduction of the blue color of the dye
to a colorless compound.
4.5: Determination of percentage of the natural loss of weight: By Calculation of weight loss as a
percentage of initial weight before storage.
5. Statistical analysis: The ANOVA method was used to determine a less significant difference
(LSD) at 5% to compare the mean of the treatments and to identify the differences between them
using SPSS (Jacob, 2005).
98
1. the firmness of the fruit:

The fruit firmness was measured periodically every 15 days during the storage period, which
lasted for 5 months. The results were as shown in the chart (1).
firmness of the fruit kg/cm2
8
6
4
2
0
Control T1 T2 T3
Chart (1) the mean of the firmness of kiwifruits during 2 seasons (2013-2014)
It is noticed that the firmness of the kiwifruits decreased significantly during the storage period,
due to the destruction of the non-soluble pectic and cellulose substances which are responsible
for the firmness of the cells (Younes, 2004).
The largest decrease in the firmness of the fruit was in the control sample. This indicates that
calcium chloride treatment significantly reduces the speed of loss of firmness of kiwifruits during
the initial storage and controlled storage as (Antunes and Sfakiotakis, 1997) found. From the
chart (1) it is estimated that the concentrations (3% & 4%) are very suitable for maintaining the
firmness of the kiwifruits during the refrigerated storage, and this is compatible with (Hopkirk et
al., 1990) who found that a concentration of 3% will provide the maximum retention of firmness
without causing external shell damage. And it is also corresponding to the results of (Franco et
al., 2008) and (Antunes et al., 2005).
The three treatments were significantly higher than the control samples during the storage period.
This corresponds to (Franco et al., 2008), but is not consistent with the results of (Antunes et al.,
2005) who showed no effect of calcium chloride treatment on the firmness of the kiwifruits in the
last two months of storage.
2. Vitamin C:
The vitamin C content of the kiwifruit was estimated during the storage period periodically every
15 days and the chart (2) shows the obtained results:
200 Vitamin C (mg/100g)
100
control T1 T2 T3
Chart (2) The mean of the content of Vitamin C (mg/100g) during 2 seasons (2013-2014)
99
The chart shows a gradual decrease in the content of vitamin C in the kiwifruits. The largest
decline was in the first three months of storage, and this corresponds to the results of (Kazemi et
al., 2011) and (Zolfaghari et al., 2010).
The results of the statistical analysis showed that there were significant differences between the
treatments, where treatment T3 (4%) exceeds the other treatments followed by treatment T2 (3%).
This shows the importance of the treatment of calcium chloride in reducing the loss of ascorbic
acid during the storage period. These results correspond to the results of (Kazemi et al., 2011)
and (Franco et al., 2008).
3. Weight loss:
The amount of natural losses by weight of stored kiwifruits was calculated monthly, and the
results were arranged in the chart (3)
20 Weight loss %
0
after 1 month after 2 months after 3 months
control T1 T2 T3
after 4 months after 5 months
Chart (3) The mean of the weight loss % during 2 seasons (2013-2014)
The chart shows clearly an increase in the percentage of natural loss of kiwifruit's weight during
storage period of all treatments and this corresponds to what (Younes, 2004) and (Antunes et al.,
2005) found, this loss is either caused by breathing or by transpiration (Khan and Ahmad, 2005).
The statistical analysis showed significant differences between the treatments and the control
sample.
CONCLUSION
By reviewing the results, we can conclude the following:

1.Treatment of Cacl2 resulted a significant increase in the storage period of "Heyward" kiwi.
2.Concentration (3%) Cacl2 is the most appropriate treatment.
REFERENCES
ABDULLAH, HASAN & ALI, ALI. Packing and storage of fruits and vegetables - Practical part
-, Directorate of books and university publications, university of Tishreen, Latakia, Syria, 2010,
153.
ANTUNES, M. D. C., SFAKIOTAKIS, E. M., 1997. The effect of controlled atmosphere and
ultra low oxygen on storage ability and quality of ‘Hayward’ kiwifruit. Acta Hort. 444, 613-618.
100
ANTUNES, M.D.C., N. NEVES, F. CURADO, S. RODRIGUES, T. PANAGOPOULOS. The
effect of pre and postharvest calcium applications on ‘Hayward’ kiwifruit storage ability. Act.
Hort., 682, 2005,909-916.
ANTUNES, M.D.C., N. NEVES, F. CURADO, S. RODRIGUES, S., FRANCO, J. AND
PANAGOPOULOS, T. (2007). The effect of calicium application on kiwifruit quality
preservation during storage. Acta Hortic. 753, 727-732.
HOPKIRK, G & HARKER, F & E. HARMAN, J. (1990). Calcium and the firmness of kiwifruit.
New Zealand Journal of Crop and Horticultural Science - N Z J CROP HORTICULT SCI. 18.
215-219. 10.1080/01140671.1990.10428097.
J. FRANCO, D. ANTUNES, F. MELO, R. GUILHERME, N. NEVES, F. CURADO, S.
RODRIGUES, “The influence of pre and post-harvest calcium applications on storage capability
and quality of ‘Hayward’ kiwifruit”. In: New aspects of energy, environment, ecosystems and
sustainable development. T. Panagopoulos, T. Vaz, M.D. Antunes (eds.) WSEAS Press, Athens,
2008, pp 512-516.
JACOB, GHASSAN. The Basics of Experimental Design, Directorate of books and university
publications, university of Tishreen, Latakia, Syria, 2005, 328.
KHAN, M. A., and I. AHMAD (2005). Morphological studies on physical changes in apple fruit
after storage at room temperature. J. Agri. and Social Sciences. 1(2): 102–104.
M. KAZEMI, M. ARAN and S. ZAMANI, 2011. Effect of Calcium Chloride and
Salicylic Acid Treatments on Quality Characteristics of Kiwifruit (Actinidia
deliciosa cv. Hayward) During Storage. American Journal of Plant Physiology, 6: 183-
189.
NAVA, GILBERTO & ROQUE DECHEN, ANTONIO & RIBEIRO NACHTIGALL, GILMAR.
(2008). Nitrogen and Potassium Fertilization Affect Apple Fruit Quality in Southern Brazil.
Communications in Soil Science and Plant Analysis. 39. 96-107. 10.1080/00103620701759038.
SOUTY, M.; REICH, M.; BREUILS, L.; CHAMBROY, Y.; JACQUEMIN, G.; AUDERGON, J.
M. 1995. Effects of Postharvest Calcium Treatments on Shelf-life and Quality of Apricot Fruit.
Acta Horticulturae. 384: 619-623.
TAGLIENTI, A., MASSANTINI, R., BOTONDI, R., MENCARELLI, F., & VALENTINI,
M. (2009). Postharvest structural changes of Hayward kiwifruit by means of magnetic resonance
imaging spectroscopy. Food Chemistry, 114(4), 1583-1589.
YOUNES, AHMAD (2004). Effect of spraying some fungicides and calcium chloride in the
storage capacity of mandarin fruits. Bassel Al Assad Journal of Engineering Sciences, Damascus,
Syria, 2004, 19.205-225.
ZOLFAGHARI, MAHBOUBE & SAHARI, MOHAMMAD ALI & BARZEGAR, MOHSEN &
SAMADLOUIE, HAMID-REZA, (2010). Physicochemical and Enzymatic Properties of Five
Kiwifruit Cultivars during Cold Storage. Food and Bioprocess Technology. 3. 239-246.
10.1007/s11947-008-0114-6.
101
QUALITY OF RED WINES PRODUCED WITH DIFFERENT OENOLOGICAL
PRODUCTS
Fumica Orbanić, Maja Sošić, Lovre Stulić, Nikola Šember
Mentor: Assist. Prof. Ana-Marija Jagatić Korenika, PhD

The University of Zagreb, Faculty of Agriculture, Department of Viticulture and Enology,
Croatia
Summary: Due to the constant need for improving the red wines quality, both sensory
and bioactive properties, which means enriching wine with compounds of positive effect on
human health, a technological experiment was set up including the grapes of cv. 'Syrah', 'Petit
Verdot' and 'Cabernet Sauvignon' (Vitis vinifera L.) grown on experimental stationJazbina,
Faculty of Agriculture. Based on numerous literary references and results obtained in similar
researches, different oenological protocols for wine production were tested. Total and individual
phenolic compounds which contribute to the color, taste quality and to the health value, i.e. the
bioactive properties of the wine, as well as the sensory properties of the wine, were analyzed. The
difference in the concentration of total phenols between all samples was statistically significant
and the highest concentration was recorded in the wine T4 ('Petit Verdot'). Although the
differences in the polyphenolic composition of the wine can be explained by the different
biological potential of the varieties, in the case of wines T1 and T2 (´Syrah´), a significant
difference due to the application of different oenological protocols was noticed.
Keywords: red wine, polyphenols, oenological products, sensory properties
INTRODUCTION
During the harvest 2017, in experimental station Jazbina, Faculty of Agriculture, students
carried out a vinification of grapes of three grape varieties, 'Syrah', 'Petit Verdot' and 'Cabernet
Sauvignon' (VitisviniferaL.) to produce four single variety wines and one blend using five
different oenological protocols. During the production of wine, samples were taken for basic
physicochemical analysis, individual organic acids, free amino nitrogen (FAN), total and
individual phenolic compounds. At the end of this experiment, sensory analysis was performed
using 100 points evaluation method (OIV).
Polyphenol compounds are a large and complex group of secondary metabolites which have
bioactive properties and are of crucial importance for the wine quality. Their great influence on
the sensory properties of wine (Hernanz et al., 2007) and the beneficial effect on human health
(Babu and Liu, 2009) makes them an interesting subject of research today as well as at the very
beginning, late 19th century (Kennedy et al., 2006). The polyphenolic compounds in wine
originate from different parts of the grape: skins, seeds and stems (Soleas et al., 1997), and their
concentration depends on variety, environmental conditions, ampelo technical procedures in
102
vineyards and technology of wine production (Mazza et al., 1999; Rodriguez-Delgado et al.,
2002). Generally, red wines have a greater concentration of polyphenolic compounds compared
to white wines. The technology of production of red wines demands an obligatory process which
is known as maceration and it allows better extraction of polyphenolic compounds. Many
polyphenolic compounds are responsible for a positive health effect, mainly flavonoids
(anthocyanins and flavan-3-old) and non-flavonoids (resveratrol, hydroxymethyl, and galactic
acids), this effect is based on antioxidant capacity that can suspend the negative effect of free
radicals. Many factors affect the polyphenol composition in wine, but the biggest differences
occur during vinification, and are consequence of a certain fermentation temperature, maceration
time, the use of clarification agents, the aging in wooden barrels and the storage methods
(Guilford and Pezzuto, 2011; Ribéreau-Gayon et al., 2006; El Darra et al., 2016; Muller and
Fugelsang, 1997). In a fact, enzymatic reactions and yeast metabolism modify or create new
polyphenolic compounds. Due to these reactions, the process of vinification, especially
fermentation enriches wine with compounds with special chemical and biological properties,
resulting in a greater number of bioactive compounds in wine than in grapes (Morata et al., 2003;
Romboli et al., 2015). Some authors argue that the benefits of polyphenol in wine are enhanced
by the formation of ethanol during fermentation that increases their bioavailability (Guilford and
Pezzuto, 2011). Therefore, wine, especially red, is considered a complex mixture of polyphenols
and alcohol of beneficial properties in which their synergistic effect has a positive effect on
human health (Ruf, 2003; Fernandes et al., 2017; Lindberg, 2008). This paper deals with the
determination of polyphenols, respectively with a bioactive potential of red wines depending on
grape variety, different oenological procedures (different enzymes, yeast strains, yeast nutrition
and antioxidants) and with sensory evaluation of wine.
Grape harvest of 'Syrah', 'Petit Verdot' and 'Cabernet Sauvignon' varieties was done
manually, at the time of phenolic maturity, in amounts of 100 kg. After crushing and destemming
of the grapes with a mechanical crushing machine, must samples were taken for analysis.
Crushed grapes were arranged in 5 treatments with 2 repetitions: T1- 'Syrah', T2- 'Syrah', T3-
'Cabernet Sauvignon', T4- 'Petit Verdot', T5- 50% 'Syrah' + 25% Cabernet Sauvignon + 25 %
'Petit Verdot'. Red grape juice was macerated in 100 L stainless steel vats with a sieve for soaking
pomace cap for 8 days lasting at a temperature of 22 °C. Oenological antioxidant additive and
enzymes were added in each vat and in two categories oenological chips were added (table 1).
Also, a few different enzymes for maceration and for extraction of bioactive and aromatic
compounds were added (table 1). After 24 hours, rehydration and inoculation of selected yeasts
and yeast's activator were performed. Medium toasted oak chips was added only in sample T2
(Table 1). Pressing of the must was carried out with a hydraulic press and after that, during
fermentation, complex yeast's nutrition was added in order to make alcoholic fermentation as
efficient as possible, especially when the nitrogen composition is below the optimum level. After
the end of fermentation first racking of wine was done with the addition of sulphites in a dose of
103
100 mL H2SO3/100 L. After that samples of wine were taken for basic physical-chemical and
instrumental analysis.
Table 1. Oenological products in the production of red wines in 2017.
Prefermentativeaditives Fermentation Yeast nutrients
T1 H2SO3 100 mL/100 L+ Vinozyme Excellence XR® 20 Vitaferment PH 50
Vintage FCE® 2 g/hL(Novozymes) g/hL + OptiFerm / g/hL(Lamothe-Abiet)
Vitactif 30
g/hL(Lamothe-Abiet)
T2 H2SO3 100 mL/100 L+ Vinozyme Excellence® DS 20 Vitaferment PH 50
Vintage FCE® 2 g/hL(Novozymes) g/hL+ Vitactif 30 g/hL(Lamothe-Abiet)
g/hL(Lamothe-Abiet)+
Pro Tanin R® 20 g/hL +
A2M 200 g/hL(AEB)
T3 H2SO3 100 mL/100 L+ Uvaferm BDX® 30 g/hL Fermaid E 40
LallyzimeOe® 1,5 g/100L+ + GoFerm® 30 g/hL(Lallemand)
oak chips 200 g/hL g/hL(Lallemand)
T4 Sumpovin Uvaferm BDX® 30 g/hL Fermaid E 40 g/hL
100 mL/100 L+ + GoFerm® 30 g/hL (Lallemand)
LallzymeOe® 1,5 g/100 kg (Lallemand)
T5 Aromax® 20 g/hL+ Fermol® Rouge20 g/hL Fermaid E 40 g/hL
Endozym Rouge® 5 g/hL + Fermoplus® Energy (Lallemand)
+A2M 200 g/hL Glu 5 g/hL(AEB)
Concentration of sugar in must was determined by a refractometer (°Oe), the pH value of the
must and wine was determined by pH meter Beckman expandomatic type SS 2. Organic acids
(tartaric, malic, lactic, citric, and succinic) were determined by high-performance liquid
chromatography (HPLC), Agilent 1050 (Palo Alto, USA). Identification and quantification was
performed at wavelength λ = 210 nm at Aminex HPX-87H (BioRad, Hercules, CA, USA). The
free α-amino nitrogen (FAN) was determined by spectrophotometer Specord 400 (Analytik Jen,
Jen, Germany) according to Dukes and Butzke (1998). The total phenolic compounds in must and
wine were determined spectrophotometrically by Singelton and Rossi (1965). The results are
expressed in the equivalents of gallic acid, mg GAE/L. Determination of the proportion of
individual polyphenolic compounds in must and wine: the volume of 2 mL of wine was
previously filtered through a 0.22 μm PTFE membrane filter in the liquid chromatography system
(HPLC Agilent 1100). The analysis was performed on a phenyl-hexyl colony (Phenomenex,
USA) with a diode array detector. Identification of wine components was performed by
comparing their retention times on the chromatographic column and the associated UV-Vis
spectars and fluorescence on the characteristic wavelengths of excitation and emission with the
retention times, spectars and fluorescence of the standard compounds. Mass concentrations of the
identified compounds were determined by calibration curves made by external standard analysis.
The sensory analysis of the wine has been conducted with the method of 100 positive points
(OIV) based on the evaluation of the individual characteristics of the wine, depending on the
intensity and the quality assigned a certain number of points. The total amount of points obtained
per individual category is added up and may range from a minimum of 40 to a maximum of 100
points.
Research of the polyphenolic composition of wine was carried out in two repetitions, and
the mass concentrations are shown as the average value with the standard deviation. The results
were statistically analyzed by variance analysis (ANOVA) using SAS 9.4 (SAS Institute, Cary
104
NC) statistical program. The significance of differences was tested at the significance level p
<0.05. The main component analysis (PCA) was performed to determine the possible grouping of
the samples depending on the enological
ological procedures applied. PCA analysis was performed on the
correlation matrix with respect to individual polyphenolic compounds.

The average pH values in the musts were within a preferred range of 3.1 to 3.4 (Jackson,
2014) and expected in terms of variety and climate, and a good indicator of freshness and future
microbiological stability of wine. Initial concentrations of total phenols expressed as equivalents
of gallic acid in the vats (Table 2) are much lower than the total coconcentration
ncentration in wines because
most of the phenolic compounds are extracted in the maceration process that followed after the
initial samples of musts were taken. The highest concentration of polyphenols in the must was
recorded in the T5 sample, in the blen
blend of all varieties (Table 2).
Concentrations of the most important organic acids in the must and wine (Ribéreau-Gayon
(Ribéreau et al.,
2006) and the total acidity of the wine as an important factor of quality (data not shown) are
found in the wanted and expected ra ranges and ratios (Jackson, 2014).
The basic physicochemical parameters in the wine (data not shown) are in line with the baseline
conditions of the must and the expected changes due to maceration and fermentation.
Table 2. The concentration of sugar, free α-amino

amino nitrogen (FAN), total phenols and pH values in musts
Sugars FAN Total phenols
Sample pH
(°Oe) γ(AN)/mg/L γ(UF)/mg/L
T1 110 3.34 31.33 1141.05

110
T2 3.33 32.56 1113.42
97
T3 3.24 22.21 581.91
97
T4 3.03 27.77 511.72
99
T5 3.3 28.70 1710.88
The
he results shown are the arithmetic mean of two repetitions
Fig. 1 shows statistically significant differences in concentrations of total phenols that are almost
in all wines above 2000 mg/L, dominated by the T4 ('Petit Verdot') and then T5 (blend). All
concentrations
ncentrations are within the range of 800
800-4000
4000 mg/L, characteristically for red wines (Cheynier
et al., 1998), with surprisingly difference between samples T1 and T2, which is an obvious effect
of the applied enological protocols.
T OTA L P H E N O L S
3000 ±2.58
2.58c ±3.73a ±3.81b
±2.40e ±4.24d
g/L as gallic
2000
1000
0
T1 T2 T3 T4 T5
Red wines vintage 2017.
Mean values marked with different letters are significantly different with p <0.05, one
one-way
way ANOVA and Tukey test
Fig 1.. Total phenols in red wine 2017.in g/L (as gallic acid)
105
Table 6. Individual polyphenol compounds in red wines 2017.
Compounds (mg/L) T1 T2 T3 T4 T5
Anthocyanins
Delphinidin-3-glucoside 31.39±0.06d 31.72±0.06c 32.23±0.02b 35.45±0.01a 35.45±0.04a
Cyanidin-3- glucoside n.d. n.d. n.d. n.d. n.d.
a c e d
Petunidin-3- glucoside 23.43±0.02 19.7±0.21 14.7±0.01 18.32±0.02 20.83±0.03b
Peonidin-3- glucoside 6.5±0.01a 5.14±0.01b 2.04±0.01d 1.69±0.01e 3.6±0.04c
Malvidin-3- glucoside 290.77±0.05a 246.94±0.06c 240.74±0.04d 192.38±0.23e 270.98±1.88b
Flavonols
Myricetin-3-O-glucoside 12.03±0.02e 13.95±0.05d 15.39±0.02b 24.97±0.04a 14.62±0.06c
Rutin n.d. n.d. n.d. n.d. n.d.
b a e d
Hiperoside 4.26±0.04 4.42±0.02 2.49±0.01 3.75±0.05 3.89±0.01b
Quercetin-3-O-glucoside 18.63±0.09d 20.39±0.01b 17.78±0.08e 28.83±0.06a 20.04±0.04c
Isorhamnetin-3-O-glucoside n.d. n.d. n.d. n.d. n.d.
Myricetin n.d. n.d. n.d. n.d. n.d.
c b a
Quercetin 0.77±0.05 n.d. 1.21±0.01 1.92±0.02 0.69±0.01c
Kaempferol 0.3±0.02b n.d. 0.33±0.01a 0.3±0ab 0.22±0.01c
Isorhamnetin 0.31±0.01a n.d. n.d. n.d. n.d.
Hydroxycinnamic acids
Caftaric acid 38.07±0.12e 40.01±0.03d 44.52±0.02c 103.36±0.45a 61.51±0.74b
Caffeic acid 15.57±0.05d 16.57±0.01c 14.57±0.02e 43.11±0.03a 19.98±0.20b
Coutaric acid 0.94±0.04c 1.04±0b 0.76±0.01d 1.48±0.01a 0.95±0.05c
p-Coumaric acid n.d. n.d. n.d. n.d. n.d.
d b c d
Fertaric acid 1.57±0.01 2.53±0.01 2.19±0.01 1.48±0.01 2.88±0.06a
Ferulic acid n.d. n.d. n.d. n.d. n.d.
Hydroxybenzoic acids
Gallic acid 15.22±0.03a 11.21±0.03d 9.15±0.01e 13.88±0.01c 14.67±0.12b
Flavan-3-ols
Epicatechin-gallate n.d. n.d. n.d. n.d. n.d.
a d c e
Gallocatechin 5.58±0.04 3.91±0.01 4.22±0.06 1.98±0.01 4.57±0.01b
Procyanidin B1 2.04±0.01d 2.42±0.02b 3.26±0.03a 2.54±0.04b 2.2±0.02c
Catechin 20.09±0.08c 16.37±0.05d 21.27±0.09b 20.03±0.01c 22.22±0.09a
Procyanidin B2 2.39±0.02a 2±0.04b 2.25±0.03a 1.72±0.02c 2.34±0.03a
Epicatechin 13.01±0.32a 8.94±0.03d 7.89±0.04e 11.15±0.02b 10.13±0.03c
Stilbenes
Resveratrol glucoside 4.1±0.07b 3.76±0.03c 1.51±0.04e 4.74±0.04a 3.21±0.01d
Resveratrol n.d. n.d. n.d. n.d. n.d.
Mean values marked with different letters are significantly different with p <0.05, one-way ANOVA and Tukey test.
106
Concentrations of individual polyphenolic compounds divided into specific chemical groups have
shown in table 6. Table starts with a group of most important anthocyanins that are responsible
for red wine color, which through the polymerization processes with tannins contributes to color
stability. Anthocyanins are the most common flavonoids in grape berry, what is confirmed by
presented results, with a multitude of biological functions such as protection against insolation
and UV radiation, inactivation of free radicals and antioxidant capacity, defense against a large
number of pathogens, as well as attracting predators for the purpose of spreading seeds and also
used as natural pigments in food production (He et al., 2010). The most common among them, in
all samples, was malvidin-3-glucoside that is in accordance with the literature (Ribéreau-Gayon
et al., 2000) and the highest concentration was determined in the sample T1. In the flavonol
group, among the yellow pigments in the glycoside form, which are found in the berry skin with
the role of UV protection, quercetin-3-O-glucoside and myricetin-3-0-glucosidedominated, which
matches the other researches (Mattivi et al., 2006), with the highest concentrations being
recorded in sample T4.Flavan-3-ols in wine can be in the form of free monomers (catechins),
oligomers or polymers (proanthocyanidins or condensed tannins). The main monomers that
dominate in the skin and in the seeds are catechin and its isomer epicatechin,
respectively(Kennedy et al., 2006), which is confirmed by the results obtained, with the highest
concentrations in the samples T5 and T1. Both compounds have a bitter taste when they are in the
form of monomers. Generally, the bitterness decreases, and the astringency increases with the
polymerization degree, i.e. increasing the size of the molecule (Cheynier et al., 2006). During
berry ripening, the concentration of tannins with the smallest molecular weight decreases, so it
can explain why with a good ripeness of grapes the quality of tannins for wine production also
increases (Kennedy, 2008). Differences between the samples in this experiment can be attributed
to variety properties; however, T1 and T2 samples also demonstrated that production technology
has a statistically significant effect on the concentrations of certain-flavan-3-old.
Fig 2. Principal components analysis (PCA) based on the individual polyphenols analysis of red wines in 2017.
107
Two basic groups of non-flavonoids in wine are hydroxycinnamic and hydroxybenzoic acids.
They are located in cellular vacuoles of pulp and skins and can be easily extracted into the wine
by pressing, which is why there is no big difference in the concentration between red and white
wines (Kennedy et al., 2006). Caftaric acid, as the main substrate of white wine browning and
dominant hydroxycinnamic acid (Jackson, 2014),that was confirmed by this research, with
significantly the highest concentrations in sample T4, followed by the caffeic acid.
Concentrations of hydroxybenzoic acids are much less in wines, and the most significant
representative is gallic acid, present in free form, but also as a precursor of hydrolysis and a part
of condensed tannins (Garrido and Borges, 2013). The highest concentration of gallic acid was
determined in samples T1 and T5. Stilbenes are compounds synthesized in berry as a plant
reaction to stress (pathogen attack or UV radiation), and considering its antioxidant, anti
cancerogenic and antimutagenic potential, have a beneficial effect on human health (Moreno-
Arribas and Polo, 2009). Red wines contain higher concentrations of stilbene than white wines,
due to longer skin contact (maceration) during fermentation and generally higher content of
phenols in red varieties (Perrone et al., 2007). The most important representative is resveratrol,
phytoalexin that vine produces as a reaction to fungal infections (Botrytis cinerea or
Plasmoparaviticola), and comes as a cis- and trans-isomer (Moreno-Arribas and Polo, 2009).
The presence of stilbenes in wine has recently become an extremely interesting topic of research,
with an emphasis on the correlation between their profile and enological procedures and various
grape varieties (Garrido and Borges, 2013). In terms of health effect the most intensively studied
is trans-resveratrol, whose biological activity prevents cardiovascular disease and cancer (Ito et
al., 2003). Ribéreau-Gayon et al. (2000) cited average concentrations of 1-3 mg/L of stilbenes,
and the results in this study included higher concentrations only for resveratrol-glucoside, except
in T3, where the highest concentration was determined in sample T4.
Fig. 2 shows the distribution of red wines of 2017. in a two-dimensional coordinate system
defined by the main components (PC1 and PC2) according to the composition of the individual
polyphenols, the first major component explaining 46% of the total variability, and the other
main component is 30% of the total variability of wine samples. As it is evident, the wines were
separated due to the variety. Sample T1 is in the positive part of component 1 such as petunidin-
3-glucoside, malvidin-3-glucoside, isorhamnetin and peonidine-3-glucoside, while sample T3 is
in the negative part of component 1 with tartaric acid and procyanidin B2. Sample T4 is
distinguished from the first major component due to the high concentrations of myricetin,
quercetin-3-O-glucoside, quercetin as well as caftaric and coutaric acids. Sensory evaluation of
wine was conducted by the 100-point method (OIV) by the students of Graduate study of
Viticulture and Enology, Faculty of Agriculture, Zagreb. The best evaluated sensory properties
based on the 100-point method were in sample T3 ('Cabernet Sauvignon ').
CONCLUSION
Based on a one-year experiment on 'Syrah', 'Petit Verdot' and 'Cabernet Sauvignon' (Vitis
vinifera L.), conducted on experimental station 'Jazbina', after vinification with various
108
oenological products and the analysis of the grape juice and wine, the difference in the
concentration of total phenols between all samples was statistically significant, with the highest
value in 'Petit Verdot' wine (T4). The most common anthocyanin in all samples was malvidin-3-
glycoside with the highest concentration in the wine 'Syrah' (T1) while the most frequent flavonol
was quercetin-3-O-glucoside with the highest concentration in the' 'Petit Verdot' wine (T4). 'Petit
Verdot' wine showed the highest concentration of caftaric acid and total stilbenes as well, which
indicates this is the grape variety of very significant polyphenolic potential. The highest
concentration of catechin was determined in the sample T5 (blend) while the highest
concentration of epicatechin was in the 'Syrah' wine (T1). Differences in polyphenolic
composition of wines can be explained by different biological potential of the varieties, however,
in the case of samples T1 and T2 ('Syrah'), it can be noticed the significant impact of applied
technology, or enological protocol, which should certainly be the subject of further
investigations.
REFERENCES
BABU, P.V.A., LIU, D. (2009): Flavonoids and cardiovascular health. In: Watson RR (ed),
Complementary and alternative therapies and the aging population. Academic press, San Diego.
CHEYNIER, V., DUEÑAS-PATON, M., SALAS, E., MAURY, C., SOUQUET, J. M., SARNI-
MANCHADO, P., FULCRAND, H. (2006): Structure and properties of wine pigments and
tannins. Am. J. Enol. Vitic., 57: 298-305.
DUKES, B.C., BUTZKE, C.E. (1998): Rapid determination of primary amino acids in grape
juice using an o-phthaldialdehyde/N-acetyl-L-cysteine spectrophotometric assay. American
Journal of Enology and Viticulture, 49(2), 125-134.
EL DARRA, N., TURK, M.F.; DUCASSE, M.A., GRIMI, N.; MAROUN, R.G., LOUKA, N.,
VOROBIEV, E. (2016): Changes in polyphenol profiles and color composition of freshly
fermented model wine due to pulsed electric field, enzymes and thermovinification pretreatments.
Food Chem. 194, 944–950.
FERNANDES, I., PÉREZ-GREGORIO, R., SOARES, S., MATEUS, N., DE FREITAS, V.
(2017): Fermented FoodsinHealthand Disease Prevention; Frías, J., Martínez-Villaluenga, C.,
Peñas, E., Eds.; Elsevier Inc.: New York, NY, USA; pp. 593–621,
GARRIDO, J., BORGES, F. (2013): Wine and grape polyphenols – A chemical perspective.
Food Res. Int. 54: 1844-1858.
GUILFORD, J.M., PEZZUTO, J.M. (2011): Wine and health: A review. Am. J. Enol. Vitic. 62,
471–486.
HE, F., MU, L., YAN, G.L., LIANG, N.N., PAN, Q.H., WANG, Y., REEVES, M.J., DUAN,
C.Q. (2010): Biosynthesis of anthocyanins and their regulation in colored grapes. Molecules 15:
9057-9091.
HERNANZ, D., RECAMALES, A.F., GONZALES-MIRET, M.L., GOMEZ-MIGUEZ, M.J.,
VICARIO, I.M., HEREDIA, F.J. (2007): Phenolic composition of white wines with a
109
prefermentative maceration at experimental and industrial scale. Journal of Food Engineering 80:
327-335.
ITO, T., AKAO, Y., YI, H., OHGUCHI, K., MATSUMOTO, K., TANAKA, T., IINUMA, M.,
NOZAWA Y. (2003): Antitumor effect of resveratrol oligomers against human cancer cell lines
and the molecular mechanism of apoptosis induced by vaticanol C. Carcinogesis, 24, 1489–1497.
JACKSON, R.S. (2014): Wine science Principles and Applications. Fourth Edition, Academic
Press Elsevier: Burlington,MA, USA.
KENNEDY, J.A. (2008). Grape and wine phenolics: Observations and recent findings. Cien. Inv.
Agr. 35: 107-120.
KENNEDY, J.A., SAUCIER, C., GLORIES, Y. (2006): Grape and wine phenolics: History and
perspective. Am. J. Enol. Vitic. 3, 20–21.
LINDBERG, M.L., AMSTERDAM, E.A. (2008): Alcohol, wine, and cardiovascular health. Clin.
Cardiol. 31, 347–351.
MATTIVI, F., GUZZON, R., VRHOVSEK, U., STEFANINI, M., VELASCO, R. (2006):
Metabolite profiling of grape: Flavonols and anthocyanins. Journal of Agricultural and Food
Chemistry, 54(20), 7692–7702.
MAZZA, G., FUKUMOTO, L., DELAQUIS, P., GIRARD, B., EWERT B.
(1999):Anthocyanins, phenolics, and color of Cabernet Franc, Merlot, and Pinot noir wines from
British Columbia. Journal of Agricultural and Food Chemistry, 47, 4009–4017.
MORATA, A., GÓMEZ-CORDOVÉS, M.C., COLOMO, B., SUÁREZ, J.A. (2003): Pyruvic
acid and acetaldehyde production by different strains of Saccharomyces cerevisiae: Relationship
with vitisin A and B formation in red wines. J.Agric. Food Chem. 51, 7402–7409.
MORENO-ARRIBAS, M.V., POLO, M.C. (2009): Wine chemistry and biochemistry. Springer
science+Business media, LLC, New York, USA.
MULLER C.J., FUGELSANG K.C. (1997). Red wine but not white: The importance of fully
characterizing wines used in health studies. Am. J. Clin. Nutr. 66, 447.
O.I.V., 2012. International code of oenological practices, 01, Paris.
PERRONE, G., NICOLETTI, I., PASCALE, M., DE ROSSI, A., DE GIROLAMO, A.,
VISCONTI, A. (2007): Positive correlation between high levels of ochratoxin A and resveratrol-
related compounds in red wines. J. Agric. Food Chem. 55: 6807-6812.
RIBÉREAU-GAYON, P., GLORIES, Y., MAUJEAN, A., I DUBOURDIEU, D. (2006):
Handbook of Enology Volume 2, Second Edition. The Chemistry of Wine Stabilization and
Treatments. John Wiley & Sons, Ltd: Chichester, West Sussex, England
RIBÉREAU-GAYON, P., GLORIES, Y., MAUJEAN, A., DUBOURDIEU, D. (2000): Phenolic
Compounds. In Handbook of Enology, the Chemistry of Wine Stabilization and Treatments, 2nd
ed.; Ribéreau-Gayon, P., Glories, Y., Maujean, A., Dubourdieu, D., Eds.; John Wiley and Sons
Ltd.: Chichester, UK; Volume 2, pp. 141–201.
RODRIGUEZ-DELGADO, M.-A., GONZALEZ-HERNANDEZ, G., CONDE-GONZALEZ, J.-
E., PEREZ-TRUJILLO, J.-P. (2002): Principal component analysis of the polyphenol content in
young red wines. Food Chemistry, 78, 523–532.
110
ROMBOLI, Y., MANGANI, S., BUSCIONI, G., GRANCHI, L., VINCENZINI, M. (2015):
Effect of Saccharomyces cerevisiae and Candida zemplinina on quercetin, vitisin A and
hydroxytyrosol contents in Sangiovese wines. World J. Microbiol. Biotechnol. 31, 1137–1145.
RUF, J.C. (2003): Overview of epidemiological studies on wine, health and mortality. Drugs
Exp. Clin. Res. 29, 173–179.
Singleton, V.L., Rossi, J.A. (1965). Colorimetry of Total Phenolics with Phosphomolybdic-
Phosphotungstic Acid Reagents. Am. J. Enol. Vitic. 16: 144-158.
SOLEAS, J.G., DIAMANDIS,E.P., GOLDBERG D.M. (1997): Wine as a Biological Fluid:
History, Production and Role in Disease Prevention. Journal of Clinical Laboratory Analysis
11:287-313.
111
EFFECTS OF LOW LEVEL WCR EGG INFESTATION ON MAIZE PLANTS IN THE
FIELD*
Branka POPOVIĆ,
Mentor: Snežana TANASKOVIĆ, Associate Professor
University of Kragujevac, Faculty of Agronomy in Čačak, Cara Dušana 34, Čačak, Serbia.
Summary: The western corn rootworm, Diabrotica virgifera sp. virgifera (Col., Chrysomelidae),
is economically the most important threat to field maize production in Serbia. The aim of the
study was to examine the impact of WCR larvae under low levels of artificial egg infestation on
maize morphology. A field experiment with the Serbian cultivar ’NS-640’ was carried out in
Bečej, Vojvodina Province (Serbia), during 2016. In the experimental field, 96 maize plants were
selected, marked and arranged in 48 pairs. Each pair consisted of an infested plant (WCR eggs
injected in the root zone) and an uninfested plant (distillate water injected in the root zone).
During the observation, number of leaves, plant height and stem diameter were recorded. Root
damage and root weight were measured and evaluated in September 2016. For statistical
analysis, the non-parametric Kruskal-Wallis one-way analysis of variance and a correlation
matrix were used. The statistical analysis showed highly significant differences in the number of
leaves and plant height between infested and uninfested maize plants in the third field
observation. During the last field inspection, significant differences were registered only in stem
diameter between infested and uninfested maize plants. There was a negative correlation
between root damage and root weight in plant pairs. Infested plants exhibited positive
correlations between stem diameter and plant height and number of leaves, while uninfested
plants showed positive correlations between root damage and plant height.
Keywords: WCR, infestation, maize, root system, height, leaves, stem diameter
INTRODUCTION
Maize, together with wheat and rice, provides at least 30% of the food supply (FAOSTAT,
2010). As estimated by Rosegrant et al. (2009), the demand for maize production in the
developing world will double. Apart from being an important food crop for human consumption,
the demand for maize as livestock feed is increasing rapidly (Bekele et al., 2011). In Serbia,
agricultural production has undergone qualitative changes, which reflected on productivity
(Stevanović et al., 2012). Maize production in the country is one of the most important
agricultural segments for human consumption, cattle production and the processing industry
(Zubović et al., 2018). In the field, maize is exposed to a large number of pests, whose presence
contributes to yield and stem quality losses (Oerke, 2006). One of the most destructive maize
pests is the western corn rootworm (WCR), Diabrotica virgifera sp. virgifera Le Conte (Col.,
112
Chrysomelidae), which has severe consequences for the root, stem architecture and leaf surface,
and causes yield losses (Bača, 1993; Ciobanu et al., 2009). It is an oligophagous, univoltine and
the most economically important maize pest native to America (Bermond et al., 2012). Since the
end of the 20th century, WCR has become the most economically important maize pest in Europe
(Mahmoud et al., 2016). The first identification of WCR plant damage in maize fields in Europe
was in Serbia, near the Belgrade airport, in 1992 (Bača, 1993). WCR larval attack on the root
system is the most important maize field damage (Ciobanu et al., 2009; Wesseler and Fall, 2010).
Larval attack on the maize root system (nodal and lateral roots) can cause the inability of maize
to uptake water and nutrients, leading to plant logging (Dun et al., 2010). Plant logging, called
“goose neck”, is the primary manner of WCR presence in the field (Chiang, 1973; Wesseler and
Fall, 2010). Under severe larval attack, lodging can reduce yield by 11–34% (Estes et al., 2015;
Pereira, 2015). Maize has the ability to decrease plant lodging and increase the root system
(Riedell and Evenson, 1993; Gray and Steffey, 1998), and to decrease yield losses in favourable
climatic conditions (Zubović et al., 2018). WCR adults, compared to larvae, cause less damage
feeding on young leaves and maize silk (Raspudić et al., 2013). The aim of this research was to
examine the impact of WCR larvae under a low level of artificial egg infestation in causing root
damage, and their effect on root weight, number of leaves, plant height, and stem diameter.
The field experiment was carried out in Bečej, the Province of Vojvodina, Northern Serbia,
during 2016. The field chosen for the experiment displayed a low level of natural WCR
infestation. The experiment was set up on 17th May and it lasted until 30th September. Serbian
cultivar ’NS-640’ was sown in the experimental field. During the experiment, 96 maize plants
were selected, labelled and arranged into pairs. There was a spacing of 1 m between the labelled
plants, with 7 plants on average. In each pair, one plant was artificially infested in the root zone
with 4 mL of WCR eggs in 0.125% agar suspension (D plants). One mL of the suspension
contained 136 WCR eggs. A total of 48 maize plants were infested with 544 eggs per plant i.e.
26,122 eggs per year in the experimental field. The other plant in the pair was the control plant
(C). In the root zone of the C plant, the same amount of distilled water (4 mL) was injected.
During the growing season, the field experiment was regularly inspected, on a weekly basis,
for five months, totalling 14 inspections starting from the last week of May until the end of
September. Inspection of the plants involved measurement of plant height, stem diameter and
number of leaves. During the field inspection, leaves were counted and plant height was
recorded. Stem diameter was measured during the last inspection and the experiment ended on
30th September. Stem diameter measurements were taken in the field using a ± 0.05 mm precision
Caliper, Pro-Max 67 IP Sylvac System USA.
Root inspection was performed as follows: all marked plants were excavated; the soil was
removed from the roots (by manual shaking). Root damage was rated from 1 to 6 (Image 1),
according to the scale recommended by Ostlie and Notzel (1987). Thereafter, the roots were
113
rinsed with water in the lab. Maize root was dried under room climatic conditions and then dry
root weight was measured on a technical balance (Kern EW 1500-2 M, Germany).
Differences in root damage, root weight, plant height, stem diameter and number of leaves
between D and C plants were analysed using the non-parametric Kruskal-Wallis one-way
analysis of variance. Correlations among root damage, root weight, number of leaves, plant
height and plant diameter in D and C plants were determined by basic statistics (correlation
matrix) at the significance level p< 0.05.
Root rating scale (Ostlie and Notzel, 1987)

1 – No feeding damage
2 – Visible feeding scars present
3 – At least one root chewed to within
1.1/2 inches of plant
4 – One entire node of roots destroyed
Image 1. Root damage scale (Ostlie and Notzel, 1987)
RESULTS
According to data, in 2016, only three D plants i.e. 6.25% had healthy root systems (rate 1)
or no registered root damage. Root injuries rated as level 2 were recorded on seven D plants i. e.
14.58%. The highest number of D plants, 12 i.e. 25%, was with rate 3. Rate 4 was registered on 7
D plants i.e. 14.58%, and rate 5 on eight plants i.e. 16.6%. The strongest and most visible
damage, rate 6, was recorded on 10 D plants i.e. 20.83% (Figure 1).
Of the total number (48) of C plants, only three C plants i.e. 6.25% were rated as level 1 i.e.
healthy root system. Root injuries rated as 2 were recorded on three C plants i.e. 6.25%. Rate 3
damage was registered on 13 C plants i.e. 27.08%. Five C plants i.e. 10.41% were rated level 4,
while 13 C plants i.e. 27.08% were rated level 5. The most destroyed root system, rate 6, was
registered on 10 C plants i.e. 20.83% (Figure 1).
114
14 D plants C plants
12
10
0
1 2 3 4 5 6
Root injuries scale
Figure 1. The level of root damage on infested (D) and uninfested (C) plants
according to the traditional scale (Ostlie and Notzel 1987) during 2016
According to the Ostlie and Notzel (1987) root scale, of the total number of plants (96 i.e. 48
D and 48 C plants) during 2016, 93.75% in both categories were diagnosed with different root
injuries caused by WCR larvae.
The statistical analysis performed in 2016 using the non-parametric Kruskal-Wallis test
showed no significant differences in root damage between D and C plants (Table 1).
Table 1. Statistical analysis based on root damage, root weight and stem diameter between D and C plants
Mean values ± Sd
Year Parameter Sig.
D plants C plants
Root damage 3.851±1.574 4.106±1.507 0.421ns
Root weight (g) 263.8 233.7 0.164ns
2016 ±123.9 ±134.03
Stem diameter 20.48±2.759 26.32±4.170 0.001**
(mm)
The smallest measured root weight during 2016 in D and C plants was 93.12 g and 67.7 g,
respectively. In the same year, the biggest measured root weight of D and C plants was 563.44 g
and 546.03 g, respectively. The average root weight in 2016 on D and C plants was 263.8 g and
233.7 g, respectively (Figure 2). The statistical analysis performed in 2016 using the non-
parametric Kruskal-Wallis test showed no significant differences in root weight between D and C
plants (Table 1).
Stem diameter was in the range of 13.28–31.29 mm in D plants and 19.32–35.26 mm in C
plants. The average values of stem diameter for D and C plants were 20.48 and 26.32 mm,
respectively (Figure 3).
The statistical analysis revealed statistically highly significant differences in stem diameter
between D and C plants (Table 1).
115
400 35
350 30
300
25
250
20
200
15
150
10
100
50 5
0 0
D plants C plants D plants C plants
* Bars represent ±Sd value * Bars represent ±Sd value

Figure 2. Root weight (g) on infested (D) and Figure 3. Stem diameter (mm) on infested (D) and
uninfested (C) plants in 2016 uninfested (C) plants in 2016
In the third field observation, the number of leaves on D plants ranged from 6 to 9, while that
on C plants from 7 to 10. Mean values of leaf numbers on D and C plants were 7.708 and 9.042,
respectively. During the VIII and last field observation, D plants had 8 to 16 leaves, while leaf
number on C plants ranged from 11 to 15 (Figure 4). Mean values of leaf number on D and C
plants were 12.94 and 13.02, respectively.
The non-parametric Kruskal-Wallis test employed for statistical analysis in 2016 showed
significant differences in the number of leaves between D and C plants during the third field
observation, and no significant differences during the last observation (Table 2).
16 D plants C plants
350
14
300
12
250
10
200
8
6 150
4 100
2 50
0
0
III observation VIII observation
III observation VIII observation
D plants C plants
* Bars represent ±Sd value * Bars represent ±Sd value

Figure 4. Number of leaves on infested (D) and Figure 5. Plant height (cm) on infested (D) and
uninfested (C) plants in 2016 uninfested (C) plants in 2016
116
Table 2. Statistical analysis based on number of leaves and plant height between D and C plants
Mean values ± Sd Sig.

Year Parameter Observation
D plants C plants
Number of III 7.708±0.743 9.042±0.824 0.001**
leaves VIII 12.94±1.227 13.02±0.956 0.711ns
2016
III 45.23±10.94 63.56±17.28 0.001**
Plant height
VIII 283.4±31.13 290.4±21.36 0.204ns
During the VIII (21st July) and last field inspection, the maximum measured height of D
plants was 310 cm and their minimum height was 130 cm. The maximum height of C plants was
the same as in D plants, 310 cm, while their minimum height was 220 cm. The average height of
D plants was 283.4 cm and that of C plants 290.4 cm (Figure 5). During the third field
observation, the maximum and minimum height of D plants was 75 cm and 25 cm, respectively.
The maximum and minimum height of C plants during the third observation was 90 cm and 28
cm, respectively. The average height of D and C plants during the third observation was 45.23
cm and 63.56 cm, respectively (Figure 5).
The statistical analysis performed in 2016 using the non-parametric Kruskal-Wallis test
showed significant differences in plant height between D and C plants during the third
observation, and no significant differences during the last (VIII) observation (Table 2).
The statistical analysis revealed a negative correlation between root damage and root weight,
and also between root damage and number of leaves in D plants (Table 3).
Table 3. A correlation matrix based on observed parameters in D and C plants
D plants C plants
Root Number Root Plant
Variable Root Plant Stem Root Stem No. of
weigh of weigh heigh
damage height diameter damage diameter leaves
t leaves t t
Root damage 1.00 -0.59 -0.22 -0.28 -0.31 1.00 -0.73 -0.36 -0.17 -0.27
Root weight -0.59 1.00 0.28 0.09 0.07 -0.73 1.00 0.41 0.06 0.28
Plant height -0.22 0.28 1.00 0.37 0.50 -0.36 0.41 1.00 0.15 0.23
Stem diameter -0.28 0.09 0.37 1.00 0.35 -0.17 0.06 0.15 1.00 0.09
No. of leaves -0.31 0.07 0.50 0.35 1.00 -0.27 0.28 0.23 0.09 1.00
A positive correlation on D plants was determined between plant height and stem diameter and
also between stem diameter and number of leaves (Table 3).
As shown by the statistical analysis for C plants, negative correlations were found between
root damage and root weight, and between root damage and plant height, whereas only root
weight and plant height were positively correlated (Table 3).
117
DISCUSSION
The comparison of the present results on root injury with those of Popović et al. (2017) and
Tanasković et al. (2017) indicated that soil moisture i.e. rainfall and temperature had a crucial
impact on the development and harmfulness of WCR larvae as soil dwelling insects. This finding
is consistent with the results of Cagáň et al. (2016). Also, Grozea et al. (2009) reported that dry
and warm weather generally increased insect numbers. This supported the present results, which
showed that the 2016 growing season was favourable for maize production, unlike 2017 or 2015.
In this regard, we can analyse the results related to the other parameters in this study as well. The
results on plant height in 2016 show that environmental conditions play a very important role in
maize physiology as well as in determining the level of root injury. According to the presented
data, the level of root injury recorded in 2016 was smaller than in the research conducted in 2017
(Popović, 2017).
Stem diameter is a direct consequence of plant nutrition, and it reflects the level of root
injury i. e. the ability of maize plants to utilise water and nitrogen. Popović (2017) published the
results on average stem diameters in infested and uninfested maize plants (29.4 mm and 30.43
mm, respectively), and found no statistically significant differences. The results of this research
indicate the average values for D (20.48 mm) and C (26.32 mm) plants and statistically highly
significant differences in stem diameter between the two groups of plants.
The comparison of 10 plants with highly damaged roots (rate 6) with the 23 plants registered
in the research by Popović (2017) suggested that the growing season of 2016 had less favourable
conditions for WCR larval development. This finding is completely consistent with the results in
Slovakia (Cagáň et al., 2016).
CONCLUSION
According to the statistical analysis (non-parametric Kruskal-Wallis test), there were

statistically significant differences during the third field observation in number of leaves and
plant height between D and C maize plants (p<0.001). During the last field observation, the
statistical analysis showed highly significant differences only in stem diameter between D and C
plants. The statistical analysis revealed a negative correlation between root damage and root
weight in D and C maize plants. A positive correlation in D plants was observed between stem
diameter and plant height and leaf number. C plants exhibited a positive correlation between root
damage and plant height.
REFERENCES
BAČA, F.: New member of harmful entomofauna of Yugoslavia Diabrotica virgifera virgifera
Le Conte (Coleoptera: Chrysomelidae). IWGO, News Letter, XII (1-2), 21, 1993.
118
BEKELE S., BODDUPALLI M. P., JONATHAN H. AND MARIANNE B.: Crops that feed the
world 6. Past successes and future challenges to the role played by maize in global food
security. Food Sec.3:307–327, 2011.
BERMOND, G., M. CIOSI, E. LOMBAERT, A. BLIN, M. BORIANI, L. FURLAN, S.
TOEPFER, AND T. GUILLEMAUD.: Secondary Contact and Admixture between
Independently Invading Populations of the Western Corn Rootworm, Diabrotica virgifera
virgifera in Europe, Plos One7, e 50129, 2012.
CAGÁŇ, L., ŠTEVO, J., PEŤOVSKÁ.: Development of the Western corn rootworm, Diabrotica
virgifera virgifera in soil. Journal of Central European Agriculture, 17(4):1050-1069, 2016.
CHIANG, H. C.: Bionomics of the northern and western corn rootworms. Ann. Rev. of
Entomology, 18: 47-72, 1973.
CIOBANU, C., ŞANDOR, M., CIOBANU, G., DOMUŢA, C., SAMUEL, A. D., VUŞCAN, A.,
CHEREJI, I.: Research regarding Diabrotica Virgifera virgifera Le Conte (The Western Root
Worm) control in sustainable agriculture. Romanian Agricultural Research 26:79-84, 2009.
DUN Z, MITCHELL PD, AGOSTI M.: Estimating Diabrotica virgifera virgifera damage
functions with field trial data: applying an unbalanced nested error component model. J Appl
Entomol.; 134: 409–419, 2010.
ESTES, R. E., TINSLEY, N. A., GRAY, M. E.: Evaluation of soil-applied insecticides with Bt
maize for managing corn rootworm larval injury. Journal of Applied Entomology, 140(1–
):19-27, 2015.
FAOSTAT. Statistical databases and data-sets of the Food and Agriculture Organization of the
United Nations 2010. http://faostat.fao. org/default.aspx. Accessed April 2010.
GRAY, M.E., AND STEFFEY, K.L.: Corn Rootworm (Coleoptera: Chrysomelidae) Larval
Injury and Root Compensation of 12 Maize Hybrids: Assessment of the Economic Injury
Index. J. Econ. Entomol. 91(3):723-740, 1998.
GROZEA, I., STEF, R., CARABET, A., VIRTEIU, A. M., DINNSEN, S., CHIS, C., MOLNAR,
L.: The influence of weather and geographical conditions on flight dynamics of WCR adults.
Communication in Agricultural and Applied Biological Science, 75 (3), 315-322, 2009.
MAHMOUD, M. A. B., SHARP, R. E., OLIVER, M. J., FINKE, D. L., ELLERSIECK, M. R.,
HIBBARD, B. E.: The effect of western corn rootworm (Coleoptera: Chrysomelidae) and
water deficit on maize performance under controlled conditions. Journal of Economic
Entomology, 109 (2), 648-698, 2016.
OERKE, E.C.: Crop losses to pests. J. Agric. Sci. 144, 31–43, 2006.
PEREIRA, A. E., WANG, H., ZUKOFF, S. N., MINKE, L. J., FRENCH, W. B., SIEGFRIED, B.
D.: Evidence of field-evolved resistance to bifenthrin in western corn rootworm (Diabrotica
virgifera virgifera LeConte) populations in western Nebraska and Kansas. Plos One, 10 (2):1-
16. 2015.
POPOVIĆ B, TANASKOVIĆ, S., GVOZDENAC, S.: Root damages and root mass in conditions
of artificial infestation with western corn rootworm eggs. Research Journal of Agricultural
Science, 49(4): 261-268, 2017.
119
POPOVIĆ B.: Some morphological characteristics and yield parameter of maize in conditions of
artificial western corn rootworm eggs infestation. Proceedings of the X Conference of
agronomy students, with international participation. Čačak, Serbia, 24-27 August, 2017. Vol.
X, pp.102-110.
RASPUDIĆ E., SARAJLIĆ A., IVEZIĆ M., MAJIĆ I., BRMEŽ M., GUMZE A.: Učinkovitost
kemijskoga suzbijanja kukuruznoga moljca u sjemenskome kukuruzu,
Poljoprivreda/Agriculture, 19: 11-15, 2013.
RIEDELL, W.E., AND EVENSON, W. E.: Rootworm feeding tolerance in single-cross maize
hybrids from different eras. Crop Sci. 33: 951-955, 1993.
ROSEGRANT, M. R., RINGLER, C., SULSER, T. B., EWING, M., PALAZZO, A., ZHU, T.:
Agriculture and food security under global change: Prospects for 2025/2050. Washington,
D.C: International Food Policy Research Institute, 2009.
STEVANOVIĆ, S., ĐOROVIĆ, M., MILANOVIĆ, M.: The development of the market
production of cereals in Serbia: example wheat and corn. Economics of agriculture 59(4):617-
632, 2012.
TANASKOVIĆ, S., GVOZDENAC, S., POPOVIĆ, B., ĐUROVIĆ, V., ERB, M.: Maize
Tolerance to Natural and Artificial Infestation with Diabrotica virgifera virgifera Eggs.
International Journal of Agricultural and Biosystems Engineering, 11(12): 787-792, 2017.
WESSELER J. FALL E.H.: Potential damage costs of Diabrotica virgifera virgifera infestation
in Europe – the “no control”. J. Appl. Entomol. 134(5):385-394, 2010.
ZUBOVIĆ, J., JELOČNIK, M., ZDRAVKOVIĆ, A., SUBIĆ, J., RADOVANOVIĆ, S.: Using
Spatial and Seasonal Panel Model to Determine Impact of Climatic Factors on Maize Yields
in Serbia. Romanian Biotechnological Letters, 23(2):13383- 13393, 2018.
120
ELIMINATION OF IRON FROM GROUNDWATER FOR IRRIGATION PURPOSE
Senka Bubulj, Radoš Zemunac
Mentor: Doc. dr Jasna Grabić

University of Novi Sad, Faculty of Agriculture Department of Water Management, Republic of
Serbia
Summary: The agriculture sector is by far the largest water consumer at global level. The iron
values in groundwater present the negative influence on the irrigation systems operation and
production quality. When iron is present in a water supply at concentrations exceeding the
permitted limits of 1,5−5 mg/L, they are objectionable because their presence gives water a
reddish and brown-black color when is in contact with air, iron gives water an unpleasant
metallic taste and deposits of iron precipitate in the distribution system reducing the pipe
diameter and eventually clog the pipe. Today, iron removal processes are known worldwide.
However, there is still effectively simple process of iron removal: sedimentation, filtration and
aeration. The main goal of the research was determination of iron concentrations in well water,
used for irrigation, from Serbia and neighbouring countries. The second part of research was
focused upon elimination of iron from groundwater by low-cost and simple methods. In collected
well water was measured groundwater temperature, concentrations of dissolved iron,
concentrations of dissolved oxygen, pH values and electrical conductivity. Experimental results
strongly suggest that sedimentation and filtration techniques of iron removal are economical
alternatives. The laboratory analysis of dissolved iron concentrations showed that iron
minimization is possible to quality for irrigation purpose for iron enriched groundwater.
Key words: groundwater, irrigation, iron removal, sedimentation, sand filtration
INTRODUCTION
In large parts of the world, agriculture without irrigation is impossible, so irrigated agriculture
is playing a critical role in the economic and social development in almost every country.
Irrigation systems with advanced technologies along with good practices can increase irrigation
efficiency and annualy stable crop production. Groundwater is using because of availability and
consistently good quality. Variation of well water quality in an area is a function of physical and
chemical parameters that are greatly influenced by geological formations and anthropogenic
activities (Selvakumar et al., 2017). Due to its simplicity and cost-effectiveness, the analyzes are
carried out by methods of sedimentation and filtration. Sedimentation is the simplest method to
eliminate species with different physical-chemical characteristics. As the simplest, there are
results that shown it has also high efficiency of iron removal. Iron needed to be oxidized to a state
in which it can form insoluble complexes that can be filtered. Filtration is the process by which
water is separated from suspended and colloidal impurities, where chemical characteristics can be
121
changed. A slow sand filter is a bed of sand and gravel through which the water passes
downward, where is main process removing the particles of suspended matter that are too large to
pass through the gaps between the sand grains. In fast sand filtration, the removal of iron is
largely by physical action, although physicochemical processes may also occur.
Iron occurs in groundwater mainly due to the dissolution of naturally-existing iron-containing

minerals in aquifers and bedrocks and partially from industrial discharges. Iron being the fourth
most abundant element and the second most abundant metal in lithosphere, it is a common
constituent of groundwater (Ityel 2011). Iron compounds that can be found in the nature are as
the iron ions Fe2+, and Fe3+ as product of chemical reaction between Fe2+ and oxygen (1). Iron
dissolved in groundwater is in the reduced Fe2+ form. This form is soluble and normally does not
cause any problem by itself. Fe2+ form disappear in oxic conditions, and it easily turns into Fe 3+
form (2). Homogeneous oxidation of iron is described in a simplified manner by equations
describing oxidation and hydrolysis (Beek et al., 2012):
Oxidation process: Fe2+ + O2 + H+ → Fe3+ + H2O (1)
3+ +
Hydrolysis: Fe + 3H2O → Fe(OH)3↓ + 3H (2)
2+ +
Leading to the overall reaction: Fe + O2 +2 H2O→ Fe(OH)3↓ + 2H (3)
3+
Iron form (3), Fe , is not soluble in the water and it can cause various aesthetic and operational
problems. Water pH values and dissolved oxygen play an important role in the quantity of
dissolved iron.
In order to determine iron concentration in well water of different user, samples were taken
from 33 locations in Serbia and neighbouring countries, in order to obtain an overview of the
quality of water for irrigation, focusing on iron. The locations are presented on maps, which are
produced using Quantum GIS 2.16.3. Analyzing collected water samples determinate that well
water for irrigation with the highest concentration of iron, was the sample collected at University
of Novi Sad (N 45,247180; E 19,850792), and further laboratory tests were carried out at that
location. The laboratory well water tests included the determination of the physico-chemical
parameters: well water temperature, concentration of dissolved oxygen, electrical conductivity,
pH value of the wellwater and concentration of dissolved iron. The measurements were
conducted using: oxymeter for determining dissolved oxygen and temperature, portable
conductometer for determining conductivity, pH measuring instrument for determining pH
values, multiparameter photometer for determining dissolved iron. Well water was sampled in
1,5 L clean sampling plastic bottles, and the bottles were completely filled. The experiment of
minimizing and removing iron was conducted on the same day as sampling, except for
sedimentation experiment.
122
The results of analyzing iron concentrations from well water are presented on Figure 7, where
different colors are indicating water quality class acording to the Regulation (Official Gazette No.
50/12).
Figure 7. Iron concentrations in well water in Serbia and neighbouring countries
Sedimentation
The concentration of iron in collected well water, in area of Agriculture Faculty, was in the range
of 12,5−13,6 mg/L, and flow rates were in range of 0,22−1 L/s. The samples were stored in
bottles, and the iron concentration and other parameters were analyzed: on 1 st day, 2nd, 3rd, 11th,
17th and after 27 days of sedimentation. After 2nd day of sedimentation, the iron concentration in
sampled well water minimize to concentration of 1,15 mg/L, and that is acceptable iron
conncentration for irrigation purposes, i.e lower than 1,5 mg/L (Bortolini et al., 2018). The results
after 27 days of sedimentation shows concentration of dissolved iron in groundwater under 0,2
mg/L. Disadvantage of this method is that requires a certain time for sedimentation and certain
capacities for water storage. This can be challenging for practical application, but efficiency
(99,8%) is high, and minimize iron concentration in 27 days for 10 times. On Figure 8 is
presented efficiency of iron removal based on experimental data for different starting iron
concentrations in samples:
123
Figure 8. Iron removal efficacy using sedimentation
Slow sand filtration

A pilot-scale
scale filter was used for iron removal in well water after collecting. The filter media
was made of 2 cloth filters
lters and 2 layers of fine sand and gravel. Cloth filters were installed
between sand and gravel, and on the top of filter media. The most efficient form of media grading
for maximum capture of solids in gravity filters would be to have the sand decreasing in size in
the direction to flow (Malcolm et al., 2017). The filter media was constructed in the bottle with
fine sand (diameter between 0,315
0,315−0,4
−0,4 mm) with a total depth of about 11cm, and gravel with a
diameter over 6,3 mm and total depth of 11cm. The ai aim
m of research was to determine iron
concentration in well water before and after filter media, where initial iron concentration was
12,6 mg/L.
Figure 9.. Efficacy of iron removal using slow sand filtration
During experiment, 9,5 L of sampled well water were treated through a filter media to determine
maximum volume of well water that can be filtered. After pouring 5 L of well water, efficacy of
124
iron removal begins to decline ((Figure 9), so
o these informations can be basic for correction of
dimension and for determining properly ratio between sizes of grains.
Fast sand filtration

The support material was gravel and fine sand. Filter media with 4 different sizes of grain
were designed into a bottle. The aim of this stage of research was to check if this filter can be
aflow if filter is installed right after water source, so it can be usable without occupying space. In
sampled well water iron concentration was 13,6 mg/L. The properties of pil pilot
ot-scale filters are
classified in Table 2,, and each of two filters was slowly filled and had the total height of 29 cm to
achieve identical porosity for each pilot
pilot-scale filter.
Table 2. Properties of pilot-scale filter

Granulation Fine sand Gravel Gravel Gravel
Size [mm] 0,315
0,315−0,4 2,5−4,0 5,0−6,3 over 6,3
Weight [g] 1248,6 120 470 153,7
Height [cm] 17 2 4 6
After 1 L of well water that is pass through pilot

pilot-scale aflow filter, total iron removal was 98,6%
for aerated water with flow rate 0,0553 L/s, and iron removal efficiency for non-aerated
non water
with flow rate 0,0224 L/s was 96,2%. Results showed at Figure 10 suggest that aerated water
(water with
th much more dissolved oxygen in flow) and fast sand filtration can be one of
combination for further research in process of iron removing from well water. Dissolved oxygen
is respond for efficacy of filtration and total dissolved iron concentration.
Figure 10.. Relation between dissolved oxygen and iron concentration in well water using fast sand filtration
125
CONCLUSION
The research includes collecting well water samples, determining water quality for irrigation
especially concerning iron, as well as performing experiments on minimizing and optimizing
concentrations of iron using simple methods. When iron concentration is generally high in the
well water thereby, it cause that the well water is unsuitable for irrigation. Interpretation of
hydrochemical analysis at 33 locations in Serbia and neighbouring countries, reveals that the well
water at University of Novi Sad contains the highest concentrations, over 12,6 mg/L of dissolved
iron, and results shows that concentration can be reduced. Laboratory tests applying
sedimentation and filtration confirm that iron concentrations can be reduced, up to the level
suitable for irrigation (under 3 mg/L). With simple methods, the results presented in this research
suggest how is easy and economical to minimize iron concentration, and prepare well water for
irrigation purpose. The obtained results could be used and applied in real conditions at University
of Novi Sad applying one of three suggested methods, because all gave good results of iron
minimizing from well water. Future research could be conducted on scaling and properly
dimensions of presented pilot-scale filters at certain locations.
REFERENCES
SELVAKUMAR, S., CHANDRASEKAR, N., & KUMAR, G. (2017): Hydrogeochemical

characteristics and groundwater contamination in the rapid urban development areas of
Coimbatore, India. Water resources and industry, 17:26-33.
ITYEL, D. (2011): Ground water: dealing with iron contamination. Filtration & Separation,
48(1): 26-28.
BEEK, C.G.E.M., VAN, HIEMSTRA, T., HOFS, B., NEDERLOF, M.M., PAASSEN, J.A.M.,
VAN, REIJNEN, G.K. (2012): Homogeneous, heterogeneous and biological oxidation of iron (II)
in rapid sand filtration. J. Water Services Research and Technology-Aqua 61 (1): 1-13.
BORTOLINI, L., MAUCIERI, C., & BORIN, M. (2018): A Tool for the Evaluation of Irrigation
Water Quality in the Arid and Semi-Arid Regions. Agronomy, 8(2): 23.
MALCOLM J. BRANDT, K. MICHAEL JOHNSON, ANDREW J. ELPHINSTON, DON D.
RATNAYAKA. (2017): Twort's Water Supply, 367-406.
Regulation 50/2012/RS of 18th May 2012 on limit values for pollutants in surface waters,
groundwater and sediments, and deadlines for their achievement, Republic of Serbia, Official
Gazette No. 50/12, (in Serbian).
126
COMPARISON OF ANTIOXIDANT PROPERTIES AMONG TWO APPLE
CULTIVARS
Emir ŠAHINOVIĆ
Mentor: Senad MURTIĆ, PhD

The University of Sarajevo, Faculty of Agriculture and Food Sciences, Department of Plant
Physiology, Bosnia and Herzegovina
SUMMARY: Apple (Malus domestica Borkh.) possess many health-promoting properties.

The main beneficial effects of apple fruits on health are related to the antioxidant properties of
components synthesizing in fruits during their growth and development. Phenolic and flavonoids
are certainly components that contribute significantly to the antioxidant capacity of fruits and
hence their health properties. The aim of this study was to compare the antioxidant properties of
apple (antioxidant capacity, ascorbic acid, total phenolic and flavonoid contents) among
cultivars Idared and Braeburn, grown under same conditions. Ascorbic acid was estimated by
2,6-dichlorophenolindophenol titration method, total phenolics byFolin-Ciocalteu method, total
flavonoids according to aluminum chloride colorimetric assay, and the ferric
reducing/antioxidant power (FRAP) assay was used to determine the total antioxidant capacity of
apple fruits. Average value of ascorbic acid, total phenolic and flavonoids and total antioxidant
capacity of apple cv. Idared were 8.09 mg100 g-1, 34.56 mg gallic acid equivalents100g-1, 12.62
mg catechin equivalents 100 g-1 and 323.28 mol Fe2+100 g-1 fresh weight, while the average
value of total phenolic and flavonoids contents and antioxidant capacity of apple cv. Braeburn
were as follows: 8.89 mg100 g-1, 33.4 mg gallic acid equivalents100 g-1, 13.56 mg catechin
equivalents 100 g-1 and 367.01 mol Fe2+100 g-1 fresh weight, respectively.One-wayANOVA
analysis showed that the observed variations in examined parameters between cv. Idared and
Braeburn were not statistically significant. Correlation analysis of data confirmed that phenolic
compounds have a significant contribution to the total antioxidant capacity of apple.
Key words: health, phenolic, flavonoids, antioxidant capacity
INTRODUCTION
The health-promoting properties of apples are mainly related to their chemical composition,
primarily to antioxidants i.e. substances that prevent or delay the oxidative degradation of active
ingredients (Hayson, 2011). Phenolic compounds are the most abundant antioxidants of apple and
their antioxidant activity is associated with a large number of conjugated double bonds and
hydroxyl groups in one or more phenolic rings of annular structure of the molecule (Del Rio et
al., 2013). Owing to this structure, phenolic compounds have the ability to stabilize free radicals
127
by delocalization of the unpaired electron around the phenol ring to form a stable resonance, i.e.
low-energy radical (Tsao, 2010).
Flavonoids comprise the largest group of phenolic compounds in apples and generally in
plants and are considered as the most effective antioxidants in plant cells (Arct and Pytkowska,
2008). Ascorbic acid is also important antioxidant substances in plants. It reacts rapidly with
reactive forms of oxygen which are generated during aerobic metabolism or during exposure of
plants to stress conditions and thus participating in their neutralization or removal from plant
cells (Li et al., 2014).
Many studies have found that the ascorbic acid, phenolic and flavonoid contents, as well as
the antioxidant capacity of apples, are results of the impact of different factors, such as
agroecological conditions, harvest time, storage and cultivars (Kevers et al., 2011; Kschonsek et
al., 2018). The past two decades, relatively new apple cultivars, such as Braeburn, have become
more popular among consumers in Bosnia and Herzegovina, resulting in a gradual increase in
presence of this cultivar in apple orchards. Notwithstanding, Idared is still the most widely apple
cultivar in orchards in our country.
Considering that the most popular apple cultivars at Gorazde area are Braeburn and Idared, as
well as the fact that the current trend in apple production is increasing the quality of apples in
terms of their antioxidant properties, the aim of this study was to compare the antioxidant
properties of apple (antioxidant capacity, ascorbic acid, total phenolic and flavonoid contents)
among these two cultivars, grown under same conditions.
The experiment was performed in 2018 in the intensive apple orchard at Gorazde area
(43°38′N 18°55′E; altitude of 455 m). The experimental site has a humid continental climate,
with an annual precipitation of about 1137 mm, average annual temperature of 8.7 °C, and a
sandy loam soil. Apple orchard was established in the autumn of 2002, covers about 1 ha area,
and a half (50%) of the total production area relates toIdared and Braeburn cultivars. Idared and
Braeburn apple trees on M9 rootstock were planted at 1.3 m x 3.5 spacing with an east-west row
orientation. The experimental plot included a row of six apple trees for each cultivar and three
such plots were the subject of study. Cultivation form (slender spindle) on all experimental plots
were identical. Also, all agricultural measures (fertilization, pruning, pest and disease
management, irrigation) were applied identically to all examined plots during the growing
season.
Idared and Braeburn apples were collected at the stage of their commercial maturity. Three
fruits were taken from the central zone of the crown of each experimental tree and transported to
the laboratory in a portable refrigerator. The following antioxidant properties of apple fruits were
determined: ascorbic acid, total phenolic and flavonoids contents, and total antioxidant capacity.
128
Ascorbic acid was estimated by 2,6-dichlorophenolindophenol titration method (AOAC,
2006). Triplicate titration was conducted for all samples and results were expressed as mg
ascorbic acid 100 g-1 fresh fruit.
Extraction of phenolic compounds from apple samples was performed using 30% aqueous
ethanol as follows: 1 g of dried and grinded apple sample was placed in a 100 ml glass bottle and
mixed with 40 ml ethanol solution. Thereafter, the mixture was placed in an ultrasonic bath with
following conditions: time of 30 min, the temperature of 35°C, and power of 150 W (the mixture
was vortexed thoroughly once every five minutes). After cooling down to room temperature, the
mixture was filtered through filter paper Whatman, 11µm pore size) in 50 ml flask and diluted to
the mark with 30% aqueous ethanol. Extract thus obtained was used for the estimation of the total
phenolic and flavonoids contents, and total antioxidant capacity.
Total phenolic content (TPC) was determined according to the Folin-Ciocalteu method (Ough
and Amerine, 1988) as follows: 0.25 ml of extract was placed in 25 ml flask, then 15 ml distilled
water, 1.25 ml Folin-Ciocalteu’s phenol reagent, and 3.75 ml saturated sodium carbonate solution
was added (Folin-Ciocalteu’s reagent was diluted with distilled water in ratio 1:2 before use).
Thereafter, the flask was filled with 30% aqueous ethanol to the mark, and heated in a water bath
at 50 ºC, for 30 min. After cooling to room temperature absorbance was measured at 765 nm
using UV/VIS spectrophotometer (Schimadzu, Japan). Quantification was done on the base of a
standard curve of gallic acid (0 -500 mg l-1), and results were expressed as mg of gallic acid
equivalent per 100 g fresh weight (mg eq. GA 100 g-1 FW).
Total flavonoids content (TFC) was determined according to Aluminium chloride
colorimetric assay (Zhishen et al., 1999) as follows: 1 ml of extract was placed in 10 ml flask,
then 4 ml distilled water, 0.3 ml 5%NaNO2 0.3 ml 10% AlCl3 and 2 ml 1mol l-1 NaOH was
added. The flask was filled with distilled water to the mark and after 15 min absorbance was
measured at 510 nm.TFC quantification was done on the base of a standard curve of catechin (0 -
100 mg l-1), and results were expressed as mg of catechin equivalent per 100 g fresh weight (mg
eq. C 100 g-1 FW).
Total antioxidant capacity (TAC) was determined according to Ferric reducing ability power
(FRAP) method (Benzie and Strain, 1996) as follows: 240 µl distilled water, 80 µl extract, and
2080 µl FRAP reagent (obtained by mixing 0.3 mol l -1 acetate buffer (pH = 3.6), 10 mmol l-1
TPTZ (2,4,6-tripyridyl-s-triazine) and 20 mmol l-1 FeCl3 x 6 H2O in ratio 10 : 1 : 1) were
added in 10 ml flask and heated in water bath at 37 ºC for 5 min. After cooling to room
temperature absorbance was measured at 595 nm.TAC quantification was done on the base of a
standard curve of FeSO4 x 7 H2O (0-2000 µmol l-1) and results were expressed as µmol Fe2+ per
100 g fresh weight (µmol Fe2+ 100 g-1 FW).
All experimental measurements were done in triplicate and the results were expressed as the
average of three analyses ± standard deviation. One-way ANOVA was used to determine whether
there are any statistically significant differences between the variants.Data were evaluated using
Pearson's correlation coefficients to identify relationships between antioxidant substances
(phenolic, flavonoids, and ascorbic acid) and total antioxidant activity (FRAP).
129
Phenolic compounds are secondary metabolites that play important roles in plant defense
system against reactive oxygen species (ROS), and hence their larger content is highly desirable
in apple fruits (Pandey and Rizvi, 2009). Different factors such as genetic, morphogenetic and
environmental conditions can influence the biosynthesis and accumulation of phenolics. A
change in any of the above factors may alter the content of phenolics even if other factors remain
constant (Yang et al., 2018). For most plants, external variables of environmental factors
(drought, salinity, light, temperature, nutrition etc.) can significantly affect some process
associated with secondary metabolism, leading to the increase of both phenolics compounds and
antioxidant activity (Verma and Shuhla, 2015). Several studies have demonstrated that the
phenolic content in apple fruits may differ significantly, depending on the growth conditions
(Zupan et al., 2014; Mditshwa et al., 2015). Veberic(2005) noted that genetic factors i.e. cultivars
also have an important role in the biosynthesis of phenolic compounds and, consequently, in the
antioxidant activity of apples. In this study, there was no significant difference in the phenolics
content of apple among cv. Idared and Braeburn. Also, the ascorbic acid and flavonoid contents
as well as the total antioxidant capacity of apple did not differ significantly among the tested
cultivars (Table 1), which is not consistent with the hypothesis mentioned above. However, only
two cultivars were covered by this study, and thus the possibilities to form a valid conclusion
regarding the influence of cultivars on the synthesis and accumulation of phenolic compounds in
the apple within this work are considerably limited.
Table 1. Ascorbic acid (AA), total phenolic content (TPC), total flavonoid content (TFC) and total antioxidant
capacity (TAC) of apple fruits (fresh weight)
Cultivar AA TPC TFC TAC

-1 -1 -1
mg 100 g mg 100 g mg 100 g µmol Fe2+ 100 g-1
Idared 8.09 ± 0.83 34.56 ± 19.02 12.62 ± 5.76 323.28 ± 220.09
Braeburn 8.89 ± 2.50 33.4 ± 20.25 13.56 ± 2.86 367.01 ± 210.11
F test n.s. n.s. n.s. n.s.
n.s. - no significant
In this study is undeniably confirmed a very high positive correlation between the content of
phenolic/flavonoids and the total antioxidant capacity of apple, regardless of cultivars. For both,
Idared and Braeburn cultivars, the correlations between the content of ascorbic acid and the total
antioxidant capacity of apple were also high, but the strength of that correlation was slightly
lower compared to the correlation between phenolic/flavonoids and total antioxidant
capacity(Fig. 1).
These results indicate that the antioxidant capacity of apple may be principally caused by the
phenolic compounds. A significant correlation between the content of phenolic/flavonoids
compounds and the total antioxidant activity shown by the apples has been observed in many
other studies (Lee et al., 2003;Wojdylo et al., 2008; Stracke et al., 2009).
130
AA - FRAP (Braeburn) 0.75
TFC - FRAP (Braeburn) 0.96
TPC - FRAP (Braeburn) 0.97
AA - FRAP (Idared) 0.68
TFC - FRAP (Idared) 0.82
TPC - FRAP (Idared) 0.95
-1 -0.5 0 0.5 1
Coefficient correlation
Fig. 1. Correlation coefficients between the antioxidant substances and total antioxidant capacity (FRAP) for both
cultivars
According to Huang et al. (2005), antioxidant capacity can be monitored by a wide variety of
assays with different mechanisms, including hydrogen atom transfer (HAT) reaction and electron
transfer (ET) reaction-based methods, depending on how the free radicals are deactivated by the
antioxidants. HAT-based methods measure the ability of an antioxidant to scavenge free radicals
by hydrogen donation and form stable compounds, while the ET-based assays measure the
capacity of an antioxidant to transfer one electron and reduce any compound, which changes
color when reduced (Apak et al., 2007).
The TPC estimation by the Folin-Ciocalteumethod, the TFC estimation by the Aluminium
chloride colorimetric assay, as well as the antioxidant capacity by FRAP method is considered
ET methods. Considering that all methods act through the same mechanism, it was expected that
the correlations between phenolic/flavonoids and the total antioxidant capacity are high, and the
results of this study confirm that observation.
CONCLUSION
The results of this study showed that the determined differences in antioxidant properties
(antioxidant capacity, ascorbic acid, total phenolic and flavonoid contents) among cv. Idared and
Braeburn apples, grown under same conditions were not statistically significant.
This study revealed that phenolic compounds significantly contribute to the antioxidant
capacity of apple.
REFERENCES
APAK,R., GÜÇLÜ, K., DEMIRATA, B., ÖZYÜREK, M., ÇELIK, S.E., BEKTAŞOĞLU, B.,
BERKER, K.I., ÖZYURT, D.(2007):Comparative Evaluation of Various Total Antioxidant
131
Capacity Assays Applied to Phenolic Compounds with the CUPRAC Assay. Molecules, 12:
1496-1547.
ARCT, J., PYTKOWSKA, K. (2008): Flavonoids as components of biologically active
cosmeceuticals. Clin. Dermatol., 26:347-357.
ASSOCIATION OF OFFICIAL ANALYTICAL CHEMISTS (AOAC) (2006): Official Methods
of Analysis: Vitamin C in juices and vitamin preparations, method 967.21. 18th edition.
Arlington: USA.
BENZIE,I.F., STRAIN,J.J. (1996): Ferric reducing ability of plasma (FRAP) as a measure of
antioxidant power: The FRAP assay.Anal.Biochem.,239: 70-76.
DEL RIO, D., RODRIGUEZ-MATEOS, A., SPENCER, J.P., TOGNOLINI, M., BORGES, G.,
CROZIER, A. (2013): Dietary (poly)phenolics in human health: Structures, bioavailability, and
evidence of protective effects against chronic diseases. Antioxid. Redox Signal., 18: 1818-1892.
HUANG, D., OU, B., PRIOR, RL.(2005):The chemistry behind antioxidant capacity assays.J.
Agric. Food Chem., 53(6):1841-1856.
HYSON, D.A. (2011): A comprehensive review of apples and apple components and their
relationship to human health. Adv Nutr., 2(5): 408-20.
KEVERS, C., PINCEMAIL, J., TABART, J., DEFRAIGNE, J.O., DOMMES, J. (2011):
Influence of cultivar, harvest time, storage conditions, and peeling on the antioxidant capacity
and phenolic and ascorbic acid contents of apples and pears.J. Agric. Food Chem., 59(11):6165-
6171.
KSCHONSEK, J., WOLFRAM, T., STÖCKL, A., BÖHM, V. (2018): Polyphenolic Compounds
Analysis of Old and New Apple Cultivars and Contribution of Polyphenolic Profile to the In
Vitro Antioxidant Capacity. Antioxidants, 7(1):20.
LEE, K.W., KIM Y.J., KIM D., LEE H.J., LEE C.Y. (2003):Major phenolics in apple and their
contribution to the total antioxidant capacity. J. Agric. Food. Chem., 51:6516-6520.
LI, Z., ZHANG, H., YANG, O., KOMLA M.G., ZHANG, X., ZHU, S. (2014): Ascorbic Acid
Enhances Oxidative Stress Tolerance and Biological Control Efficacy of Pichia caribbica against
Postharvest Blue Mold Decay of Apples. J. Agric. Food Chem., 62(30): 7612-7621.
MDITSHWA, A., VRIES, F., VAN DER MERWE, K., CROUCH, E., OPARA, U.L.
(2015):Antioxidant content and phytochemical properties of apple ‘Granny Smith’ at different
harvest times. S. Afr. J. Plant Soil, 32(4): 221-226.
OUGH,C.S., AMERINE, M.A. (1988): Phenolic compounds. In: Methods for analysis of must
and wines, New York: Wiley, pp. 50-70.
PANDEY, K.B., RIZVI, S.I. (2009): Plant polyphenols as dietary antioxidants in human health
and disease. Oxid. Med. Cell.Longev., 22(5):270-278.
STRACKE, AB., RÜFER, C.E., BUB, A., WEIBEL, F.P., KUNZ, C., WATZL, B. (2009):Three-
year comparison of the polyphenol contens and antioxidant capacities in organically and
conventionally produced apples (Malus domestica Bork. Cultivar Golden Delicious). J. Agric.
Food Chem., 57:4598-4605.
TSAO, R. (2010): Chemistry and biochemistry of dietary polyphenols. Nutrients, 2(12):1231-46.
132
VEBERIC, R., TROBEC, M., HERBINGER, K., HOFER, M., GRILL, D., STAMPAR, F.
(2005): Phenolic compounds in some apple(Malus domesticaBorkh.) cultivars of organic and
integrated production. J. Sci. Food Agric., 85: 1687-1694.
VERMA, N., SHUKLA, S. (2015): Impact of various factors responsible for fluctuation in plant
secondary metabolites. J. Appl. Res. Med. Aromat. Plants, 2: 105-113.
WOJDYLO,A., OSZMIAŃSKI, J., LASKOWSKI, P. (2008): Polyphenolic compounds and
antioxidant activity of new and old apple varieties. J. Agric. Food. Chem., 56:6520-6530.
YANG, L., WEN, K.S., RUAN, X., ZHAO, Y.X., WEI, F., WANG, Q. (2018): Response of
Plant Secondary Metabolites to Environmental Factors. Molecules, 23: 762.
ZHISHEN,J., MENGCHENG,T., JIANMING,W. (1999): The determination of flavonoid
contents in mulberry and their scavenging effects onsuperoxide radicals. Food Chem., 64(4):
555-559.
ZUPAN, A., MIKULIC-PETKOVSEK, M., SLATNAR, A., STAMPAR, F., VEBERIC, R.
(2014):Individual phenolic response andperoxidase activity in peel of differently sun-exposed
apples in the period favorable for sunburn occurrence.J. Plant Physiol., 171: 1706-1712.
133
THE INFLUENCE OF MATURITY STAGE AND EXTRACTION SOLVENT ON
TOTAL PHENOLICS AND ANTIOXIDANT ACTIVITY OF STALK OF SWEET
CHERRY
Marijana Peić Tukuljac
Mentor: Dejan Prvulović, Associate Professor

University of Novi Sad, Faculty of Agriculture, Serbia
Summary: Cherries present one of the most important fruits in the nutrition becaouse of its high
level of polyphenols and their antioxidant potential. Also, stalks are valuable part of cherries
and its have found place in alternative medicine as natural diuretic. The aim of this study was to
determine the content of phenolic compounds and antioxidant capacity in stalk of sweet cherry
cultivar Sandor and investigated influence of ripening stage and extraction solvent on these
measured parameters. The results show that the total phenolic content and antioxidant activity
are significantly affected by maturity stage.
Key words: sweet cherry, stalk, antioxidant capacity, phenolics
INTRODUCTION
Cherry botanically belong to the genus Prunus, subgenus Cerasus, within the Rosaceae
family, section Eucerasus. Sweet cherry (Prunus avium, L.) and sour cherry (Prunus cerasus, L.)
are economicaly most imortant species of cherry fruits. The origin of these species is located in
Central Europe and areas surrounding the Caspian and Black Seas, but now are grown around the
world, especially in areas with a moderate climate (Serradilla el al., 2016).
Sweet cherries are one the most popular fruits in human dietary because of their attractive
organoleptic characteristics including red colour, firmness and delicious taste. They are usually
consumed as fresh fruits as well as frozen or processed in jams, jellies and dried forms Cherries
are also known for their rich nutritional content and broad range of bioactive compoundssuch as
phenolics compounds (Martini et al., 2017) vitamin C (Schmitz-Eiberger and Blanke, 2012) and
they are good source of potassium (McCune et al., 2011). A numerous studies show that the
concentration of these compounds depends on genotype, ripening stage, pre- and postharvest
treatments (Cao et al., 2015; Faniadis et al, 2010). Cherries’ fruits and stalks are used in
alternative medicine, because they can reduce the level of oxidative stress and according to
several studies have antimicrobial, antifungal, antioxidant, antiinflammatory, anticancer,
antidiabetic, neuroprotective, and cardioprotective effects (Gonçalves et al., 2019). This health
benefits are related with high level of different phenolic compounds. Stalks are very valuable
parts of cherry and they have traditional use in folk medicine as natural diuretic, for the treatment
of kidney stones and as sedative (Hooman el al., 2009)
134
The goal of this paper was to determine the content of phenolic compounds and antioxidant
activity in stalks of sweet cherry (cultivar Sandor) extracted with two different extraction solvents
(70% ethanol and 70% acetone) at different maturity stages.
Plant material
Fruits and stalks of sweet cherry cultivar Sandor were collected from the productive orchard
“Sloga” Kać in vicinity of Novi Sad, Serbia. Cherry fruits were picked at two maturity stages:
seven days before commercial maturity and at commercial maturity, on the basis of fruit colour.
Stalks (pedicels) were air-dried at ambiental temperature to the constant weight.
Extraction
Plant material was reduced to a fine dried powder and extracted (1g per sample) with two
different extraction solvent: 70% aqueous ethanol solution (50 ml) and 70% aqueous acetone
solution (50 ml) by sonication for 20 minutes in an ultrasonic bath at room temperature. After the
extraction, the extract was kept in cold storage until further analysis.
Determination of total phenol content and total tannin

The total phenol content (TPC) of acetone and ethanol extracts was determined by
spectrophotometry after reaction with the Folin-Ciocalteu phenol reagent (Kroyer, 2004;
Hagermann et al., 2000). 50 µl of extracts and 2.5 ml of Folin-Ciocalteu reagent (diluted using
distilled water in a 1:2 ratio) are mixed and incubated for 5 min. Then, 2 ml saturated sodium
carbonate solution was added. After 45 min, the absorbance of the solution was measured at 730
nm. For the determination of tanin content, 0.1 g PVPP (polyvinylpolypyrrolidone) was added to
1 ml of extracts. After the centrifugion, the content of total phenols in the supernatant was
measured. The obtained value was than subtracted from the value obtained for total phenolics
content. Quercetin was used for constructing the standard curve and total phenol and total tannin
content was expressed as mg gallic acid equivalents per gram of dry weight (mg QE/g DW)
Determination of total flavonoid content

The total flavonoid (TF) content was determined according to the procedure by Saha et al.
(2013). 200 µl of extracts was mixted with 3ml of 2% AlCl3 solution. After mixing, the solution
was incubated for 15min at room temperature and the absorbance was read at 430nm.
Determination of total flavonoid content was carried out in triplicate and calculated from the
calibration curve obtained with quercetin, which was used as a standard. The values obtained
were recalculated end expressed as quercetin equivalents in mg per gram of dry weight (mg QE/g
DW)
135
Determination of antioxidant activity
The DPPH (2,2-diphenyl-1-picryhydrazyl) assay was used to measure radical scavenging activity
(Lai and Lim, 2011). A 1.5ml volume of DPPH reagent dissolved in 70% methanol was
incubated with 20 μl of sample. After 30 minutes, the absorbance were read at 517nm.
The ABTS (2,2’-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) assay was based on ability
of the extract to neutralize the radical cation, ABTS˙ + ( Re et al.,1999). Reaction mix was
prepared with 1.5ml of the ABTS reagent and 20μl of the sample, and the absorbance was read at
734 nm after 2 h.
The determination of ferric reducing antioxidant power (FRAP) was performed as described by
Valentão at. al. (2002). The reagent was prepared by mixing 300 mmol/dm3 acetate buffer pH
3.6, 10 mmol/dm3 TPTZ in 40 mM HCl and 20 mmol/dm3 FeCl3 x 6H2O in a 10:1:1 ratio.
Reaction mix was prepared by mixing 1,5 ml of the FRAP reagent i 20 µl of the sample. The
absorbance is measured at 510 nm.
The calibration curve was constructed with standard Trolox solution and the results of TRC,
DPPH, FRAP and ABTS were expressed as Trolox equivalents in mg per gram of dry weight
(mg Trolox/g DW).
The total antioxidant activity (TAA) of extracts were analised according to the
phosphomolybdenum method described by Kalaskar and Surana (2014) and the results were
expressed as mg of butylated hydroxytoluene equivalents per gram of dry weight (mg BHT/g
DW).
The nitroblue tetrazolium (NBT) assay was used to determine the superoxide free radical
scavenging activity (Kalaskar and Surana, 2014). The percent inhibition of superoxide anion
generated was calculated using the following formula:
Scavenging activity (%) = (1 - absorbance of sample/absorbance of control) x 100
Statistical analysis
All the assays were carried out in triplicate and the results of the total polyphenols, flavonoids,
tannins and antioxidant capacity assayed with the total antioxidant activity, ABTS, DPPH, FRAP
and NBT test were expressed as mean ± standard deviation (Table 1). Statistical significance was
tested using software STATISTICA ver. 13 (StatSoft, Inc., USA). The effect of maturity stage
and extraction solvent on the content of phenolics,flavonoids, tanins and antioxidant activity was
evaluated by two-way analysis of variance (Factorial ANOVA), followed by comparison of
means by Duncan’s multiple range test (p<0.05).The correlation coefficients were calculated
according to Spearman (Table 2).
The results of the total polyphenols, flavonoids, tannins and antioxidant activity assayed with
the total antioxidant activity, total reduction capacity, DPPH, FRAP, ABTS are shown in Table 1.
The contents of phenols and flavonoids were higher for the acetone extract than for the ethanolic
136
one, but content of tannins did not show significant difference between the two solvents. Levels
of phenolics are higher in ripe than in semi-ripe cherries and this is in agreement with report of
Gonçalves et al. (2004).
Table 1. Phenolic content and antioxidant activity in extracts of sweet cherry (cv. Sandor) fruits.
Maturity stage Semi ripe Ripe
Extraction 70% ethanol 70% acetone 70% ethanol 70% acetone

solvent
Total phenolics3 17.62 ± 1.53a 23.39 ± 0.76b 18.92 ± 1.12a 21.40 ± 0.64b
Total 1.91 ± 0.08a 2.74 ± 0.11b 2.61 ± 0.12b,c 2.96 ± 0.11c

flavonoids1
Total tannins3 11.97 ± 0.16a 13.90 ± 0.21b 14.16 ± 0.14b 13.94 ± 0.04b
DPPH test2 36.92 ± 3.34a 37.36 ± 2.89a 44.58 ± 2.90b 44.27 ± 0.44b
ABTS test2 125.96 ± 9.59a 135.98 ± 148.74 ± 7.71b 173.46 ± 5.93c

11.82ab
FRAP test2 20.06 ± 0.75a 28.45 ± 0.61b 13.97 ± 0.38c 15.07 ± 0.27d
NBT test5 23.87 ± 0.96a 31.53 ± 1.29b 24.40 ± 1.04a 35.99 ± 0,69c
TAA4 76,40 ± 14.55a 99.40 ± 9.44b 158.43 ± 12.50c 159.50 ± 7.02c
a-d
values without the same superscript within each column differ significantly at p< 0.05
(Duncan post-hoc test)
1
Expressed as mg quercetin equivalents (QE)/g dry weight
2
Expressed as mg trolox equivalents (TE)/g dry weight
3
Expressed as mg gallic acid equivalents (GAE)/ g dry weight
4
Expressed as mg butylated hydroxytoluene equivalents (BHT)/g dry weight
5
% of inhibition of superoxide anion generated
According to the ANOVA, the maturity stage, extraction solvent and interaction between
these two factors show statistically significant (p<0.05) influence on total flavonoid and total
tannin content, but maturity stage as a single factor do not have statistical significant effects on
137
phenolics content. The two main functions of phenolics in plants, including sweet cherry, are to
protect them against pathogens and predators, as well as to contribute to their color and
nutritional characteristics. The first function is achieved by the fact that phenolic compounds, as
secondary metabolites, are the most important non-enzymatic antioxidants which are able to
participate in the oxidative stress response. On the other hand, the second function is
accomplished during the process of fruit ripening, when the degradation of chlorophyll leads to
the substantial increase in the amount of different phenolic compounds such as anthocyanins,
which subsequently causes the change of cherry fruit color (Ferretti et al., 2010).
The antioxidant capacity of extracts from cherries and their stalks have been widely
investigated using different methodological approaches. In this study, antioxidant capacity was
determined using five different antioxidant tests: DPPH, FRAP, ABTS, TAA, and NBT. The
extracts of stalks removed from ripe fruits showed higher DPPH-radicals scavenging activities
than the extracts of stalks removed from semi-ripe fruits, which is expected since ripe cherries
have a high content of polyphenolics compounds and, because of that, high capacity for DPPH
scavenging activities. This is in accordance with the results of Prvulović et al. (2011).
Table 2. The Correlation between biochemical parameters maturity stage and extraction solvent
Maturity Extraction TP3 TF1 TT3

stage solvent
Maturity
stage
Extraction 0
solvent
TP3 -0.096 0.869*
1
TF 0.531 0.772* 0.580*
3
TT 0.676* 0.048 0.070 0.461
2
DPPH 0.869* -0.048 0.021 0.378 0.517
2
ABTS 0.821* 0.338 0.182 0.797* 0.580*
2
FRAP -0.869* 0.434 0.524 -0.168 -0.643*
4
TAA 0.869* 0.193 0.182 0.706* 0.629*
5
NBT 0.338 0.869* 0.776* 0.846* 0.189
*Statistically significant at p< 0,05 (Spearman correlation)
1
Expressed as mg quercetin equivalents (QE)/g dry weight
2
Expressed as mg trolox equivalents (TE)/g dry weight
3
Expressed as mg gallic acid equivalents (GAE)/ g dry weight
4
Expressed as mg butylated hydroxytoluene equivalents (BHT)/g dry weight
5
% of inhibition of superoxide anion generated
The total phenolic and flavonoid contents were in strong positive and significant correlation
with the extraction solvent. This result was expected, taking into consideration a high
138
antioxidative potential of the phenolic compounds (Halliwell i Gutteridge, 2015) and their direct
influence on the antioxidant activity. In addition, ABTS was in strong and significant correlation
with total flavonoids content, since ABTS tests are specific for flavonoids. When it comes to the
other antioxidant tests, it was established that the maturity stage was in a strong and significant
correlation with DPPH, ABTS, TAA and FRAP test. This could be explained by the fact that
total antioxidant activity determined by the FRAP test could be directly connected to the content
of phenolic compounds and other antioxidants, and also with their ability to neutralize free
radicals, including DPPH-radical (Sharma and Ramawat, 2013). On the other hand, the solvent
used for the extraction was in a strong and significant correlation with NBT test, which suggested
that both the maturity stage and the solvent type significantly influence the antioxidant capacity
of a given extract.
CONCLUSION
The results of this study revealed that the phenolic compounds content and antioxidant
capacity of the extracts of sweet cherry stalks are significantly affected by maturity stage. In
addition, the solvent system used for the extraction process also showed influence on all
investigated biochemical parameters, but not such significant as the ripening stage one. Based on
these findings, the analysis of total phenolic, flavonoid and tannin content and the antioxidant
activity presented in this study have shown that the stalk of sweet cherry fruits could be a potent
source of natural antioxidants and might attract new interest in evaluating their status in
terapeutical purpose. Beside that, consuming this part of fruit in a daily nutrition can reduce the
food waste.
REFERENCES
CAO, J., JIANG, Q., LIN, J., LI, X., SUN, C., & CHEN, K. (2015): Physicochemical
characterisation of four cherry species (Prunus spp.) grown in China. Food Chemistry,173, 855-
863.
FERRETTI G, BACCHETTI T, BELLEGGIA A, NERI D.,(2010): Cherry antioxidants: from
farm to table. Molecules;15:6993-7005.
GONÇALVES, A. C., BENTO, C., JESUS, F., ALVES, G., & SILVA, L. R. (2019): Sweet
Cherry Phenolic Compounds: Identification, Characterization, and Health Benefits. Studies in
Natural Products Chemistry, 31–78.
GONÇALVES, B., LANDBO, A.-K., KNUDSEN, D., SILVA, A. P., MOUTINHO-PEREIRA,
J., ROSA, E., & MEYER, A. S., (2004).: Effect of Ripeness and Postharvest Storage on the
Phenolic Profiles of Cherries (Prunus aviumL.). Journal of Agricultural and Food Chemistry,
52(3), 523–530.
HAGERMANN A., HARVEY-MUELLER I., MAKKAR H.P.S., (2000): Quantification of
tannins in tree foliagea laboratory manual. Vienna: FAO/IAEA Working Document.
HALLIWELL, B., GUTTERIDGE, J.M.,(2015): Free Radicals in Biology and Medicine. Oxford
University Press, USSA
139
HOOMAN, N., MOJAB, F., NICKAVAR, B., POURYOUSEFI-KERMANI, P., (2009): Diuretic
effect of powdered Cerasus avium (cherry) tails on healthy volunteers. Pakistan Journal of
Pharmaceutical Sciences, 22, 381–383.
KALASKAR M.G., SURANA S.J., (2014): Free radical scavenging, immunomodulatory activity
and chemical composition of Luffa acutangula var.Amara (Cucurbitaceae) pericarp. Journal of
Chilean Chemical Society, 59:2299-2302.
KROYER G.T., (2004): Red clover extracts as antioxidant active and functional food ingredient.
Innovative Food Science and Emerging Technologies, 5: 101-105.
LAI H.Y., LIM Y.Y.,( 2011) : Evaluation of antioxidant activities of the methanolic extracts of
selected ferns in Malaysia. International Journal of Environmental Science and Development,
2:442- 447.
MARTINI, S., CONTE, A., TAGLIAZUCCHI, D. (2017):Phenolic compounds profile and
antioxidant properties of six sweet cherry ( Prunus avium ) cultivars. Food Research
International, 97, 15–26.
MCCUNE, L.M., KUBOTA, C., STENDELL-HOLLIS, N.R., THOMSON, C.A.,(2011):
Cherries and health: a review. Critical Reviews in Food Science and Nutrition 51, 1–12.
PRVULOVIC, D., MALENČIĆ, Đ., POPOVIĆ, M. LJUBOJEVIĆ, M. OGNJANOV, V., (2011):
Antioxidant Properties of Sweet Cherries (Prunus avium L.) - Role of Phenolic Compounds.
World Academy of Science, Engineering and Technology. 59. 1149-1152.
RE R., PELLEGRINI N., PROTEGGENTE A., PANNALA A., YANG M., RICE-EVANS C.,
(1999): Antioxidant activity applying an improved ABTS radical cation decolorization assay.
Free Radical Biology and Medicine, 26:1231-1237.
SAHA A.K., RAHMAN MD.R., SHAHRIAR M., SAHA S.K., AL AZAD N., DAS D., (2013):
Screening of six Ayurvedic medicinal plant extracts for antioxidant and Cytotoxic activity.
Journal of Pharmacognosy and Phytochemistry, 2:181-188.
SCHMITZ-EIBERGER, M. A., BLANKE, M. M. (2012): Bioactive components in forced sweet
cherry fruit (Prunus avium L.), antioxidative capacity and allergenic potential as dependent on
cultivation under cover. LWT – Food Science and Technology, 46, 388–392.
SHARMA V., RAMAWAT K. G. (2013): Salinity-induced modulation of growth and
antioxidant activity in the callus cultures of miswak (Salvadora persica). Biotech. 3 (1), 11–17.
SERRADILLA, M. J., HERNÁNDEZ, A., LÓPEZ-CORRALES, M., RUIZ-MOYANO, S., DE
GUÍA CÓRDOBA, M., & MARTÍN, A. (2016):Composition of the Cherry ( Prunus avium L.
and Prunus cerasus L.; Rosaceae). Nutritional Composition of Fruit Cultivars, 127–147
VALENTÃO P., FERNANDES E., CARVALHO F., ANDRADE P.B., SEABRA R.M.,
BASTOS M.L., (2002): Antioxidative properties of cardoon (Cynara cardunculus L.) infusion
against superoxide radical, hydroxyl radical, and hypochlorus acid.Journal of Agricultural and
Food Chemistry, 50:4989-4993.
140
THE CHROMATOGRAPHIC ANALYSIS OF THE STAR ANISE ESSENTIAL OIL AS
THE POTENTIAL BIOPESTICIDE
Tijana Stojanović,
third year, doctoral studies, Ministry scholarship holder
Mentors: Vojislava Bursić1: PhD, associate professor, Gorica Vuković2: PhD, research
assistant
1
University of Novi Sad, Faculty of Agriculture, Department of Plant and Environmental
Protection, Serbia
2
Institute of Public Health of Belgrade, Bulevar despota Stefana 54A, Belgrade, Serbia
Summary: Since the main components of essential oils are considered responsible for
their biological activity the chromatographic analysis of star anise essential oil was done.
The identification of the essential oil compounds was carried out by matching their linear
retention indices and MS spectra with those of authentic standards from MS library data
bases. The main constituent was trans-anethole with 84.79% while 24 other constituents in
total make less than 15.00% of the studied essential oil.
Key words: biological activity, star anise, essential oil, trans-anethole, GC-MS
INTRODUCTION
The attention drawn by essential oils due to their biological effect as potential agents in pest
control is evidently growing from year to year. They are considered the by-products of plant
metabolism and regarded to as evaporable secondary metabolites of plants which are the mixture
of mono and sesquiterpenes (Stojanović et al., 2018). The biological activity of essential oils
depends on their chemical composition, the part of the plant they have been extracted from,
phenological state of the plant, environmental conditions and the extraction methods (Ukeh and
Umoetok, 2011.). A large number of isolated allelochemicals show their bioactivity in low (10 –5–
10–6 mol/dm3) or extremely low concentrations (10–10 mol/dm3) (Šućur, 2015.).
The star anise (Figure 1) is an evergreen aromatic plant from Schisandraceae family native to
southwest China. It is widely cultivated in tropical and subtropical regions and is used as food as
well as a medicine, which indicates small or no toxic effect on humans (Zhou et al., 2016). The
essential oil of star anise is applied locally for rheumatism and externaly after childbirth, it is also
used as an antiseptic, as well as for the treatment of emesis, stomach ache, pain and insomnia.
The content of essential oil in fresh fruits is 2.5-3.5% while in case of dried material is between 8
and 9 percent (Wang et al., 2011).
141
Figure 1. The plant (а) and the fruit (b) of star anise
(a) www.toxicologycentre.com, (b) original photo (Т. Stojanović)
Since the main components of essential oils are considered responsible for their biological
activity the objective was to carry out the chromatographic analysis of star anise essential oil
obtained in the distillation by water vapor.
The essential oil of star anise was extracted from commercially available fruit (fructus) by
hydrodestilation (HD) with n-hexane as an organic solvent/recipient. Thirty grams of star anise
fruit were subjected to hydrodestilation for 3 hours using a Clevenger-type apparatus according to
the method outlined by the European Pharmacopoeia (Council of Europe 2010). The essential oil
was collected over water, separated, dried over anhydrous sodium sulphate and stored in the dark
at 4 ºC.
The identification of the essential oil compounds was carried out by matching their linear
retention indices (relative to C8-C36 n-alkanes on the HP-5MSI column) and MS spectra with
those of authentic standards from NIST11 (2011) and homemade MS library data bases (Petrović
et al., in press).
142
From the obtained results it can be concluded that the main constituent of the star anise
essential oil is trans-anethole with 84.79% in content, which can be evidently seen in the
chromatogram (Figure 2). Beside trans-anethole there were 24 constituents which in total make
less than 15.00% of the studied essential oil (Table 1).
Figure 2. Chromatogram of the essential oil of star anise
143
Table 1. Constituents of the star anise essential oil
RT/ RT/ %,
No Constituents KIE KIL Area % ID RRT CI
MS FID m/m
1 a-Pinene 927,0 932 6,58 10,776 368,2 0,76 0,76 0,437 9
2 Camphene 941,3 946 7,00 11,295 15,1 0,03 0,03 0,458 0
3 b-Pinene 970,1 974 7,87 12,301 38,1 0,08 0,08 0,499 1
4 Myrcene 991,6 988 8,58 12,763 72,5 0,15 0,15 0,517 2
a-
5 1001,0 1002 8,80 13,283 94,6 0,20 0,20 0,538 2
Phellandrene
6 d3-Carene 1005,1 1008 8,94 13,504 331,7 0,69 0,69 0,547 8
7 a-Terpinene 1013,1 1014 9,21 13,741 51,2 0,11 0,11 0,557 1
8 p-Cymene 1020,4 1020 9,46 14,067 343,9 0,71 0,71 0,570 8
9 Limonene 1023,5 1024 9,56 14,257 2975,6 6,18 6,18 0,578 73
10 1,8-Cineole 1024,9 1026 9,61 14,340 468,5 0,97 0,97 0,581 11
11 g-Terpinene 1056,5 1054 10,71 15,346 61,8 0,13 0,13 0,622 2
12 n.i. 1071,8 11,22 15,922 17,1 0,04 0,645 0
13 Terpinolene 1086,3 1086 11,72 16,506 88,1 0,18 0,18 0,669 2
14 Linalool 1099,3 1095 12,16 16,990 540,4 1,12 1,12 0,689 13
15 Terpinen-4-ol 1173,4 1174 14,73 20,038 179,5 0,37 0,37 0,812 4
16 a-Terpineol 1187,9 1186 15,24 20,572 192,1 0,40 0,40 0,834 5
17 Estragole 1198,7 1195 15,62 20,817 511,1 1,06 1,06 0,844 13
18 cis-Anethole 1255,3 1249 17,56 22,882 235,1 0,49 0,49 0,927 6
p-Anis
19 1261,3 1251 17,74 23,254 524,1 1,09 1,09 0,942 13
aldehyde
trans-
20 1288,4 1282 18,65 24,673 40843,0 84,79 84,79 1,000 1000
Anethole
21 a-Copaene 1366,7 1374 21,20 27,359 33,1 0,07 0,07 1,109 1
cis-a-
22 1406,3 1411 22,48 28,632 41,0 0,09 0,09 1,160 1
bergamotene
trans-
23 caryophyllen 1408,5 1417 22,54 28,915 25,8 0,05 0,05 1,172 1
e
trans-a-
24 1427,0 1432 23,11 29,324 91,8 0,19 0,19 1,188 2
Bergamotene
trans-b-
25 1449,8 1454 23,82 30,104 25,1 0,05 0,05 1,220 1
Farnesene
48168,6 100,00 99,96
144
CONCLUSION
Based on the chromatographic analysis of the star anise essential oil obtained in the
distillation by water vapor it can be concluded as follows:
- The main constituent of the star anise essential oil is trans-anethole with 84.79% in content.
- Beside trans-anethole there were 24 constituents which in total make less than 15.00% of
the studied essential oil.
A number of studies led to the similar findings. Zhou et al. (2016) also identified trans-
anethole as the main constituent of the star anise essential oil. The same group of authors stated
that numerous studies showed the insecticidal effect of trans-anethole in case of German
cockroach (Blattella germanica), Brown-tail moth (Euproctis chrysorrhoea) and the
Mediterranean fruit fly (Ceratitis capitata). Kim and Ahn (2001) determined the trans-anethole
toxic activity towards the adults of rice weevil (Sitophilus oryzae) and cigarette beetle
(Lasioderma serricorne). In the studies conducted by Lopez et al. (2008) and Ebadollahi (2013)
trans-anethole showed the toxic effect against the lesser grain borer (Rhyzopertha dominica).
Considering the literature data essential oil of star anise has a potential as a biopesticide.
Based on the findings of this study and the assumption that the main components affect the
biological activity of essential oil trans-anethole (propenylphenol synthesized via the shikimic
acid-phenylpropanoid pathways) is considered responsible for biological activity of the star anise
essential oil and its biopesticide potential.
Acknowledgements
The authors would like to thank Jovana Šućur, PhD for the extraction of the star anise
essential oil and the Ministry of Education, Science and Technological Development for the
financial support (Project TR 31027).
REFERENCES
COUNCIL OF EUROPE (2010): European Pharmacopoeia. 7th edn. Strasbourg, France,

European Directorate for the Quaility of Medicines and Healthcare: 1090-1091.
EBADOLLAHI, A. (2013): Plant Essential Oils from Apiaceae Family as Alternatives to
Conventional Insecticides. Ecologia Balkanica, 5(1): 151.
KIM, D. H., AHN, Y. J. (2001): Contact and fumigant activities of constituents of Foeniculum
vulgare fruit against three coleopteran stored-product insects. Pest Management Science, 57: 303-
305.
PETROVIĆ, M., POPOVIĆ, A., KOJIĆ D., ŠUĆUR, J., BURSIĆ, V., AĆIMOVIĆ, M.,
MALENČIĆ, Đ., STOJANOVIĆ, T., VUKOVIĆ, G.: Assesment of toxicity and biochemical
response of Tenebrio molitor and Tribolium confusum exposed to Carum carvi essential oil. In
press.
145
STOJANOVIĆ, T., TEŠEVIĆ, V., BURSIĆ, V., VUKOVIĆ, G., ŠUĆUR, J., POPOVIĆ, A.,
PETROVIĆ, M. (2018): The study of dill essential oil chemical composition. 11th International
scientific/professional conference “Agriculture in nature and environment protection“, Vukovar
28-30 May, Proceedings and book of abstracts: 181.
ŠUĆUR, J. (2015): Biopesticide activity of the extracts of self-seeding plants of Lamiaceae
family. Doctoral thesis, Faculty of Sciences, Department of Chemistry, Biochemistry and
Environmental Protection, University of Novi Sad: 36.
UKEH, D. A., UMOETOK, S. B. A. (2011): Repellent effects of five monoterpenoid odours
against Tribolium castaneum (Herbst) and Rhyzopertha dominica (F.) in Calabar, Nigeria. Crop
Protection 30: 1352.
WANG, G. W., HU, W. T., HUANG, B. K., QIN, L. P. (2011): Illicium verum: A review on its
botany, traditional use, chemistry and pharmacology. Journal of Ethnopharmacology 136: 11-13.
ZHOU, B. G., WANG, S., DOU, T. T., LIU, S., LI, M. Y., HUA, R. M., LI, S. G., LIN, H. F.
(2016): Aphicidal Activity of Illicium verum Fruit Extracts and Their Effects on the
Acetylcholinesterase and Glutathione S-transferases Activities in Myzus persicae
(Hemiptera:Aphididae). Journal of Insect Science, 16(1): 2, 4.
LOPEZ, M. D., JORDAN, M. J., PASCUAL-VILLALOBOS, M. J. (2008): Toxic compounds in
essential oils of coriander, caraway and basil active against stored rice pests. Journal of Stored
Products Research, 44: 277.
146
SENSITIVITY OF CERCOSPORA BETICOLA ISOLATES TO AZOXYSTROBIN
Milijanka Balandžić
Mentor: Dragana Budakov, assistant professor
University of Novi Sad, Faculty of Agriculture, Departmant of Environmental and Plant

Protection, Serbia
Summary: Cercospora beticola Sacc. is causing leaf spot disease. Damages from this pathogen
in the sugar beet crop are due to decay of leaf mass and retrovegetation, which leads to a
decrease in yield and sugar content. Azoxystrobin belongs to the group of strobilurins, they
inhibit mitochondrial respiration by blocking cyt.c reductase. Group of strobilurins posses a very
high risk of resistince which results in loss of biological activity of fungicides. Foliar
applications of fungicides are extremely valuable for successful control of CLS.
INTRODUCTION
Sugar beet leaf spot, caused by the phytopathogenic fungus Cercospora beticola Sacc., is
economically the most significant and most destructive sugar beet leaf disease (Holtschulte,
2000; Budakov, 2014). It becomes more severe with avaliability of abundance of hosts, not only
sugar beet, but also vaious plants from the genera Chenopodium, Atriplex, Amaranthus,
Polygonum, Lactuca, Medicago, Glycine, Taraxacum, Malva, Spinacia, Limonium, Apium, etc.
(Ruppel, 1986; Weiland and Koch, 2004; Lartey et al., 2010), as well as with favourable
environmental conditions such as warm and humid weather during vegetative period after sugar
beet closes rows (Budakov et al., 2014).
The symptoms of disease appear firstly on older leaves as individual spots ranging from 3-5
mm in diameter (Jacobsen and Franc 2009). Tissue within lesion is tan to light brown with brown
or reddish purple margins. Necrotic area increases with appearance of new lesions which
evetually lead to complete leaf necrosis and regrowth of new leaves using already accumulated
sugar in sugar beet root (Weiland and Koch, 2004). Control of C. beticola within integrated
disease management comprises agrotechnical, cultural, biological, but also unavoidable fungicide
applications, which are being used 2-5 times each year in our environmental conditions (Budakov
et al., 2014). Since C. beticola is polycyclic parasite, each year it creates a large number of spores
that are exposed to fungicide activity several times during the vegetation. The most effective
fungicides against this disease are various systemic active ingredients, which in most cases have
specific mode of action, i.e. they target one site in fungal metabolism, which is why they are
called site-specific fungicides. According to FRAC (Fungicide Resistant Action Committee),
these fungicides possess a very high risk of resistance which results in loss of biological activity
of fungicides (Brent and Hollomon, 2007). Resistance to fungicides can be defined as a stable,
147
inheritable adaptation of a pathogen that results in a reduced sensitivity to a fungicide that allows
individuals within the population to survive the treatment with plant protection products, which
in normal conditions should result in the suppression of the harmful organism (Gallian et al.,
2001). One of fungicides with a high risk of resistance is azoxystrobin, belonging to the group of
strobilurins, which acts as inhibitor of mitochondrial respiration by blocking the transport of
electrons between cytochrome b and cytochrome c1 and inhibiting cytochrome c reductase. First
case of resistance to strobilurins among field population of C. beticola was recorded in 2011 in
the United States (Kirk et al., 2012) after failing to control CLS in sugar beet fields treated with
strobilurins over several years of application. In Serbia, resistance to stroblurins is also present
and described by Trkulja et al. (2017). When it comes to in vitro testing of susceptibility of C.
beticola to strobilurin fungicides, applied methods are based on measurement of the inhibition of
spore germination (Secor et al., 2010) or determining the reduction of mycelial growth
(Malandrakis et al., 2011). The fungus in the presence of strobilurin have the ability to use of
alternative oxidase (AOX), which enables sensitive isolates to show mycelial growth on the
medium amended with fungicide. Studies of AOX importance in strobilurin resistance have used
salicylhydroxamic acid (SHAM) to inhibit the activity of AOX during strobilurin resistance
testing (Avila-Adame and Köller 2002, 2003). By inhibiting AOX, resistance to strobilurins
becomes dependent on mutations in the cytochrome b gene, which as a consequence inhibits the
mycelial growth of sensitive isolates, and thus reveals only the resistance that is caused by
mutations in the cytochrome b (Larson, 2004).
Since one of the most important factors in the antiresistant strategy includes monitoring of the
sensitivity changes of fungus populations to various fungicides (Brent and Hollomon, 2007), the
aim of this work was to determine level of resistance of a large number of Cercospora beticola
isolates from one sugar beet field to azoxystrobin.
For the purpose of this research, from the trial at the Institute of field and vegetable crops,
Rimski Šančevi, sugar beet leaves with typical symptoms of leaf spot caused by Cercospora
beticola were collected. Sampling was carried out in June 2017, before the first application of
fungicide in order to evaluate not only level of resistance of individual isolates, but also
frequency of resistance to azoxystrobin on this site.
The leaves were then taken to the phytopathological laboratory (University of Novi Sad,
Faculty of Agriculture, Department of Environmental and Plant Protection), after which
fragments with characteristic spots caused by C. beticola were taken for the further research.
They were placed in a Petri dish with a moist filter paper on the microscope slides and incubated
at 25 °C for 24-36 hours in order to induce sporulation. Single lesion isolations of the parasite
were performed using a sterile entomological needle by transferring one or more conidia from
one lesion to the PDA (Potato Dextrose Agar).
148
Level of sensitivity of tested isolates was determined on the basis of the methods described by
Budakov (2014) where discriminative concentration of tested fungicide was used for sensitivity
testing (Karaoglanidis et al., 2003). Discriminative concentration is a concentration of a fungicide
at which the tested isolates are clearly divided in two groups: the ones whose growth was
inhibited and those not inhibited by the presence of the fungicide in the medium (Jo et al., 2006).
Discriminative concentration of 0.1 μg azoxystrobin /ml was already determined before
(Budakov, 2014) by testing reference isolates of C. beticola which were isolated from the
beetroot in organic production, where pathogen population has a baseline sensitivity since tested
fungicide was never used on this location (results not shown). In sterile conditions and in a sterile
cooled medium, a solution of technically pure azoxystrobin in methanol was added in an amount
that will provide a concentration of 0.1 μg /ml. Salicylhydroxamic acid (SHAM) at a
concentration of 0.5 mM was added in order to inhibit the activity of an alternative oxidase. As a
negative control, the PDA with the addition of SHAM, without fungicide, was used. On these
prepared plates, fragments of mycelium (5 mm diameter) were placed on the surface of the agar
with mycelia facing down in order to make the contact with the fungicide.
The isolates were incubated in an incubator at 25 °C for 6 days. Evaluation of sensitivity was
performed by calculating an average diameter of a colony based on measurements of two normal
diameters of mycelium. The obtained data were used to calculate the relative growth in the
mycelium for each isolate using the formula:
RP(%) = x 100
Wherein:
- RP is a relative growth of mycelium,
- A is the average mycelium growth on media with the addition of fungicides and SHAM,
- B is the average mycelium growth on media with SHAM, but without fungicide (negative
control).
Based on the relative growth of the mycelium, all isolates were divided into four groups:
1. Sensitive (RP <20%)
2. Decreased sensitivity (RP = 20-39.9%)
3. Moderately resistant (RP = 40-69.9%)
4. Highly resistant (RP ≥ 70%)
149
RESULTS
A total of 84 isolates of Cercospora beticola from locality Rimski Šančevi were selected for
sensitivity testing to azoxystrobin (0.1 μg/ml). The results are shown in Table 1.
Table 1. Frequency of isolates with different sensitivity

Percent
Range of Number
Sensitivity level of Isolate codes
sensitivity level of isolates
Isolates
Sensitive / / / /
Decreased 3
20.89-31.52 4% 15RŠ9, 19RŠ6, 15G2
sensitivity
15G7,15RŠ8, 14G3,18RŠ1, 18G2,18RŠ2,
Moderatly 15G4,15RŠ5, 13RŠ5,13RŠ6, 20RŠ2,19RŠ5,
40.00-69.69 22 26%
resistant 14RŠ5,19RŠ12, 19RŠ9,15RŠ4,
18G1,15RŠ10, 14RŠ7,13G3, 13RŠ2, 15RŠ3
20G1, 18G3, 15RŠ6, 18G4, 14G7,
13RŠ4,19RŠ7, 14G4, 19G3, 13RŠ3, 18G5,
14G8, 15G8, 19Rš4, 15RŠ13, 18RŠ4,
15RŠ12, 20G2, 18RŠ3, 20RŠ1, 19G2,
14RŠ2, 18G7, 19G1, 15G1, 15RŠ11, 18G8,
Highly resistant 70.12-275.00 59 70% 19G5, 15G6, 19RŠ3, 18RŠ5, 15RŠ, 13G8,
13G7, 19RŠ8, 19RŠ1, 18G6, 14G2, 15G5,
13G2, 19G6, 14RŠ3, 14RŠ6, 14G6, 14G1,
14RŠ4, 19RŠ10, 13RŠ1, 14G5, 14RŠ1,
19G4, 13G5, 15G3, 19RŠ2, 19RŠ11, 13G6,
13G1, 15RŠ7, 13G4
Results showed that none of the tested isolates was sensitive to azoxystrobin (RP <20%), 4%
isolates showed reduced sensitivity, while resistance showed a total of 96% isolates out of which
26% were moderately resistant (RP = 40-69.9%) and 70% were highly resistant (RP ≥ 70%).
The results of determination the sensitivity of Cercospora beticola isolates to azoxystrobin
indicate a high percentage of the resistance to this fungicide, which inevitably lead to the
complication of sugar beet protection since this fungicide cannot successfully control sugar beet
leaf spot. These results should help introducing new strategies to prevent the occurrence of
resistance in order to successfully control the sugar beet leaf spot caused by Cercospora beticola.
150
CONCLUSION
This study indicates the importance of monitoring changes in the sensitivity of Cercospora
beticola populations to the dominant fungicide used in our sugar beet growing area.
Since systemic fungicide that is used for CLS control is, according to FRAC, possess medium
to high risk for the emergence of resistance, it can be expected that in the future a change in the
composition of the parasite popilation occurs, i.e. to increase the proportion of resistant isolates.
Their predominance in the field population can lead to significant losses in sugar beet yield and
sugar content, but also to major economic losses due to fungicide application in which lacks the
necessary biological efficacy.Their predominance in the field population can lead to significant
losses in sugar. In order to examine the level of sensitivity of the population of Cercospora
beticol to the active substance azoxystrobin, results were obtained on the basis of which the
following conclusions can be drawn:
In the tested isolates for the use of azoxystrobin at a concentration of 0.1 μg / ml with the
addition of 0.5 mM salicylhydroxamic acid, there were no susceptible isolates, with 4% isolates
with reduced sensitivity, 26% of moderately resistant isolates, while highly resistant 70 %.
The obtained data indicate a higher proportion of resistant than sensitive isolates, as well as
the importance of monitoring the changes in the sensitivity of the population of Cercospora
beticola.
The dominance of this parasite can lead to significant economies due to the application of
fungicide, which can lead to large losses in the yield of sugar beets.
REFERENCES
AVILA-ADAME, C., KÖLLER, W. (2002): Disruption of the alternative oxidase gene in

Magnaporthe grisea and its impact on host infection. Molecular plant-microbe interactions, 15(5),
493-500.
AVILA-ADAME, C., KÖLLER, W. (2003): Characterization of spontaneous mutants of
Magnaporthe grisea expressing stable resistance to the Qo-inhibiting fungicide azoxystrobin.
Current genetics, 42(6), 332-338.
BRENT, K.J., HOLLOMON, D.J. (2007A): Fungicide resistance: the assassement of risk. FRAC
Monograph No.2. Brussels, Belgium: Fungicide Resistance Action Committee.
BRENT, K.J., HOLLOMON, D.J. (2007B): Fungicide resistance in crop pathogens: How can it
be managed. FRAC Monograph No.1. Brussels, Belgium: Fungicide Resistance Action
Committee.
BUDAKOV D. (2014): Sensitivity of Cercospora beticola (Sacc.) the causer of sugar beet leaf
spot to fungicides.
GALLIAN, J.J., NOLTE, P., MILLER, J.S. (2001): Managing fungicide resistance. University of
Idaho.
151
HOLTSCHULTE, B., ASHER, M. J. C., MOLARD, M. R., ROSSO, F., STEINRÜCKEN, G.,
BECKERS, R. (2000): Cercospora beticola-worldwide distribution and incidence. Cercospora
beticola Sacc. biology, agronomic influence and control measures in sugar beet., 5-16.
JACOBSEN, B., FRANC, G. (2009): Cercospora leaf spot. In: Harveson RM, Hanson LE, Hein
GL, eds. Compendium of beet diseases and pests. St. Paul, Minnesota, USA: APS Press, 7-10.
KIRK, W.W., HANSON. L.E., FRANC, G.D., STUMP, W.L., GACHANGO, E.N., CLARK, G.,
STEWART, J. (2012): First report of strobilurin resistance in Cercospora beticola in sugar beet
(Beta vulgaris) in Michigan and Nebraska, USA. New Disease Reports. 26, 3
LARSON, J.B. (2004): Integrated menagement of Cercospora leaf spot on sugar beet. Master of
Science thesis. Montana State University.
LARTEY, R.T., WEILAND, J.J., PANELLA, L. (2010): A Brief History of Cercospora Leaf
Spot. In: Lartey R, Weiland J, Panella L, Crous P, Windels C, eds. Cercospora leaf spot of sugar
beet and related species. St. Paul, Minnesota, USA: APS Press.
MALANDRAKIS, A.A., MARKOGLOU, A.N., NIKOU, D.C., VONTAS, J.G., ZIOGAS, B.N.
(2011): Molecular diagnostic for detecting the cytochrome b G143S–QoI resistance mutation in
Cercospora beticola. Pesticide biochemistry and physiology, 100(1), 87-92.
RUPPEL, E.G. (1986): Cercospora leaf spot. Whitney and JE Duffus, eds. American
Phytopathological Society, St. Paul, MN, 8-9.
SECOR, G.A., RIVERA-VARAS, V.V., GUDMESTAD, N.C., WEILAND, J.J. (2010):
Sensitivity of Cercospora beticola to foliar fungicides in the Red River Valley of North Dakota
and Minnesota. In: Lartey R, Weiland J, Panella L, Crous P, Windels C, eds. Cercospora leaf spot
of sugar beet and related species. St. Paul, Minnesota, USA: APS Press, 213-22.
TRKULJA, N., MILOSAVLJEVIĆ, A., MITROVIĆ, M., JOVIĆ, J. TOŠEVSKI, I., KHAN, M.,
SECOR, G. (2017): Molecular and experimental evidence of multi-resistance of Cercospora
beticola field populations to MBC, DMI and QoI fungicides. European Journal of Plant
Pathology 149, 895-910
152
THE GENETIC INTERACTION BETWEEN SUNFLOWER LINS IN THE PROCESS
OF DEVELOPING OROBANCHE.CUMANA RESISTANCE HYBRIDS
ONISAN GRIGORE-EMILIAN
MENTOR: PETRESCU IRINA

irinuta@hotmail.com
BANAT UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY MEDICINE
TIMISOARA King Michel I of România
Summary: Orobanche.cumana causes important economic damage in sunflower production in a

number of countries around the world, causing more then 70% damage in production for
susceptible hybrids Breeding programs are getting more intense due to the evolution and
phenomenal adaptation of broomrape, any information about this genomic battlefield between
Orobanche.cumana and sunflower will contribute to a higher efficiency in obtaining resistant
hybrids which makes collaborations between public research departments and private
companies crucial. . We develop lines and tested them to 0robanche.cumana in 5 different
location in Romania since 2013 and also 1 location in Bulgaria and 1 in Spain in 2018 .
Our main goal is to complete a part of the picture of how the interaction of different combination
of sunflower female lines and restoring male lines with different genetic backgrounds influence
the capability of the hybrids to obtain resistance to different Orobanche.cumana races and to
present some genetically conclusion that happen in our testing and breeding programs along
this 6 years.
Key words: Sunflower, Orobanche.cumana , genes interaction, female line ,restorer
INTRODUCTION
The are a large number of objective when it comes to sunflower breading, resistance to
herbicide , resistance to disease and genetically distance between female line and restorer line
may be one of the most important keys when it comes to developing performant sunflower
hybrids. One of the biggest issue of sunflower hybrids is Orobanche Cumana , representing up
to 90% of yield losses for the susceptible hybrids (Alcántara et al, 2006; Duca, 2015).
In Romania , it was first discovered by Savulescu si colab.(1928-1960) in the year 1940-
1941 quoted by Dumitras and Sesan (1988). The highest intensity and frequency is found in the
southern part of Moldova, Dobrogea and the eastern part of Baragan's field. It is a substantial
threat in Europe, especially in countries around the Black Sea and in Spain (Molinero-Ruiz et al.,
2013) , it is also a threat outside of Europe , one of the regions of great intensity is Jilin ,a
province from China with over 100 strains of Orobanche.cumana on a single sunflower plant in
some cases .(Tingrui si colab.,1996). In order to control this parasite there are several methods
but there still remains an extremely difficult problem in sunflower breading ,because of the very
fast development of this parasite and a rapid appearance of new and more virulent races
(Fernandez Martinez et al., 2008) , thousands of seeds are produced by a single broomrape
plant which gives a very high advantage for the Orobanche.cumana because of the mutation that
could appear and lead to different variation .
153
One of the best way to eliminate the threat of Orobanche.cumana in the field is to do
programs of breading for genetic resistance, lately it has become more and more difficult,
because of the interaction of the female line with the genes form the restore line must fit
perfectly in order to give resistance to Orobanche.cumana. The gene-for-gene model (V r â n c e
a n u et al., 1980) and different other authors agreed with monogenic and dominant inheritance
of resistance to sunflower broomrape (P o g o r l e t s k y and G e s h e l e, 1976; S u k n o et al.,
1999),but there are also reported two dominant genes (D o m i n g u e z, 1996b) , one recessive
gene (R a m a i a h, 1987) ,double dominant epistasis and dominant-recessive epistasis (B.
Akhtouch 2008),coming with a concludent conclusion that resistance must be incorporated into
both parental lines for developing resistant hybrid . There are results with different crosses
between different race F resistant lines and different susceptible parental lines, that shown that
dominance relationships and genetic control of broomrape resistance in sunflower is highly
dependent on the race of broomrape, the source of resistance and also, the susceptible parental
line used for the cross (P e r e z - V i c k et al., 2004a). It is very clearly that things started to
complicate after Orobacnhe.cumana appear more and more aggressive in the fields requiring an
stabile and complex genetic background of the sunflower lines in order to obtain resistance to
Orobanche.cumana.
We tested several hundred combinations and evaluated them in Romania, Bulgaria and Spain
and the results seems to indicate that genes interaction between different lines seems to lower the
possibility in obtaining resistance hybrids if it is not done with a suitable partner.
MATERIAL AND METHOD
Since the breading programs started we focused especially on developing A-lines and
restorer-lines that are resistant to different races of Orobanche cumana, we manage to develop
lines that show resistance and tolerance up to race G .
The new combination where first tested for the yield in 3 location in Romania and 3 of Hungary
, after that all the best combination where then tested in fields, with naturally infestation, 4
location in Romania , 1 location in Bulgaria and 1 location in Spain . In order for us to have a
better indication of the level of Orobanche.cumana we used commercial hybrids in order for us
to have a better vision and indication of the broomrape race that is present .We also tested our
material in the green house (artificial infestation conditions).
Plants are grown in soil infested with a known race and concentration of broomrape
seeds in plants pots of 5 kg and size 30 X 30 X 7 cm .Mixing soil, sand and broomrape
seed – 3 part soil X 1 part sand X 1 gr orobanche seeds. Sand has to be quartz because
the particles have a different colour to broomrape and the soil has to be sterilized to be
sure that no broomrape seed comes with it. Broomrape populations have always a mix of
races with one being dominant. In the mix there may be new races capable to infect any
new resistant source. The amount of broomrape seed is calculated according to the
volume of soil/sand mix required. The concentration of seed depends on its viability. Fill
the pots and label. Make a hole 2 cm deep and place the seeds in the hole, 1 per pot.
Close the holes and place the pots in greenhouse the following parameters.
- Day; 14 hours 24°C
- Night; 10 hours 20°C
- Humidity – 50 – 80 %
- Temperature :day 24ºC +2ºC ; night 22ºC +2ºC
154
The amount of water required will depend on the exact conditions and soil that you have.
Regular watering is recommended so that the plants don’t wilt but are not standing in water for
long periods. For each test hybrid, there will be 2 replicate trays with 20 plants. In every pots, the
1-st line is susceptible standard.After 45 days the soil is removed from the roots and the number
of broomrapes per plant is recorded. The test is suitable for ranking susceptibility of hybrids and
lines. Record the number plants/infected.
Our main objective is to create lines that are resistant to different races of
Orobanche.cumana and to develop an efficient strategy in creating and developing hybrids.
Table 1. Resistance to race G for A-1996 and Rf lines used to form hybrids
Green House (Race G Bulgaria)

Nr/susceptible
Genotype Nr/plants %
plants
A-1996 100 10 90%
Rf-101 100 98 2%
Rf-102 100 89 11%
Rf-103 100 89 11%
Rf-104 100 95 5%
Rf-105 100 90 10%
As the mother line we used A-1992 obtained by using Spain germplasm sources which is
resistant to race G from Bulgaria(Table1) . From the 100 new combination with different restorer
lines, approximately 5 hybrids where picked according to the phenotypical aspects and to the
yield performance from several location in Romania and Hungary.
We tested approximately 500 plants from each genotype that sowed very good performance, we
evaluated every genotype with a scale form 1 to 9 .One of the hybrids that sowed good tolerance
is A1992 X Rf 104 as you can see in (table 2). The hybrid A1992 X Rf 105 is significantly
different form all the others hybrids ,having in Spain all the plants attacked .In the experiment
from Spain the origin of the line A-1992 is confirmed , all the material except the hybrid that
resulted as susceptible are resistant to races form Spania . However, in other regions from
Romania and Bulgaria the hybrids weren’t so constant regarding the number of plants that where
susceptible.
155
Table 2. Orobanche.cumana result for all location tested in 2018.
Nr (G) % of
Topraisar Sevilia Radnevo
Genotype Drajna Green- plants
Rep 1 Spania Bulgaria
House susceptible
A1992 X Rf
8
1 101 3 1 3 3 37%
A1992 X Rf
8
2 102 5 1 5 3 44%
A1992 X Rf
6
3 103 3 1 1/2 1 30%
A1992 X
4
4 Rf104 1 1 1 1 21%
A1992 X Rf
9
5 105 7 7 6 7 80%
Check
2
6 HYBRID 1 1 1 1 7%
There are no major differences between hybrid A1992 X Rf 101 and A1992 X Rf 102
both of them showing similar percentages of susceptible plant . Also, between A1992 X 103 and
A1992 X 104 there is not a significant difference . Our check hybrid is on of the best on the
market for Orobanche.cumana sowing resistance to all the races in all location having only 7 %
plant that are susceptible .
We tested several other combination in this experiment but only mother A-line 1992
sowed significant difference between restorer lines. As you can see in Fig.1 it is very clear that
the most susceptible hybrid is very distant from other hybrids regarding percentage indicating
that the resistance of the line A-1992 had no effect in combination with the restorer line Rf 105.
120 A1992 X Rf 101 A1992 X Rf 102

A1992 X Rf 103 A1992 X Rf104
100 A1992 X Rf 105 100
90 90 90
80 80
70 70
60 60
50
40 40
37
30 31
30 30
20
12 10 10
0 1 1
Fig. 1 Percentage of susceptible plants
156
We tested for yield performance for this year 2018 in 4 location in Romania for
all the 5 hybrids Fig.2 . We also wanted to see the capacity of the hybrids in
different location but also in places where it is a very high intensity of
Orobanche.cumana . We selected location near Black sea in Cogealac . As you
can see in the graphic significant different is only for line A1992 X Rf 105,
indicating that the yield capacity was most affected for this hybrid in particular
.
4500
4201 4123
4120
4000 3907
3895 4021 3964
3904
3888 3943 4021 3896
3865
3754 3795 3756 3843
3609 3567 3654 3674
3643
3543
3500
3000 3100
2500
2000
1500
1000
500
0
A1992 X Rf A1992 X Rf A1992 X Rf A1992 X A1992 X Rf Mt
101 102 103 Rf104 105
Rm.Sarat Carpinis Negru Voda Constanta
Fig .2 Yield production in normal condition and Orobanche.cumana natural infestation
More costant results where obtained for the othere 4 hybrids ,remaning with a clear
conclusion that the combination of A1992 X Rf105 is susceptible .
CONCLUSION
Depending on the genetical interaction between the inbreds lines it can lead to susceptible
hybrids or to very tolerant hybrids .
Nomenclature of the races differs from one region to another finding also susceptible
plant on the commercial hybrids. Mother line - A1992 showed with in all of the 4 hybrids,except
the susceptible one, resistance in Spain. For the othere location there was a prercent of
susceptible plants that needs to noticed.
New strategies and better understanding of how gene interactions between lines work
must be develop in order to obtain maximum efficiency in the process of developing resistance
hybrids.
REFERENCES
1. Aly R., Cholakh H., Joel D. M., Leibman D., Steinitz B., Zelcer A., Et Al. (2009). Gene
silencing of mannose 6-phosphate reductase in the parasitic weed Orobanche aegyptiaca through
the production of homologous dsRNA sequences in the host plant.
157
2. Arcade A., Labourdette A.,Falque M., Mangin B.,Chardon F., Charcosset A., Et Al. (2004)
BioMercator: integrating genetic maps and QTL towards discovery of candidate genes.
3. Clark R. T., Famoso A. N., Zhao K., Shaff J. E., Craft E. J., Bustamante C. D., Et Al. (2013).
High-throughput two-dimensional root system phenotyping platform facilitates genetic analysis
of root growth and development: root phenotyping platform.
4. Cook C. E., Whichard L. P., Turner B., Wall M. E., Egley G. H. (1966). Germination of
witchweed (Striga lutea lour.): isolation and properties of a potent stimulant.
5. De Givry S., Bouchez M., Chabrier P., Milan D., Schiex T. (2005). Carhta Gene:
multipopulation integrated genetic and radiation hybrid mapping.
6. Eizenberg H. (2003) Resistance to broomrape (Orobanche spp.) in sunflower (Helianthus
annuus L.) is temperature dependent. J. Exp. Bot. 54 1305–1311. 10.1093/jxb/erg129
7. Fernández-Aparicio M., Flores F., Rubiales D. (2009A) Recognition of root exudates by seeds of
broomrape.
8. Fernández-Aparicio M., Flores F., Rubiales D. (2009B) Field response of Lathyrus cicera
germplasm to crenate broomrape (Orobanche crenata).
9. Fernández-Aparicio M., Flores F., Rubiales D. (2012a). Escape and true resistance to crenate
broomrape (Orobanche crenata Forsk.) in grass pea (Lathyrus sativus L.) germplasm. Field
Crops Res. 125 92–97. 10.1016/j.fcr.2011.09.003
10. Fernández-Aparicio M., Flores F., Rubiales D. (2009A). Low strigolactone root exudation: a
novel mechanism of broomrape (Orobanche and Phelipanche spp.) resistance available for faba
bean breeding.
11. Fernandez-Aparicio M., Flores F., Rubiales D. (2012A). Resistance against broomrapes
(Orobanche and Phelipanche spp.) in faba bean (Vicia faba) based in low induction of
broomrape seed germination.
12. Fernández-Aparicio M., Flores F., Rubiales D. (2009B) Update on breeding for resistance to
sunflower broomrape.
13. Fernandez-Martinez J. M., Pérez-Vich B., Akhtouch B., Velasco L., Muñoz-Ruz J., Melero-Vara
J. M., Et Al. (2004) Registration of four sunflower germplasms resistant to race F of broomrape.
158
THE EFFECT OF SODIUM CHLORIDE ON THE MORPHOLOGICAL
DEVELOPMENT OF ZEA MAYS L. HYBRIDS
Ruxandra Ciaglîc
Mentor: Irina Petrescu

Banat’s University of Agricultural Sciences and Veterinary Medicine “King Michael I of
Romania” from Timișoara, Faculty of Horticulture and Forestry, Department of Genetic
Engineering in Agriculture, Timiș
Despite its high nutritional needs and weed control problems, maize is a greatly relevant
cereal, cultivated in many parts of the World. It is one of the main cultivated plants in Romania,
which makes its production and economic efficiency a matter of national concern.
In this paper we study the effect of sodium chloride on several Zea Mays hybrid seedlings
after germination and growth, in particular its effect on the number of roots formed, in length
growth of roots, formation of cotyledons and the leaves length characters.
According to the literature, a high salt concentration lowers the accessibility of the soil water
for plants, making it inaccessible if too concentrated.
We reached the conclusion that an elevated concentration of salt in the soil solution has an
inhibitory effect on the studied characters.
Key words: Sodium chloride, Zea Mays, hybrid
INTRODUCTION
Whether it is grown for grain or cultivated for animal feed, maize has limited applications in
organic systems, due to its high nutritional needs, weed control problems and the risk of erosion.
It does, however, give a break between grain cereals, because it is not sensitive to the same
categories of diseases and pests.
Corn contains starch, volatile oil, vitamin C, vitamin E, B vitamins, potassium and potassium
salts, iron, vitamin K, magnesium, phosphorus and zinc. Corn silk and flour are used for
medicinal purposes. Corn silk is harvested during the blooming period, from June to August.
Maize has astringent, anti-inflammatory and antioxidant properties. Maize is primarily used as
food (cereal) and then as a medicinal herb.
In Romania, maize is the main cultivated plant, occupying the first place in arable land surface
(on average 30%), as well as production (total and on average per hectare). Therefore, the level of
production and the economic efficiency of corn crops are a matter of national concern.
The effect of salts in the soil solution can be evaluated with sufficient precision, as we found a
close correlation between the salt concentration of the soil solution and the growth of the plants.
The cultivated plants begin to suffer when the soil solution concentration reaches 0.4%. Seed
germination and plant growth are hampered by a concentration of 0.7-0.8%, and at concentrations
159
above 1-1.5% the harvest is compromised. The reduction of the accessibility of water for plants
occurs due to the increase of the soil solution concentration, as a result of the accumulation of salt
from irrigated water in the soil. Increasing the soil solution concentration immediately results in
an increase in the osmotic pressure and, implicitly, the increase in total suction and, thus, a
significant reduction in accessibility of water for plants. Experimental data from the literature is
particularly suggestive, as it clearly highlights the significant reduction in water accessibility,
because of the accumulation of salts in the soil. It is observed that at a similar humidity, on
desalinated soils, water is easily accessible to plants, meanwhile on a soil with 0,55% salts, water
accessibility is low, close to the permanent wilting point, and on a soil with 1,13% salts, soil
water becomes completely inaccessible.
The main purpose of the research was the study of the influence of sodium chloride on the
morphological development of various maize hybrids.
The main objective deriving from this goal is determining the effect of several salt solutions
on different characters of maize hybrid seedlings after germination and growth:
● the number of roots;
● the in length growth of roots;
● the formation of cotyledons;
● the length of the leaves.
Experimental variants of soil salt concentrations used:
V1 - H2O (control variant)
V2 - 5,84g NaCl/L
V3 - 7,60g NaCl/L
Observations were made on various Pioneer maize hybrids:
● P9903 — Mid-early hybrid, with a high adaptability, specifically created for excessively
continental climate. Its excellent multiannual production results from different locations in
Romania make us believe that it will surely give the best results among the group. Good
resistance to the main corn diseases and an exceptional tolerance to drought and heat.
● P0412 — Simple, semi-late hybrid with pronounced tolerance to drought and resistant to
Sorosporium and Fusarium.
● P9911 — Semi-late hybrid, high adaptation to hydric and thermal stress, great production
capability, extraordinary and stable productions in all areas of culture. It’s a tall plant with well-
developed root system, medium inserted, long and compact cob. Significant tolerance to drought
and resistant to Gibberella.
● P0023 — New generation, semi-late hybrid with superior productivity and exceptional
agronomic characters. The plant is medium-sized with a medium inserted, compact cob. It has an
exceptional tolerance to high temperatures.
160
1. The effect of sodium chloride on the roots number of Zea Mays hybrid seedlings
In figure 1, we can remark that the number of roots formed in the P9903 hybrid after the
observations made on the various concentration of salt (V2 and V3) was significantly lower as
against the control variant V1(4,02±0,08). On the variant with the concentration V2, an average
of 1.98 ± 0.15 roots were formed, while the variant with the highest concentration V3, had the
lowest number of roots formed with an average of 1,25±0,05.
For hybrid P0412, the number of roots formed was high in the control variant V1(2,34±0,18),
compared to V3 with 0,25±0,08 roots formed. In a similar situation is hybrid P0023, which had
an average of 3,56±0,06 roots formed on the control variant V1 and an average of 3,02±0,06 on
V2, while on the third variant V3 the average is significantly lower (0,85±0,03).
The number of roots in the hybrid 9911 is different depending on the salt concentration of the
variants we used, highlighting its inhibitory effect in the highest concentration variant
V3(0,18±0,02) compared to the control variant V1 with an average of 1,60±0,04 and
V2(1,18±0,12).
Figure 1. Number of roots formed in Zea Mays hybrid seedlings
2. The effect of sodium chloride on the in length growth of roots of Zea Mays hybrid seedlings
For the hybrid P9903, the in length growth of the roots is high in the variants V2 and V3,
values which vary between 2,03 and 1,92 cm and very high in the control variant V1 with a value
of 2,68 cm.
The length of roots is different for the hybrid P0412, showing maximum average values of
1.03 cm for V1 and medium values of 0,68 and 0,52 cm for V2 and V3.
The hybrid P0023 has an in length growth of 2,45 cm for the control variant V1; 1,89 cm for
V2 and 1,66 cm for the variant with the highest concentration V3.
The inhibitory effect of sodium chloride on the in length growth of roots is visible for the
hybrid P9911, where the lowest value is 0,30 cm in V3.
161
Figure 2. In length growth of roots of Zea Mays hybrid seedlings
3. The effect of sodium chloridee on the formation of cotyledons in Zea Mays hybrid seedlings
The effect of sodium chloride on the analyzed hybrids was observed at the end of the study
period. It has the same value of 1,00±0,0 for the hybrids P0023, P0412 and P9911 and a value of
2,00±0,2
,2 for P9903 on the control variant V1.
Figure 3. Formation of cotyledons in Zea Mays hybrid seedlings(P9911,

P9911, P0023 and P0412)
4. The effect of sodium chloride on the length of leaves in Zea Mays hybrid seedlings
In hybrid P9903, control variant V1 was nnoted

oted by an increase in leaf length of 0,39 ± 1.67, for
the high salt concentration V3 there is a short length of 0,14 ± 0.50 compared to variant V2 -
0,87±0,05.
The high salt concentration variants also have an inhibitory effect in the case of hybrids
P0023
23 and P9911, where the salinity had a strong impact.
162
The length of leaves for the hybrid P0412 is remarkable by its value of 0,27±1,33 for the
control variant V1, 0,39±0,67 for V2 and 0,02±1,67 for the variant with the highest concentration
V3.
Figure 4. Leaves length in Zea Mays hybrid seedlings (P9903 and P0412)
CONCLUSION
Root formation for the P9911 hybrid has been delayed. There is a very large difference
between both salt and water concentrations. The difference between V2 and V3 is large, the
inhibitory effect of the higher salt concentration being obvious.
The length of the roots formed in variants V1 and V3 after the observations made was
different. We observed a large length of roots in both hybrid P9903 and P0023 for the variant V2,
while hybrids P9911 and P0412 had a visible difference in the length of the roots.
The formation of cotyledons for the hybrids P0023, P0412 and P9911 had the same value for
V1, while for P9903 the value was doubled for the same variant.
The high salt concentration had a strong effect on hybrids P0023 and P9911 for the leaves
length character. The leaves length was higher in V1 for hybrids P9903 and P0412 compared to
V2 and V3.
REFERENCES
ŞTEFAN V., 2003. Fitotehnia plantelor tehnice. AMC – USAMV Bucureşti;

SÂULESCU, N., A., SÂULESCU, N., N., 1967 - Câmpul de experienţă. Ed. Agro-silvică,
Bucureşti, p. 193-323;
Badea, Elena, Marcela, Otiman P.I. - Plante modificate genetic în cultură, Ed.Mirton, Timişoara,
2006, pag.37-72;
Bîlteanu, G., Bîrnaure, V., - Fitotehnie, Ed. Ceres Bucureşti, 1979, pag.216-220;
163
Ciulca S., 2006, Elemente de genetică cantitativă şi genetica populaţiilor. Ed. Agroprint,
Timişoara, pag.125-130;
Cristea M. 1977, Rasele de porumb din Romania, Editura Academiei Republicii Socialiste
Romania, Bucuresti,1977;
Cristea M. 2004 – Importanţa economică, răspândirea geografică, producţia şi comerţul cu
porumb, în Porumbul – studiu monogrfic, vol.I, Ed.Academiei Române, Bucureşti, pag.189-213;
Hera C., Borlan Z., 1980, Ghid pentru alcătuirea planurilor de fertilizare, (Editia 2-a) Editura
Ceres, Bucureşti;
Mureşan, T., Şipoş, G., Paulian, F., Moga, I. 1973 - Cultura porumbului, Ed. Ceres, Bucureşti,
1973, pag.136-152;
Potlog, A.S., Suciu, Z., Lăzureanu, A., Nedelea, G., Moisuc, A. - Principii moderne în
ameliorarea plantelor, Ed.Facla, Timişoara, 1989, pag.86-93;
Savatti, M., Nedelea, G., Ardelean, M. - Tratat de ameliorarea plamtelor, Ed.Mirton Timişoara,
2004, pag. 256-264;
Hatman, M., Iacob, Viorica, Săpunaru, T., Mihai, T., Aonofriesei, Luminiţa - The behaviour of
some native and foreign maize hybrids to the attack of pests and pathogenic agents, under
conditions of chemically treated seed, Cercetări Agronomice în Moldova, Anul XXXX, Vol. 3
(131), 2007, pag.23-31;
Ho, J.C. – Extend and distribution of genetic variation in US maize, Crop.Sci., 45, 2005,
pag.1891-1900.
164
PRELIMINARY RESULTS OF DETECTION OF XANTHOMONAS ARBORICOLA
PV. CORYLINA (XAC) ON HAZELNUT IN REPUBLIC OF SRPSKA
Zorana Đekanović
Mentor: Doc.dr Duška Delić

University of Banjaluka, Faculty of Agriculture, Bosnia and Herzegovina
Summary: Hazelnut belongs to the family birch (fam. Betulaceae), the genus Corylus L.The
economic importance of cultivation is based on fruit production, which is a valuable food in both
the domestic and the world market. Plant bacteria that cause significant hazelnut disease are
Xanthomonas arboricola pv, corylina, Xanthomonas campestris pv.corylina, Pseudomonas
avellanae. The occurrence of the disease is insensitive in wet and rainy years. On semi-selective
media (YNA), were observed Yellow-mucoid, shiny and rounded with regular edges bacterial
colonies after 72 hrs of incubation.
Key words: Corylus avellane, plant bacteria, occurrence, culture medium
INTRODUCTION
The high price and favorable climatic conditions stimulated farmers in Bosnia and
Herzegovina to start hazelnuts production in several few years. However, there are no official
data on new hazel plantations in BiH. Based on the demand for seedlings, the increased interest
for the cultivation of this species is apparent (Davidović, 2015). On the other hand there is also
the uncontrolled spread of seedlings by the manufacturers through the offshoots from existing
production plants, as well as due to the import of badly or incorrectly labelled seedlings of hazel
and its reproduction, and expansion of unregistered sales by manufacturers. As a consequence all
this follow introduction of different pets where the biggest problem present the quarantine ones.
One of the biggest phytosanitary risk for the EPPO region representing Xanthomonas arboricola
pv. corylina (Xac) (Miller et al., 1949) Vauterin et al. 1995, (syn. Xanthomonas campestris pv.
corylina) a gram-negative bacterium causal agent of bacterial blight of hazelnut. The pathogen is
regulated as an EPPO A2 quarantine pest of potential economic importance in EPPO Standards,
PM 7/22 (Anonymous, 2004). The disease is now widely distributed and it causes a potentially
serious disease, resulting in economic losses to hazelnut production worldwide (Anonymous,
1986). There are no official reports of the presence and distribution of the important regulated
pathogen in Bosnia and Herzegovina therefore the main aim of this study was to preliminary
check the presence of the causal agent of of bacterial blight of hazelnut.
MATERIAL AND METHOD
Sample collection
Samples were collected during June 2018 in two hazelnut orchards in location Gradiška. Plant
parts showing characteristic symptoms of bacterial blight were cut, separately put into sterile
plastic lab bags and brought to the laboratory. The number of the samples taken was 12 and
different varieties and plants aging from to were included into sampling procedure.
165
Isolation
Sample processing was made, for the isolation of the causal agent. The samples were surface
sterilized in 1% NaOCl for 1 min followed by two rinses in sterile destilated water (SDW) (each
for 1 min). Excised and each section was then crushed in SDW, left for 5 minutes in order to
allow bacterial streaming then sample was homogenized in a PBS 0.01 M, pH 7.2. Streaking (10-
100 μl) was made by taking a loopful of the resulting suspension onto the surface of semi-
selective media (YNA) (Schaad et al., 2001). The plates were incubated at 26±1°C and examined
daily up to three days for bacterial growth.
Pathogenicity tests
Pathogenicity tests were carried out on disease- free Pelargonium zonale plants under artificial
inoculation conditions. Tests were performed with 2-day-old X. arboricola pv. corylina cultures
grown on YNA using two methods: (i) injecting leaves with a bacterial suspension (10 7 CFU
ml−1 in sterile distilled water) using a hypodermic syringe (Wainwright and Nelson 1972) and
(ii) infiltration of young leaves of pelargonium plant shoots with a 10 7 CFU ml-1 suspension of a
48 h culture grown on YNA. The inoculated shoots were covered with plastic bags and after 48 h
the appearance of brown, necrotic spots were observed.
Disease symptoms in the fields

On the symptomatic plants the most prominent symptoms were expressed as water-soaked
necrotic spots (Figure 1) appeared on the leaf surfaces.
Often, leaves showed numerous oily polygonal lesions which merged together causing a general
chlorosis of the lamina. Infected leaves sometimes showed browning of the leaf margins (Figure
1) and longitudinal browning and necrosis along the mid-ribs and secondary veins on the lower
leaf surface were frequently found (Figure 1).
Black heel symptoms with the browning of the shell and corresponding part of the involucres
were observed on the fruits. In addition, oily lesions of different size could be seen, on the
involucres and shell before lignifications (Figure 1).
Figure 1. Symptoms of Xanthomonas arboricola pv. corylina on leaf and fruit
All the examined cultivars from location Romanovci produces bacterial colony on the YNA. The
disease incidence was very high in the older orchard in Gradiska location. The GPS locations
and cultivar names are listed in the Table 1
166
Table 1. Samples code, locations, GPS coordinates and cultivars eximinade during survey
Identification of the isolates
Sample Location GPS Variety
code
H1 Romanovci/Gradiška 44°58'42"N17°17'15 White
" Lambert
" Lambert
" Lambert
" Lambert
" Lambert
H6 Romanovci/Gradiška 44°58'42"N17°17'15 Red Lambert
"
" Lambert
" Lambert
" Lambert
" Lambert
H11 Romanovci/Gradiška 44°58'42"N17°17'15 Red Lambert
"
H12 Aleksandrovac/Banjaluka Daviana
44°96'77"N17°31'71"
H13 Aleksandrovac/Banjaluka Butler
44°96'77"N17°31'71"
H14 Aleksandrovac/Banjaluka Negret
44°96'77"N17°31'71"
H15 Aleksandrovac/Banjaluk Tonda
44°96'77"N17°31'71" Gentile
Romana
Yellow-mucoid, shiny and rounded with regular edges bacterial colonies (Figure 2) were
observed on YNA media after 72 hrs of incubation.
The colonies were small of approximately 1.5 to 2 mm in diameter on the media. A larger amount
of mucoid polysacharide (xanthan gum) produced on this media, differentiated- entiates Xac from
other often commonly occurring saprophytes with dark-yellow colony pigment.
167
Figure 2. Yellow-mucoide bacterial colonies
Pathogenetic tests
All the isolates reproduced the symptoms of the disease on the plants artificially inoculated.
Water- soaked necrotic spots (Figure 3) were observed on leaves within a 3-5 days after the
artificial inoculation. Bacteria reisolated from the diseased parts of the plants had the same
characteristics of the inoculated strains.
Figure 3. Water- soaked necrotic spots on pelargonium test plant
This small scale study reports preliminary results which are significantly indicating presence of
Xac in Bosnia and Herzegovina. Xac is reported and widely distributed in European countries
where hazelnut productions is well established such Turkey (Black Sea area), UK, Italy (Civilleri
et al., 2007), former Yu- goslavia, Russia (Bradbury, 1987; Anonymous, 1996), and Iran Guilan
province (Kazempour et al., 2006; Ali et al., 2006). The planting material that coming in Bosnia
and Herzegovina is usually imported from Serbia, Slovenia and Italy. Once established, this
serious pathogen cannot be eradicated except by removal of all Corylus (hazelnut) plants.
Although more biochemical, serological and molecular tests should be performed to proved
etiology of the bacterial blight causal agents the obtained results highly indicated its presence in
the observed orchards. This is so far also and first report indicating Xac infection of hazelnut in
BiH. Therefore the most important element of control is to introduce only disease-free planting
material into an orchard (Gardan,1986; Gardan and Devaux, 1987). Standard hygienic practices
in affected orchards such as removing and destroying affected shoots and disinfecting pruning
tools may reduce the impact of the pathogen. The application of protective copper-based sprays
such as copper oxychloride may be efficacious but probably must be applied annually as a
prophylactic routine; when the disease is seen to be severe, it is unlikely that spray applications
will be effective. Treatments with copper compounds must be done in the spring, beginning at
168
bud break, and repeated in the autumn, at leaf fall (Gardan and Devaux, 1987; Scortichini, 1995).
Spring sprays are most efficacious. The number of applications varies depending on the length of
the rainy season (Noviello, 1969).
The use of resistant cultivars is recommended when planting a new orchard (cultivars Négret,
Gunslebert, Segorbe, Logued'Espagne, Merveille de Bollwiller) or the species Corylus pontica
(Gardan, 1986). The Graham filbert was reported as a Corylus species with a degree of resistance
to blight (Brooks and Olmo, 1958). Different cultivars of hazelnut are characterized to a different
degree of susceptibility, but none are immune (Noviello, 1969). However symptoms were not
observed on Negret cultivar from orchard in Aleksandrovac locality. Less vigorous plants due to
inappropriate cultural techniques are more susceptible to the disease. It is therefore important to
avoid plantations in soil with poor drainage (Noviello, 1969).
CONCLUSION
In the study isolation method and pathogenicity test indicated the presence of Xanthomonas
arboricola pv. corylina (Xac) infecting hazelnuts in the surveyed orchards. Future studies will be
dedicated to the extended surveys and to biochemical, serological and molecular methods
together with isolation on semi selective and selective media in order to get clearer picture about
Xac presence, distribution and economic impact in hazelnut orchards in Republica Srpska as well
as in Bosnia and Herzegovina.
REFERENCES
ANONYMOUS, (2004): EPPO diagnostic protocols for regulated pests PM 7/22. Xanthomonas
arboricola pv. corylina. EPPO Bulletin 34, 155–7
ANONYMOUS (1996): XANTHOMONAS CAMPESTRIS PV. CORYLINA. CAB
INTERNATIONAL, DISTRIBUTION MAPS OF PLANT DISEASES NO. 699.
BRADBURY, (1987):Xanthomonas campestris pv. corylina. IMI Descriptions of Fungi and
Bacteria, No. 896..
BROOKS and OLMO, (1958); Register of New Fruit and Nut Varieties: Brooks and Olmo List
36
CIVILLERI G., SCUDERI G., BONACCORSI A., SCORTICHINI M., (2007). Occurrence of
Pseudomonas syringae pv. coryli on hazelnut orchards in Sicily, Italy and characterization by
fluorescent amplified fragment length polymorphism. Journal of Phytopathology 155: 397-402.
DAVIDOVIĆ,J. (2015): Pomological Identifi cation of Hazel Cultivars (Corylus avellana L.) in
Plantations in Bosnia and Herzegovina.
GARDAN,L.(1986): Xanthomonas campestris pv. corylina. EPPO Data sheets on quarantine
organisms. EPPO/EPO Bull 16: 13–16.
GARDAN,L.,DEVAUX,M.(1987): La bactériose du noisetier (Xanthomonas campestris pv.
corylina): biologie de la bactérie. Bulletin OEPP/EPPO Bulletin 17, 241-250.
SCORTICHINI,M. (1995); Influence of subculturing some patogenestic bacteria on their
carbohydrate utilizing profile.
KAZEMPOUR MN., ALI B., ELAHINIA SA., (2006): First report of bacterial blight of hazelnut
caused by Xanthomonas arboricola pv. corylina in Iran. J Plant Pathol 88: 341.
MILLER PW., BOLLEN PW., SIMMONS JE., (1949): Filbert bacteriosis and its control.
Bulletin of the Oregon Agricultural Experimental Station 9, 1–107.
169
NOVIELLO,C., (1969):[Infectious diseases of hazel]. Annali della Facoltà di Scienze Agrarie
della
Università degli Studi di Napoli Portici 3, 11-39.
SCHAAD,N., JONES J., CHUN W., (2001): Laboratory Guide for Identification of Plant
Pathogenic Bacteria. 3rd edn. St Paul, MN, USA: APS Press.
WAINWIGHT,S.H.,NELSON P.E., (1972): Hiatopatology of Pelorgonium species infected with
Xantomonas pelorganii. Phitopatology,62,1337-1347.
170
PESTS INCIDENCE IN DIFFERENT BBCH STAGES IN SMALL GRAINS AGRO-
ECOSYSTEMS THROUGHOUT AGGRESSIVITY ASSESSMENT
Denis MURGU,
Mentors: Assistant dr. Ramona ȘTEF, Professor dr. Ioana GROZEA

Department of Biology and Plant Protection, Banat’s University of Agricultural Sciences and
Veterinary Medicine “Regele Mihai I al României” from Timisoara
Summary: Recent studies have highlighted the increase in damage caused by grain crop
pests, including crops that are severely affected by unfavourable climatic conditions and the
farming system with incomplete or incorrect crop technologies. Also, the data from the literature
highlights the climate change and the cultivation technologies that have caused changes in the
importance of certain insect pests towards the cereals. In particular, climate warming, drought
and desertification favoured increases in the abundance and aggressivity of some insect pest
groups that requires special attention. The research was carried out in the Western part of
Romania in 2017/2018
The experiment included 3 replications and 3 variants, each variant consists from one crop
and the data obtained were interpreted statistically. Observations were performed on two
different growth stages: BBCH 23-25 and BBCH 63-69. The highest frequency of attack in this
phase of vegetation (BBCH 23-25) was exerted by the species Phyllotreta vittula. Agriotes sp.
presented the lowest aggressivity in the experimental variants. The dominant species in this
growth stage (BBCH 63-69) was Oulema melanopa, the second being the leaf mining fly
(Hydrellia griseola).
Key words: attack aggressivity, pests, triticale, oat, barley
INTRODUCTION
Wheat is the most important plant grown in the world, leading to the existence of 22
species and several thousand varieties. Currently this plant occupies an important place in the
security strategy. In Romania, occupying the 1st place as a share of the arable land area (Muntean
S., 1995, cited by Guș P. et. col., 2008). In recent years, in severely affected crops, unfavorable
climatic conditions and the farming system with incomplete or incorrect crop technologies, an
increase in damages caused by pests was registered (Malschi end al., 2012). Studies conducted by
researchers in Romania highlight the main pests of grain cereals: diptera, homoptera,
thysanoptera, coleoptera, etc. (Malschi, 2007, 2008, 2009; Malschi et al., 2012).
Climate changes and culture technologies used have caused changes in the importance of
certain species of insect pests to the wheat. Climatic warming, drought in 2007-2012 have
favored increases in the abundance and aggressiveness of some insect pest groups that require
special attention.
171
Cultivation technologies, the partition of arable land in strips and small areas, the non-
application of phytosanitary measures have led to an even more pronounced increase in the
biological reserve of pests.
The research carried out during the period 2007-2018 revealed that for the current period the
most important groups of wheat pests are: the wheat trip (Haplothrips tritici) - which has become
eudominant (32-100%), the cicadas : 10-31.9%), aphids (dominant: 10-31.9%), cereal stink bugs,
some diptera species (dominant: 10-31.9%) (Chloropidae flyfish, Oscinella frit, Erychiptera
cornuta, Meromyza nigriventris, Anthomyiidae: Phorbia securis, Delia platura, Delia coarctata)
and soil flea Chrysomelidae: Chaetocnema aridula, soil pests (Agriotes spp., Zabrus
tenebrioides, Agrotis spp., Anisoplia spp.) .
Long-term research on the phytosanitary status of field crops is at the basis of the
development of pest control technologies (Bărbulescu et al., 1987, 2002, Popov et al., 2002,
2004, 2005, 2007, 2010) suitable for climatic changes and desertification (Popov et al., 2003,
2006, Malschi, 2007; Malschi et al., 2012).
MATERIAL AND METHOD
The research was carried out in 2018 on the Western part of Romania, Şag, belonging to the
Timiş County.
The experiment included 3 replications and 3 variants, each variant consists from one crop
and the data obtained were interpreted statistically. Observations were performed on two different
phenophases: BBCH 23-25 and BBCH 63-69.
The variants had a surface area of 30 m2 (length 10 m and 3 m width). In this trial following
breeds have been used: triticale Mv Medal; Barley Laverda; Oat Lovrin 1. The calculation
method used was that of monofactorial experiences, in order to determine the frequency of attack
caused by the harmful species present in the three agroecosystems. To determine the
aggressiveness, 100 plants / variant were studied, so the frequency of each replicate is an average
of the 100 plants observed..
Three replicates were counted for each crop to eliminate any errors. The aggressiveness of
attack was calculated according to the following formula:
n  100 where:
F
N
F - expressed as a percentage represents the frequency of pathogen attack;
n – number of attacked plants;
N – the total number of plants analyzed;
172
In Romania, small grains cereals (wheat, barley, oats, triticale) occupy the first place in the
cultivated agricultural area. The success of the crops studied is conditioned by the presence of
their lowest or even the absence of pests.
The first evaluation was carried out in April 2018, in BBCH 23-25 growth stage, the results of
the present species and the aggressivity determined in barley, oat and triticale agroecosystems are
reported in Table 1. At tillering stage of barley and oat plants, larval attack of Zabrus
tenebrioides was observed. The attack did not show statistical differences compared to the
average of the experience, although mathematically the barley plants showed an aggressivity of
11% higher than that recorded in oat drilled variants (6.67%).
The statistical interpretation of data on the number of plants attacked at BBCH 23-25 stage by
Phyllotreta vittula shows that barley plants were the most attacked, recording a difference of
19.11 compared to the average of experience (Table 1).
Comparing the number of triticale plants showing longitudinal lesions produced by
Phyllotreta vittula, with the average of experience, it is noticed that there are no statistical
differences. In this growth stage, the attack was produced by adults.
The experimental year 2018 was favourable for the development of Oscinella frit species,
which is present in barley, oat and triticale agroecosystems (table 1). The number of plants in the
experimental variants, which showed the dry central leaf, ranged between 1.33 (triticale) - 12.33
(barley). The highest aggressivity of Swedish fly attack was observed in barley drilled variants.
The climatic conditions of 2018 caused barley, oats and triticale plants to be attacked by
Agriotes sp. in growth stage BBCH 23-25. The data analysis shows that the presence of the pest
in the experimental variants was sporadic, the aggressivity of the attack values ranged between
1.0-7.0%. The maximum aggressivity was reported in triticale plants.
It is observed that in the experimental variants the present species were: Zabrus tenebrioides,
Phyllotreta vittula, Oscinella sp. and Agriotes sp. In this phenophase (BBCH 23-25) the
dominant species was Phyllotreta vittula. Among harmful species present in agroecosystems
studied, Agriotes sp. was reported sporadically, in some variants drilled with oats the attack was
absent.
Throughout macroscopic observations, made directly in experimental groups, the composition
of epigeal fauna in BBCH 65-68 growth stage was determined. The species present in this growth
stage were: Oulema melanopa, Aphidae sp., Eurygaster sp., Epicometis hirta, Hydrellia griseola.
The triticale crop showed the highest number (448) of plants attacked by epigeal pests. Barley
plants showed a lower attack frequency (332 attacked plants) than oat and triticale.
The aggression of the Oulema melanopa attack in the experimental year 2018 showed an
average of 60%. The oat plants showed the highest frequency of Oulema melanopa attack, the
results being distinctly significantly negative compared to the control. The barley drilled variants
showed an average aggressivity of 42.33% (Table 2), we mention that in this growth stage the
attack was produced by both larvae and adults.
173
Table 1. Aggressivity of species present in cereal agroecosystems in BBCH 23-25 growth stages
Attack frequency % of Zabrus
Diff.
tenebrioides Diff. Signifi
Variant Mean relativ
Repetiti Repetitio Repetiti Abs. cance
e.
on I n II on III
Orz 10 14 9 11 124,52 2,17 -
Ovăz - - - - - -
Triticale 8 5 7 6,67 75,47 -2,17 -
Mean of
experien 9,0 9,5 8,0 8,83 100 0 -
ce
DL5% = 4,58; DL1% =7,58; DL0,1% = 14,20;
Attack frequency % of Mean Diff. Diff. Signifi
Phyllotreta vittula relativ Abs. cance
Variant
Repetiti Repetitio Repetiti e.
on I n II on III
Orz 50 61 57 56,0 151,80 19,11 00
15 10 11 -
Ovăz 12,0 32,53 ***
24,89
Triticale 47 39 42 42,67 115,65 5,77 -
Mean of
experienc 37,33 36,67 36,67 36,89 100 0 -
e
DL5% = 8,47; DL1% =12,84; DL0,1% = 20,62;
Attack frequency % of Mean Diff. Diff. Signifi
Oscinella frit relativ Abs. cance
Variant
Repetiti Repetitio Repetiti e.
on I n II on III
Orz 15 10 12 12,33 198,17 6,11 000
Ovăz 7 5 3 5,0 80,34 -1,22 -
Triticale 2 0 2 1,33 21,42 -4,89 **
Mean of
experienc 8,0 5,0 5,67 6,22 100 0 -
e
DL5% = 2,37; DL1% =3,59; DL0,1% = 5,77;
Attack frequency % Agriotes Mean Diff. Diff. Signifi
sp. relativ Abs. cance
Variant
Repetit Repetitio Repetiti e.
ion I n II on III
Orz 5 3 6 4,66 110,49 0,44 -
Ovăz 2 1 0 1,0 23,67 -3,22 *
Triticale 6 7 8 7,0 165,74 2,77 0
Mean of
experienc 4,33 3,67 4,67 4,22 100 0 -
e
DL5% = 2,37; DL1% =3,59; DL0,1% = 5,77;
174
Table 2. Aggressivity evaluation by pest species in barley, oats and triticale agroecosystems in BBCH 63-69 growth
stage
Attack frequency % Oulema melanopa Diff. Diff.
Variant Mean Significance
relative Abs.
Repetition I Repetition II Repetition III
Orz 37 49 41 42,33 101,11 17,66 -
Ovăz 90 86 82 86,0 47,22 26,00 00
Triticale 52 48 55 51,66 86,11 -8,33 -
Mean of
59,67 61 59,33 60 100 0 -
experience
DL5% = 18,79; DL1% =28,45; DL0,1% = 45,70;
Variant Attack frequency % Aphidae sp. Dif. Diff.
Mean Significance
Repetition I Repetition II Repetition III relative Abs.
Orz 12 8 10 10 58,82 -7,0 -
Ovăz 15 21 23 19,66 115,68 2,66 -
Triticale 20 19 25 21,33 125,49 4,33 -
Mean of
15,67 16,0 19,33 17,0 100 0 -
experience
DL5% = 32,74; DL1% =49,58; DL0,1% = 79,66;

Variant Attack frequency % Eurygaster sp. Dif. Diff.
Mean Significance
Orz 11 10 9 10,0 65,68 -5,22 *
Ovăz 13 15 17 15,0 98,53 -0,22 -
Triticale 17 21 24 20,66 135,75 5,44 0
Mean of
13,67 15,33 16,67 15,22 100 0 -
experience
DL5% = 3,75; DL1% =5,68; DL0,1% = 9,12;
Attack frequency % Epicometis hirta Dif. Diff.
Orz 15 13 14 14,0 79,24 -3,66 **
Ovăz -
7 5 6 6,0 33,96 ***
11,66
Triticale 35 33 31 33,0 186,79 15,33 000
Mean of
19 17 17 17,66 100 0 -
experience
DL5% = 1,63; DL1% =2,47; DL0,1% = 3,97;

Attack frequency % Hydrellia griseola Dif. Diff.
relative Abs.
Repetition I Repetition II Repetition III
Orz 28 33 36 32,33 140,57 9,33 00
Ovăz 16 12 14 14,0 60,86 -9,0 **
Triticale 25 22 21 22,67 98,55 -0,33 -
Mean of
23 22,33 23,67 23,0 100 0 -
experience
DL5% = 5,57; DL1% =8,44; DL0,1% = 13,57;
175
In the control variant there was an average of 17 plants attacked by aphids. In May, triticale
plants showed the highest attack of aphids compared to barley and oats.
In the BBCH 63-69 growth stage, Eurygaster sp. From the analysis of the 100 plants/variant it
turned out that the aggressivity of the attack oscillated between 10 and 20.66%.
Following the phytosanitary control carried out in the three experimental variants, the attack
of the species Epicometis hirta was also revealed. This pest is not specific on grain crop, but
more and more present in the past few years in more and more crops. The attack observed in the
experimental variants was produced by adults who consumed floral organs.
The analysis of the data presented in Table 2 leads us to affirm that the triticale plants were
the most sensitive, based on difference from the mean of experience of 15.33%, this difference
being very significantly negative due to the statistical calculations. The aggressivity of
Epicometis hirta species on oat plants showed a difference of -11.66 compared to the average of
the experience, showing very significant differences. The aggressivity of Hydrellia griseola, in
barley variants, was 32.33%. The attack was also observed in oat and triticale cultures.
CONCLUSION
The following conclusions have been drawn on the composition and aggressivity of the
epigeal fauna in cereal agroecosystems: at tillering the composition of harmful invertebrate fauna
was represented by the species: Agriotes sp, Zabrus tenebrioides, Phyllotreta vittula, Oscinella
frit. The highest frequency of attack in this phase of vegetation was exerted by the species
Phyllotreta vittula. Agriotes sp. presented the lowest aggressivity in the experimental variants. At
tillering barley plants had the highest frequency of attack of harmful fauna. The oat was less
attack by the harmful organisms
At flowering - end of flowering, the following species were found in the experimental
variants: Oulema melanopa, Aphidae sp., Eurygaster sp., Epicometis hirta, Hydrellia griseola.
The dominant species in this growth stage was Oulema melanopa, the second being the leaf
mining fly (Hydrellia griseola). In BBCH 63-69, the most attacked crop was triticale, followed
by oat.
REFERENCES
BĂRBULESCU, A., BÂGIU, LILIANA, BRATU, R., CIURDĂRESCU, G., CRAICIU,
MIHAELA, GHEORGHE, MAGDALENA, MATEIAŞ, M.C., PELMUŞ, ANICUTA,
PELMUŞ, V., POPOV, C., RUGINĂ, MARTA, TUŞA, CORINA, VOINESCU, I., VONICA, I.,
KOZINSCHI, T. (1987): Evoluţia unor boli şi dăunători ai cerealelor, plantelor tehnice şi furajere
în ţara noastră în anul 1988. Probl. prot. pl., XVII (1): 67-87.
BĂRBULESCU, A., POPOV, C., MATEIAŞ, M. C. (2002): Bolile şi dăunătorii culturilor de
câmp. Editura Ceres, Bucureşti, 279 pag.
GUŞ P., RUSU T. (2008): Sisteme minime de lucrare a solului-alternative pentru protectia
mediului. In: Sisteme de lucrari minime ale solului: al Vlea Simpozion cu participare
internationala. Ed. RISOPRINT, Cluj-Napoca, p. 9-18.
176
MALSCHI DANA (2007): Wheat pest entomofauna in climatic changes conditions of central
Transylvania. The 20thSIEEC Conference, May 2007, Cluj-Napoca. Section: Global climate
change, fauna change and Entomofaunistics. Universitatea Babes-Bolyai Cluj-Napoca.
Entomologica romanica, nr.12, 2007, p.185-193, ISSN 1224-2594, Cluj-Napoca, 2008.
MALSCHI DANA (2007): Mediu-agricultura-dezvoltare durabila şi managementul integrat al
dăunătorilor agroecosistemelor cerealiere. Ed. Argonaut, 2007. Cluj-Napoca: 186 pp. ISBN 978-
973-109-086-3.
MALSCHI DANA (2008): Mediu-agricultură-dezvoltare durabilă. Optimizarea tehnologiilor de
management integrat al damnatorilor grâului in dinamica modificărilor agroecologice din
Transilvania. Ed. Argonaut. 2008. ISBN 978-973-109-115-0.
MALSCHI DANA (2009): Integrated pest management in relation to environmental
sustainability. Part I. Ecological management of wheat pests. Manual online. Faculty of
Environmental Sciences, Babeş-Bolyai University, Cluj-Napoca. Bioflux Publishing House, Cluj-
Napoca, Editor: Malschi Dana, 2009, p. 200. ISBN 978-606-92028-3-8,
http://www.editura.bioflux.com.ro/carti-2009/
MALSCHI DANA, MIRCEA IGNEA, ADINA IVAS, ALINA ŞIMON, CORNEL CHEŢAN
(2012): Dinamica şi combaterea integrată a dăunătorilor, grâului în tehnologia conservativă a
solului fără arătură, adecvată schimbărilor climatice actuale, AN. I.N.C.D.A. Fundulea, vol.
LXXVIII, NR. 2, 2013
POPOV C., LIDIA CANĂ, ELENA TROTUŞ, VALERIAN STOICA, FLORICA VILĂU,
GEORGETA TRAŞCĂ, CORNELIA CIOBANU (2010): Cercetări privind îmbunătăţirea
tehnologiei de combatere a gândacului Zabrus tenebrioides Goeze., prin tratarea seminţelor de
grâu şi orz, AN. I.N.C.D.A. Fundulea, VOL. LXXVIII, NR. 2, 2013
POPOV C., LIDIA CANĂ, EMIL GEORGESCU (2009): Rolul indicatorilor de biodiversitate în
aprecierea managementului dăunătorilor din cultura grâului. În: Analele Institutului de Cercetare-
Dezvoltare Agricolă Fundulea, Vol. LXXVII, Ed. New Agris – Revistele agricole, Bucureşti,
p.199-210.
POPOV, C. (2002): Cercetări privind protecţia cerealelor, leguminoaselor pentru boabe, plantelor
tehnice şi furajere faţă de agenţii patogeni şi dăunători, efectuate în anul 2001. Probl. prot. pl.,
XXX (2): 109-190.
POPOV, C., GURAN, MARIA, RARANCIUC, STELUŢA, ROTĂRESCU-MINCU,
MIHAELA, SPIRIDON, CRISTINA, VASILESCU, S., GOGU, FLORICA (2003): Starea
fitosanitară a culturilor de cereale, leguminoase pentru boabe, plante tehnice şi furajere din
România, în anul 2002. Probl. prot. pl., XXXI (1): 1-22
POPOV, C., GURAN, MARIA, RARANCIUC, STELUŢA, ROTĂRESCU-MINCU,
MIHAELA, SPIRIDON, CRISTINA, VASILESCU, S., GOGU, FLORICA (2006 a): Starea
fitosanitară a culturilor de cereale, leguminoase pentru boabe, plante tehnice şi furajere din
România, în anul 2005. Probl. prot. pl., XXXIV (1-2): 15-38.
177
EFFICACY ASSESSMENT OF DIFFERENT CONTROL METHODS OF WEEDS IN
SOYBEAN AGRO-ECOSYSTEM
ROMINA MAZĂRE,
Mentors: Assistant dr. Ramona ȘTEF, Lecturer dr. Alin CĂRĂBEȚ
Department of Biology and Plant Protection, Banat’s University of Agricultural Sciences and
Veterinary Medicine “Regele Mihai I al României” from Timisoara
Summary: The research was carried out in the Western part of Romania (Sag, Timis
County). The study aimed primarily at testing herbicides in control of weeds in soybean
agroecosistem, which is very sensitive to the presence of weeds because it has a slow growth and
a long vegetation period. The main weeds were Echinochloa crus galli (52.0 plants / m 2) and
Cirsium arvense (31.0 plants / m2). To assess the effectiveness of herbicides in weed control,
have been applied in pre-emergence and post-emergence, alone or in combinations. Applied
herbicides reduced weed infestation from soybean crops to 59.83% to 98.5%. Application of 50%
flumioxazine + quizalofop-p-tefyl herbicide + 360 g / l glyphosate 480 g / l glyphosate
isopropylamine + 480 g / l bentazone + 22.4 g / l imazamox conduct to weed reduction of
93.25% - 98.5%. Soybean production averaged 1.05 t / ha - 2.97 t / ha.
Key words: weeds, control degree, herbicide, soybeans
INTRODUCTION
Soy is one of the main crops in the Western Plain of Romania, due to soil and climate
favourable conditions (Popa, 2006 cited by Manea D et al., 2013). Soy contributes decisively to
providing the protein on a planetary scale and producing the highest quality vegetable oil
(Mohammadi, Amiri, 2011). It is a plant that contributes substantially to the soil fertility rate, by
the fixed amount of nitrogen, being valuable in any system of crop rotation (Rusu T., 2014).
In view of the uses of the soybean plants, attention should be paid to this crop because it has a
particular sensitivity to enrichment in the first growth stages. Once invaded by weeds, especially
in the first stages of vegetation, culture does not recover even if the weeds are subsequently
fought because soy is a medium-sized and easy to race weed plant. Damage produced by weeds
in culture can be 30% to 80%, sometimes even compromising culture (Berca, 2004).
The most common weeds encountered in soybean culture are, in particular, those with late
spring germination but not only (Gus et al., 2004, cited by Rusu et al., 2014): Echinochloa
crusgalli, Setaria sp., Digitaria sanguinalis, Sorghum halepense, Agropyron repens, Solanum
nigrum, Amaranthus retroflexus, Chenopodium album, Galinsoga parviflora, Xanthium sp.,
Abutilon teophrasti, Datura stramonium, Polygonum sp., Cirsium arvense, Convolvulus arvensis
etc.
Weed management is one of the most important aspects of soybean production and includes a
wide range of herbicides. The correct choice of herbicides according to the weed spectrum,
178
growth stage, it is very important for the good control of soybean weeds (Barić and Ostojić,
2000; Bilandžić et al., 2003; Djurkić et al., 2004 cited by Mira Knežević, 2004).
If we do not consider the high costs, weeds could be controlled in soybeans using good
management of all practical methods available, combining them into integrated weed
management (IWM) (Rafael Vivian, 2013).
MATERIAL AND METHOD
The research was carried out in 2018, in the western part of Romania, the village Şag anului,
Timiş county.The field of experience was placed according to the Latin rectangle with 10 variants
in three replicates.
The length of the parcels was 10 m, and the width of 3.0 m. The width of the pathway was 1.0
m.
The herbicides used in the experience were: Corum + Dash, Listego, Pantera 40 EC, Pleg e 50
WP, Dominator SL. The only pre-emergent herbicide was Dominator (SL). Phytosanitary
products were applied in two doses (one recommended by the manufacturer and one larger).
Before applying the phytosanitary treatments, the degree of weed infestation was established by
the numerical quantitative method. The method consists in counting weeds by species from a
sample surface, with the expression of results at 1m 2.
After determining the species present in the experimental variants, participation (%) was
calculated.
"P- represents the percentage expression of measures in which one species participates in
the general infestation.
It is obtained through the relationship:
p% - =mx100/M
in which:
m = the average number of individuals of a weed;
M = average number of weeds per 1 m2
After application of herbicides, observations were made to assess the efficacy exerted, the
evaluation was made after the EWRS scale.
Soybean production was determined by harvesting 1 m2 of each of the variants, and then the
samples were transported to the laboratory where weights were made (observation sheet,
analytical balance).
179
Table 1. Trial protocol
Commercial
Active ingredient Doze
product
1. Control unhoed - -
2. Manual hoeing - -
480 g/l bentazon + 22,4 g/l
3. Corum + Dash 1,9 l/ha
imazamox
4. Listego (Pulsar) 40 g/l imazamox 1,0 l/ha
Plege 50 WP + flumioxazin 50%+ quizalofop- 0,09 kg/ha
5.
Pantera 40 EC p-tefuril 40 gr/l + 0,75 l/ha
360 g / l glifosat, 480 g / l
Dominator (SL) + glifosat-isopropylamine + 480 1,0 l/ha +
6.
Variant
Corum g/l bentazon + 22,4 g/l 1,9 l/ha

imazamox
480 g/l bentazon + 22,4 g/l
7. Corum + Dash 2,5 l/ha
imazamox
8. Listego (Pulsar) 40 g/l imazamox 2,0 l/ha
0,100
Plege 50 WP + flumioxazin 50%+ quizalofop-
9. kg/ha + 1,0
Pantera 40 EC p-tefuril 40 gr/l
l/ha
360 g / l glifosat, 480 g / l
Dominator (SL) + glifosat-isopropylamine + 480 2,0 l/ha +
10.
Corum g/l bentazon + 22,4 g/l 2,5 l/ha
imazamox
As a result of the weed screening in the control variant (non-herbicidated-no hoeing) it was
observed that the following species were present in the soybean agroecosystem: Echinochloa
crus galli, Cirsium arvense, Convolvulus arvensis, Xanthium strumarium, Sorghum halepense,
Hibiscus trionum, Polygonum persicaria, Digitaria sanguinalis, Ambrosia artemisiifolia. Species
Echinochloa crus-galli was dominant, recording the highest percent of presence (36,62%),
second placed the species Cirsium arvense with 21,83% (table 2).
180
Table 2. Weed species present in control plot (no herbicide application) in soybean crop
No. Weeds no % Botanical
Weed species
crt. /m2 participation class
1. Echinochloa crus 52,00 M.a.
galli 36,62
2. Cirsium arvense 31,00 21,83 D.p.
3. Convolvulus 9,00 D.p.
arvensis 6,34
4. Xanthium 16,00 D.a.
strumarium 11,27
5. Sorghum halepense 12,00 8,45 M.p.
6. Hibiscus trionum 6,00 4,23 D.a.
7. Polygonum 4,00 D.a.
persicaria 2,82
8. Digitaria 8,00 M.a.
sanguinalis 5,63
9. Ambrosia 4,00 D.a
artemisifolia 2,82
Total 142,00 100,00
From the data presented in Table 2, it is observed that: annual monocotyledonous were
dominant, with a degree of participation (42.25%); perennial dicotyledonous were present at
28.17%; annual dicotyledonous have registered a share of 21.14%, the last place being held by
perennial monocotyledons (8.45%). Noteworthy is the presence of the invasive species,
Ambrosia artemisiifolia, which is in a continuous expense, being more and more frequently
encountered in agricultural crops.
Figure 1 shows the data on the effectiveness of weed control chemicals from soybean culture.
The effectiveness percentages of herbicides used in the experiment showed values ranging from
59.83 to 98.5%. Compared to the non-herbicidal control, all herbicides showed very significant
positive differences
181
Figure 1 - Graphical representation of weed control results from soybean agroecosystems
DL5%- 6,15; DL1% - 8,45; DL0,1 - 11,50

Figure 2 - Representation of the absolute differences regarding the control percent recorded in the experimental
variants compared to the hoed variant
For the hierarchy of herbicides used in weed control we compared the results with manual
hoeing variant 2 (Figure 2). As a result of the statistical interpretation, the herbicide Dominator
(SL) + Corum showed the highest weed control ratio in soybean culture (dose I - 96.16% and
98.50% at the second dose). The lowest rate of control was recorded in Listego (Pulsar) 1.0 l / ha
(59.83%) and Listego (Pulsar) 2.0 l / ha (63.5%).
Following studies, it was observed that by combining the 50 WP + Pantera 40 EC herbicide
weeds were reduced by 89.83% and 93.5%.
From figure 2 we can see the superiority of the hoeing variant to the Corum + Dash and
Listego (Pulsar) herbicides, this statement being statistically assured, with the percentage of
control showing very significant negative differences. In herbicide variants with Plege 50 WP +
Pantera 40 EC and Dominator (SL) + Corum the control rates did not differ statistically
182
Table 3. Results on the influence of treatments on soybean production
Tr yields t/ha Sign
t. Repli Repli Mea Dif. iff
Herbicide Repli
No cate cate n abs.
cate I
. II III
Corum + Dash 1,9
1. 1.821 1.877 1.802 1.83 0.78 ***
l/ha
Listego (Pulsar)
2. 1.883 1.912 1.892 1.90 0.85 ***
1,0 l/ha
Plege 50 WP 0,09
3. kg/ha + Pantera 40 2.278 2.436 2.449 2.39 1.34 ***
EC 0,75 l/ha
Dominator (SL)
4. 1,0 l/ha + Corum 2.688 2.799 2.750 2.75 1.70 ***
1,9 l/ha
Corum + Dash 2,5
5. 1.811 1.897 1.883 1.86 0.81 ***
l/ha
Listego (Pulsar)
6. 1.969 1.952 1.998 1.97 0.92 ***
2,0 l/ha
Plege 50 WP
0,100 kg/ha +
7. 2.657 2.648 2.700 2.67 1.62 ***
Pantera 40 EC 1,0
l/ha
Dominator (SL)
8. 2,0 l/ha + Corum 2.950 2.993 2.954 2.97 1.92 ***
2,5 l/ha
9. Prășit - 2 manuale 2.831 2.902 2.931 2.89 1.84 ***
Neerbicidat ppi +
10. 0.930 1.13 1.09 1.05 0.00 -
Neerbicidat post.
DL5% 0.07257852
0.09953625
DL1% 6
0.13547990
DL0.1% 4
The soybean yield, in the experimental variants, ranged from 1.05 t / ha to 2.97 t / ha (Table
3). The yield increases recorded in the herbicide variants showed very significant differences
compared to those obtained in the control variant (non-herbicidated / no- hoeing). The analysis of
the soybean yields data from the experimental variants highlights the herbicides Dominator (SL)
183
+ Corum and Plege 50 WP + Pantera 40 EC produced the highest yields (1.62 t / ha respectively
1.92 t / ha). Good results on soybean production were also recorded in hoed variants (2.89t / ha).
CONCLUSIONS
Considering the results obtained in the present study, the following conclusions were drawn:
The dominant species in the experimental variants were: Echinochloa crus-galli and Cirsium
arvense. The number of weeds/m2 in the control variant was 142 plants.
The weed control rates in the experimental variants fluctuated in the range of 59.83% -
98.50%
The best results in reducing the number of weeds were recorded in herbicide-treated variants:
Dominator (SL) + Corum and Plege 50 WP + Pantera 40 EC.
Increasing the herbicide application rate has increased the degree of control, but the
differences were not significant.
Soybean production correlated with the effectiveness of herbicides and applied agro-technical
measures (manual hoeing).
REFERENCES
Berca M., 2004, Managementul integrat al buruienilor, Ed. Ceres, Bucureşti

Bilandžić, M., Sudarić, A., Duvnjak, T., Mijić, A. (2003.): Učinkovitost različitih načina
suzbijanja korova u soji. Fragmenta phytomedica et herbologica, 28 (1,2):33.-40.
Coble, H.D., Williams, F.M., Ritter, R.L. (1981): Common ragweed (Ambrosia artemisiifolia)
interference in soybean (Glycine max (L.) Merr.). Weed Sci. 29: 339 –342.
Đurkić, M., Knežević, M., Hajba, K. (2004.): Utjecaj obrade tla i herbicida na korove i prinos
soje. XXXIX. Znanstveni skup hrvatskih agronoma s meĎunarodnim sudjelovanjem, Opatija 17.
do 20. veljače 2004., 527.– 530.
influenced by starter fertilizer, AJCS 5(11):1350-135
Manea Dan Nicolae, Ienciu Anişoara Aurelia, Peţ Ioan, Ştef Ramona, Cărăbeţ Alin (2013):
Research on the influence of weed control on the soybean yield, Analele Universitatii din Oradea,
Fascicula Protectia Mediului, vol XXI
Mira Knežević, Manda Antunović, Ljubica Ranogajec, Renata Baličević (2008): Effectiveness of
some post-emergence herbicides in soybean, Poljoprivreda, vol. 14, no. 2, Prosinac
Mohammadi G.R., Amiri F., 2011, Critical period of weed control in soybean (Glycine max) as
Rafael Vivian, André Reis, Pablo A. Kálnay, Leandro Vargas, Ana Carolina Camara Ferreira,
Franciele Mariani(2013): Weed Management in Soybean — Issues and Practices,
http://dx.doi.org/10.5772/54595
Rusu, T., Cheţan, C., Bogdan, Ileana, Cheţan, Felicia, Moraru, Paula Ioana, Pop, A.I., Duda, B.,
Coste, Camelia, 2014 ‒ Strategia de control a buruienilor din cultura de soia. “Lucrări ştiintifice”,
volumul 41, Universitatea Agrară de Stat din Moldova, Facultatea de Agronomie, Chişinău.
184
INFLUENCE OF SALINITY ON GROWTH AND DEVELOPMENT OF PANSY (VIOLA
X WITTROCKIANA)
Magdalena Pušić
Mentor: Doc. dr. Emina Mladenović

University of Novi Sad, Faculty of Agriculture, Department of fruit growing, viticulture,
horticulture and landscape architecture, Serbia
Summery: The purpose of this paper is to determinate influence of salinity on growth and
development of pansy (Viola x wittrockiana). One of the results of this work was to look at how
to use the pansy in the autumn-winter aspects, as well as the more correct choice of area on which
to use. For the purpose of this research, a total used 40 seeds of pansy was divided into four
treatments of ten seedlings. The tretments were labeled: control, treatment I, where was used 3 g/l
NaCl, treatment II, where was used 5 g/l NaCl and treatment III, where was used 7 g/l NaCl.
Only control treatment was not performed with concentrated water. During the test, certain
parameters were measured, such as: height, number of leaf, number of flowers, diameter of
flowers, length of leaf, width of leaf as well as vitality and decorativeness. Using these
parameters, it was noted how a certain amount of salt influenced on the growth of pansy during a
certain period of time. The results of this study show that pansy can tolerate a certain
concentration of salt, but it will affect the reduction of growth and development, as well as the
vitality and decorativeness.
Key words: development, growth, pansy, salinity, treatments, Viola x wittrockiana.
INTRODUCTION
Pollution of the pedosphere is a major challenge for the development of agroculture in the
modern world. One of the aspects of soil contamination is happening in urban areas, when during
the winter streets abundantly fall into industrial salt (NaCl) (Anastasijević, 2007). Due to the
protection de-icing during the winter period, in the land of city zones and the most effective
roads, very often there are very damaging processes, such as salinization, alkalinization and
accumulation havy metals (Nešić, 2004). Salinization is widespread today, is a growing problem.
During the winter period, roads in Serbia are sprayed with sodium chloride (NaCl). It is most
commonly used because of their low market value and great accessibility. It’s concentration has a
negative impact on the ground and the plant. This makes it difficult to adopt nutrients and water,
which can be toxic to plants. The most endangered are those plants belonging to the group of
flowering plants since they have a very shallow root system, gently tree and sheets, and can adopt
a higher amount of sodium-chloride. The optimal concentration of sodium chloride salt in the soil
solution is 0.001-0.01%.
185
High salt levels not only cause harmful effects on plants, but also increase the pH of soil
reaction. High salt concentrations can affect the deterioration of the soil structure, resulting in a
difficult flow of air and water, which are important for the biological processes occurring in the
root sistem of the plant. As a results of the harmful effects of salinization, reduced plant
productivity, while arable land irretrievably lost. Salinization land causes different effects on the
physiology of plants, such as increased rate of respiration, ion toxicity, changes in plant growth,
reducing photosynthesis and other changes which are due to replacement of calcium with sodium
(Khalid et al.,2010). The accumulation of salts in morphological organism of plants causing it’s
premature aging. This reduced supply of assimilates of plant parts that are the fastest growing and
thus the growth of plant tissue. On the sheets, dark necrotic surfaces appear first at the edges of
the leaf and later spread to the inside of the leaf surface. As sheets die photosynthetic capacity of
the plant is reduced and the end result is that the sheets prematurely dying and dried. The same
thing happens with flowers whose petals first begin to curl and fall of later.
MATERIAL AND METHOD
Pansy (Viola x wittrockiana) is a two year plant, most commonly grown as a one year old.
It’s quite durable and has a bloom that flashes troughout the season. Grows best during the cold
days of spring and autumn, but it also grows and bloom during the winter. There are many
varieties with shiny flowers and with or without a characteristic ’’face’’ or stain. They are very
suitable for planting in parks, gardens and on other green areas.
The experiment was set in the Botanical garden of Faculty of Agriculture in Novi Sad.
Testing experiment lasted eight weeks. For the purpose of this research, a total used 40 seeds of
pansy was divided into four treatments of ten seedlings. The tretments were labeled: control,
treatment I where was used 3 g/l NaCl, treatment II where was used 5 g/l NaCl and treatment III
where was used 7 g/l NaCl. Only control treatment was not performed with concentrated water.
Before it started testing all seedlings were examined in detail, especially the root system and
concluded that they all had a healthy and well developed root system without any damage. The
transplanted seedlings were in the greenhouse during the entire research in order to facilitate
aclimatization and preservation from cold and adverse weather conditions. During the test,
certain parameters were measured, such as: height, number of leaf, number of flowers, diameter
of flowers, length of leaf, width of leaf as well as vitality and decorativity. Using these
parameters, it was noted how a certain amount of salt influenced the growth of violet during a
certain period of time.
RESULTS
The morphological parametars examined in this paper will be shown in table 1.The
experiment lasted eight weeks. The results are shown for the first, fourth and eight weeks of the
study.
186
Table 1. Morphological parameters examined in treated pansy
One week
Number Number Diameter Lenght Width Vitality
Height of of of of of and
leaves flowers flowers leaves leaves decorative
K 13 31 3 7 5 3 5
T1 13 29 2 8 5 3 4
T2 13 28 2 7 5 3 4
T3 14 30 2 8 5 3 4
Fourth week
K 11 36 2 8 5 3 5
T1 10 39 1 8 4 3 4
T2 11 37 3 8 5 3 4
T3 9 35 2 8 4 3 4
Eight week
K 7 29 2 6 4 3 4
T1 6 28 2 8 4 3 3
T2 9 30 3 7 4 3 3
T3 6 25 2 / 4 2 2
In the first week, watering with concentrated water was done, each treatment was filled with a
certain salt concentration. During the first week there were no significant changes from the aspect
of the measured parameters (height, number of leaf, number of flowers, diametar of flowers,
length and width of leaf and vitality and decorativity), but there were some changes to the sheets
such as mild depressions, mild curling sheets, brown spots due to the influence of salt. In the
treatment I, where the plants were treated with 3 g/l NaCl, the leaves began to lose their glow and
they became mildly rough. The plants which had been treated with 5 g/l NaCl, the sheets received
mild depressions on the surface began to yellow and slightly curl. Significant changes did not
occurred in plants treated with 7 g/l NaCl as well as in plants not treated.
The plants which had been treated with 7 g/l NaCl in the fourth week, there was a large
change. Drastically reduced the measured parameters ( height, number of leaf, number of flowers,
diameter of flowers, length of leaf, width of leaf as well as vitality and decorativity). While plants
treated with 3 and 5 g/l NaCl remained in average values. In this week the plants which had been
treated, in addition to mild depressions, have also appeared brown spots on the sheets, while
some plants had a brownish-black edges on the sheets.
At the end of the experiment, in the eight week, the measured parameters dropped drastically.
The height ranges from 6-9cm, while the number of sheets reaches up to 25 sheets per plant.
Also, the number of flowers has drastically decreased, a large number of plants in this week have
no flowers, and the length and width of the leaves remained within the optimal range. In the
eighth week plants treated with 3, 5 and 7 g/l NaCl, in addition to changes on the leaves,
appeared on the flowers. At the flowers, there was drying, and at the end of their decline.
187
Figure 1. The seedling at the end of experiment
After the last treatment, soil control was carried out. Measurement of electrical resistance, pH
value and electrical conductivity of the extract in all treatments was carried out.
Table 2. The content

ent of total water
water-soluble salts in the soil
Control Treatment I Treatment II Treatment III
3 g/l NaCl 5 g/l NaCl 7 g/l NaCl
% salt 0.12 0.32 0.38 0.44
From the results shown in table 2., we can conclude that the highest percentage of salt occurs
in treatment III, where plants are treated with 7 g/l NaCl, amount to 0.44% and the lowest
percentage occurs in the control where it is 0.12%.
Table 3. pH value of the tested samples

pH 7.01 7.30 7.31 7.25
Table 3. shows the average values of the pH of the tested substrate samples. The pH values
measured in the saturation land fall usually is between 7.01 to 7.31, which means that such a
substrate has a neutral to slightly alkaline reaction. Plants that are found in treatment II, treated
with 5 g/l NaCl, had a maximum pH value to 7.31.
Table 4. The value of ECe at 25oC (mS/cm) of the water extract of the substrate
ECe (mS/cm) 0.53 2.8 3.67 6.54
The values of the obtained results of the electrical conductivity of the saturated substrate
extract are shown in table 3. From this we conlude that the highest value of ECe has a treatment
in whichh the plants are treated with 7 g/l NaCl, it is 6.54 mS/cm. When this value ECe plant
species will suffer a greater drop in the yield, it is necessary to ensure their adequate care in order
to have good vitality and decorative.
188
DISCUSSION
As is well known, salinity is one of the most important agricultural and environmental factors
that drastically affects large areas, causing reduction of crop productivity throughout the world.
Salinity and alkalinity are the two most important factors in water quality and if not
monitored carefully, can affect the reduction of growth and development in plants up to 50%
(Roberts, 1991). The results of this study show that concentrated water can drastically affect the
growth and development of plants and what kind of consequences it leaves on them. Reserch on
influence of salinity to pansy and impatiens in different growth media by Kuehni and Morales
(1998) also proved that necrotic area on the leaves and the number of leaves and flowers is
reduced. The results of the Sayyed et al. (2014), which was done on a cadiz (Tagetes erecta),
show similar changes that occurred with pansy under the influence of salt. There was damage to
the root system, to reduce the number of leaf and flowers. Salinization is today one of the biggest
problems in the agricultural world, every day doing new research and testing various plants in
order to figure out how to reduce it. Some plant species are more tolerant of high concentrations
of salt and some less. Depending on the plant species and of the same age and the types and
amounts of salts which are present in the soil and the time of exposure, it affects the adoption in
salts as well as the rate of plant growth and development, but also affects the difficulty in
adoption in mineral substances that are required plants to develop normally and grow (Bulut et
al.,2010).
CONCLUSION
On the basis of the examined in this paper, we can notice that larger quantities of salt in the
autumn-winter period damage the pansy. Although it is a very durable flower species, it is still
susceptible to a higher concentration of salt, which adversely affects it’s growth and
development. From the results shown in this paper, we can conluded that the parameters tested
were drastically reduced in plants treated with 5 and 7 g/l NaCl. Also, in these plants, changes
occurred mostly on the surface of the leaf in the form of depressions, brown flea, curling sheets
and the appearance of yellow leaf, as far as the flowers are concerned, their drying and falling.
Based on the pedological analysis we can conclude that the highest pH value of the soil solution
have treatment II, where the plants are treated with 5 g/l NaCl. Plant species which are treated
with 7 g/l NaCl have the highest percentage of water-soluble salts as well as the highest value
ECe water extract.
Recommendation for the maintenance of healthy and decorative pansy seedlings would be
during the melting of ice and snow during the winter period using less salt concentrations on
those surfaces where flower beds are found, as well as different materials (from fabric or plastic)
for covering the seedlings in order to maintained it’s vitality and decorativity
189
REFERENCES
ANASTASIJEVIĆ. (2007). Podizanje i negovanje zelenih površina. Šumarski fakultet,

Beograd ISBN 978-86-7299-321-9.
ASHRAF, M. (2004). Some important physiological selection criteria for salt tolerance in
plants. U Flora (str. 361-370).
KHALID NAWAZ, K. H. (Jun 2010). Fatality of salt stress to plants: Morphological,
physiological and biochemical aspects.
KUEHNY S. JEFF AND MORALES BLANCA. (1998). Effects of salinity and alkalinity
on pansy and impatiens in three different growing media. Journal of Plant Nutrition, 21:5,
1011-1023, DOI: 10.1080/01904169809365460.
MUNNS R. (2002). Comparatiiv physiology of salt and water stres. U Plant Cell Envirion
(str. 25: 239-250).
MUNNS R. (2005). Genes and salt tolerance: bringing them together. U New Phytol (str.
167: 645-663).
NEŠIĆ LJILJANA. (2004). Uticaj zaslanjenosti zemljišta i vode na proizvodnju povrća u
zaštićenom prostoru. Savremeni povrtar- naučno-stručni časopis iz oblasti povrtarstva.UDK:
635.1/.8(05) ISSN: 1451-2793, godine III;, broj 9, str. 7-8.
PLAZA M. BLANCA, F. F. (2013). Effects of treated and Untreated wastewater from
Municipal Solid Waste (MSW) leachates on the nutritional state of viola spp.: sodium,
potassium, calcium and magnesium. Communications in Soil Science and Plant Analysis,
44:1-4, 480-491, DOI:10.1080/00103624.2013.744148.
SAYYED AQIB, G. H. (2014). Effect of salt stress on growth of Tagetes erecta L.
Pakhtunkhwa J. Life Sci. Volume 02, Issue 03/04, 2014, P 96-106, 96-106.
SUSAN, V. Z. (2002). Delyed and carryover effects of salinity on flowering in Iris
hexagona (Iridaceae). American Journal of Botany, 89(11): 1847–1851.
190
EFFICIENCY OF IMMOBILIZED BRADYRHIZOBIUM JAPONICUM STRAIN USING
ENCAPSULATION METHOD
Dora Krznarić, Bernarda Lenkert, Ana Puljko
Mentor: Sanja Kajić, mag. mol. biol
University of Zagreb, Faculty of Agriculture, Department of Microbiology, Croatia
Summary: Inoculation method in this work was performed with ionic gelation encapsulation.
The alginate based microcapsules (300 μm) contained viable B. japonicum strain were solifided
in CaCl2. Initial number of viable bacteria in every sample was 9,0 log CFU/ml. Chitozan coated
capsules had higher mortality rate than alginate capsules with 1,3 log CFU/ml in lyophyilisated
and wet microcapsules. The high viability of B. japonicum was registered in wet capsules
storaged at constant -20°C for thirty days with viability rate of 8,84 log CFU/ml.
Keywords: Encapsulation, Bradyrhizobium japonicum, storage, viabilty, biofertilizers
INTRODUCTION
Seed inoculation by microorganisms is one of the basic methods in agriculture production. This
method is known from 19th century when Beijernick isolated and describe the microorganisms
that form nodules on legume roots. (Crawford et al., 2000). The goal of inoculation technique is
providing adequte numbers of live and effective rhizobial bacteria for colonisation of soil and
plant root which will result in rapid start of root nodulation. The better nodulation will have
effect in increased N2 fixation and better crop yields (Catroux et al., 2001; Deaker et al., 2004).
As for now, the majority of commercial bacterial inoculums are peat-based (Denton et al., 2009).
The peat as carrier for microorganisms can be easily applied as coating on legume seeds or used
for direct application in the soil. The peat-based formulations yield a good results, especially with
rhizobial inoculums (Thompson 1980; Denton et al 2009). Disadvantage of convencional peat-
based inoculums is its longevity and protection from environmental factors during storage before
use and during application in the soil (Fallik and Okon, 1996; Bashan 1998). In the last decades,
there is increased tendency in searching for a formulations of the carriers. The aim of
bioencapsulation is the protection of living bacteria before and during introduction into soil, as
well as their slowly and prolonged release in the seedbed (Bashan, 1986; Kim et al., 2012,
Schoebitz et al., 2013). Encapsulation advantages are: better protection from biotic and abiotic
stresses, shelflife stability and decreased contamination during storage time and transportation
and controled release of microorganisms by soil degradation (Bashan et al., 2002). However, it is
very costly when compared to peat-based carriers and it requires more biotechnological expertise
(Fages 1992). The difference between convencional and encapsulation methods are that in the
microcapsules, whole bacterial cells are immobilized in defined space where the cells are
191
protected from the environment (Karel et al., 1985; Rathore et al., 2012). The viable bacterial
cultures are directly put and mix into commerically available carriers and they do not provide
sufficient protection against external conditons. With encapsulation method the cells are
immobilized within the capsule which is mostly made from biodegradable polymer materials.
The capsule itself has permeabile structure that allows access of nutrients, exchange of the gases
and metabolites for maintaining the viability of immobilized cells (Ding and Shah, 2009; John et
al., 2011; Rathore et al., 2012). Alginate is the most common polymer for construction of macro-
or microcapsules in many industries and it is extracted from brown algae. The main purpose of
the matrix in the capsules is to protect bacterial cells from environment and maintance of their
viability during storage time (Young et al., 2006). The alginate is linear macromolecule consisted
of two monomers linked via β - (1-4)-D-Mannuronic acid to α - (1-4) -L-Guluoronic acid
(Schoebit et al., 2013). There is a various formulations and methods for making microcapsules. In
agricultural and food industry the ionic gelation method is the most recognized. For pre-
ecapsulation process the alginate solution is thoroughly mixed with cell culture. After formation
of the droplets in the machine, they are mixed with CaCl2 (usually 0,05 – 0,1 M) where capsules
obtain their recognizable shape (Cassidy et al., 1996). The formation of the capsule is happening
in the first few second of contact between Na-alginate and Ca2+ ion whereas induration of the
capsule occurs. Na-alginate droplet solidification is possible when Ca2+ react with negatively
charged alginate chains and the three-dimensional structure is made. This structure contains water
as hydrogel through which Ca2+ is diffused into the liquid droplet and coats it via crosslinking
“from outside to inside” (Vemmer and Patel, 2013). One more protection against mechanical
stress during storage is adding outer layer or extra coating on the capsules (Zaiem et al., 2017).
With ionic polymer coating the morphology of the capsule is define as suspension of polyanionic
beads – core of the capsule and polycationic solution – shell of the capsule. In this case, the
polyanionic solution is chitosan (0,2 % solution). Between chitosans amine groups and alginates
carboxyl groups complex formation is made, that add more protection to living cells inside beads
(Poncelet et al., 2001).
The aim of this study was to make small sized (300 μm) sodium alginate microcapsules which
contain viable cells of commercial Bradyrhizobium japonicum 344 strain. By supplementing
microcapsule with an extra chitosan layer it is consideret that the capsules would get an extra
protection during storage time. The utter aim was to determine the encapsulated cell viability at
different storage temperature and which one is most adequate because one of the most restrictive
effect in bacterial inoculation production is high cell morality of bacteria during storage time.
Medium and bacterial strain. Medium used for bacterial growth was YMB (Yeast Mannitol
Broth). The composition per liter was: 0.5 g K2HPO4; 0.1 g NaCl; 0,2 g MgSO4; 10 g mannitol;
5 ml bromothymol blue; 0.4 g Yeast extract. Rhizobial strain used for encapsulation was
192
Bradyrhizobium japonicum 344 and was obtained from strain collection of Univeristy of Zagreb,
Faculty of Agriculture, Department of Microbiology.
Preparation of strain sample for encapsulation. For preparation of bacterial sample for
encapsulation itself, in 40 ml of sterile YMB solution it was added pure culture of B. japonicum
344 strain with sterile loop. The bacterial sample was placed in incubation shaker (Orbital
Shaker-Incubator ES-20, Biosan) for 7 days (150-200 rpm, 28 °C) for its growth.
The encapsulation method and microcapsule formation. The initial step of encapsulation process
was adding 40 ml of bacterial suspension grown in YMB into 500 ml Erlenmayer flask with 160
ml sodium alginate. Sodium alginate was prepared ahead by dissolving it in water for 30 minutes.
The suspension with Na-alginate was thorougly mixed. A tube from Encapsulator B-390 (Büchi,
Switzerland; provided from Department of Chemistry, Faculty of Agriculture, University of
Zagreb) was placed inside Erlenmayer flask and it sucked the Na-alginate bacterial suspension.
The formation of microcapsules was conducted in this conditions: air pressure was 27 – 28 mbar,
at frequency of 800 Hz in sterile environment. Diameter of the nozzle was 300 μm which was
also the size of final microcapsules. Under the nozzle was placed 500 ml glass flask which
contained 0.1 M CaCl2. When drops of Na-alginate bacterial suspension was dripping into CaCl2
they immediately formed bead shape droplets. For fully formation of microcapsules, the glass
flask was placed on magnetic stirrer (Tehtnica Železniki MM- 540) and stirred on low speed for
40 minutes. The encapsulation method yielded 50 grams of Na-alginate microcapsules. The beads
were transfered from CaCl2 on sterile gauze and carefully rinsed with saline solution (0.85 %
(w/v) NaCl). One half of the capsules were coated with 100 ml of 0.2 % polysaccharide chitozan
solution, aditionally. The microcapsules with only Na-alginate and with added chitosan were
placed at various temperatures in Petri dishes.
Microcapsule storage at different temperatures. Wet sodium alginate microcapsules were stored
at – 20 °C and 7 °C, likewise they were stored at room temperature as wet and as lyophyilisated
microcapsules. The growth rate of rhizobial bacteria was determined from zero until thirtieth day
(days of observation were 0., 1., 2., 4., 6., 8., 10., 12., 14., 20., 22., 30.) One part of sodium
alginate capsule was stored at -20 °C and bacterial viability was observed only on zero and
thirtieth day. Wet chitosan microcapsules and lyophyilisated ones were stored at the same
conditions described above.
Cell count and morphology of microcapsules.The cell viability was determined for thirty days
after process of microcapsulation except the one sample that was stored at -20 °C where
enumeration of cells were conducted only first and last day. The capsules with entrapped bacteria
had to be desolved in the tube that contained 8 ml of sodium solution with added 1 ml Na-
tricitrate for easier count of viable bacteria. The enumeration was carry out only with living cells
by serial dilution method. Liquid bacterial inoculums (0,1 ml of bacterial solution) was plated on
YMA (Yeast Mannitol Agar) and incubated 5-7 days at 30 °C. After incubation it was determined
the growth rate of B. japonicum 344 and the results were asserted as CFU (Colony Forming
Units)/ml. The results were carried out as an average of three repetition. Morphology of
microcapsules were observed under fluorescence microscop (Olympus BX60).
193
RESULTS AND DISCCUSION
Number determination of B. japonicum 344 and capsule microscopy

Initial culture concentration of B. japonicum 344 after encapsulation in Na-alginate
microcapsules as well as Na-alginate microcapsules treated with chitosan was 9,0 log CFU/mL.
Samples from different storage temperatures were taken from zero to 30 days and the
microcapsules morphology and bacteria viability were determined. It was noted that Na-alginate
capsules had a well-defined membrane (Fig 1a-d.).
Fig 1. Microcapsules stored at + 7 ° C, (a) day zero, (b) tenth day, (c) eighteen day, (d) twentieth day (a-d: Na-
alginate); (e) first day, (f) twentieth day (e-f: chitozan coated)
Encapsulation process decreases viability of a certain amount of bacteria (Ivanova et al.2005;

Bashan et al., 2002). With the method of encapsulation the spheric microcapsules with the
diameter of 300 μm were obtained. This capsule size has a higher bacterial survival rate, because
they have good mechanical strength, better diffusionas well as nutrient and metabolite
modification and this size of capsule are optimal for oxygen intake (Uludag et al., 2000; Rathore
et al., 2013). Chitosan is added as an additional layer protector around the capsule membrane to
improve the storage capability of the capsule itself and directly affects the survival of the
bacteria. Under microscope a slight deformation of the membrane of the capsule was shown,
already after the first day (Fig 1e.). It is evident that with the increase of the day of storage, the
chitosan-treated capsule membranes have become more deformed, affecting the survival of the
bacteria inside them (Fig 1f.).
194
Viability of B. japonicum 344 in Na-alginate microcapsules at different temperatures
It is shown in Fig 2. that after initial number of bacteria (9,0 log CFU/ml), every sample had
same number of bacteria on the first day, except wet Na-alginate capsules stored at -20 °C, with
numbers dropping for 1 log. The decreasing trend was observed in every given sample but the
most significant decline in Na-alginate microcapsules was deteced in wet bacterial samples stored
at -20 °C. After ten days in this sample number of bacteria dropped to 5,67 log CFU/ml. On 20.
day viability dropped to almost 63 % of theirs initial number. Last day of sampling (30. day) the
viability of bacteria was 2,33 log CFU/ml. Wet microcapsules and lyophilized microcapsules
stored at room temperature had similar results in reduction of number in bacterial viability. On
second day of sampling, their microcapsules contained 8,67 log CFU/ml of bacteria. In wet Na-
alginate capsules was slightly greater decline in bacterial numbers. More noticable drop in
numbers was observed in lyophilized microcapsules between 14. and 20. days. Number of
bacteria dropped from 6,33 log to 5,33 log CFU/ml. At the last day of determination above
mentoned samples final number of bacteria were 4,33 log CFU/ml for both samples. The best
results in observed Na-alginate microcapsules which were subject to constant environmental
changes during storage was in wet sample stored at +7°C. The decline of bacterial numbers was
noticed on forth day (8,67 log CFU/ml). A large decrease was observed between day 6 and 8
where the viability dropped from 8.67 log to 7.7 log CFU/ml. The constant CFU was shown
between 20. and 22. days (6,33 log CFU/ml). At the last day there was slightly decreas in
bacterial number with result of 6,00 log CFU/ml.
Fig 2. B. japonicum 344 viability during 30 days of storage in Na-alginate microcapsules at different temperatures
and different storage conditions (log CFU/mL)
The best results had wet Na-alginate microcapsules stored at -20 °C from zero to thirtied day.
The decline in viability was recorded for only 1 log and with the initial 9.0 log dropped to 8.48
log CFU/ml. The capsules were stored at constant temperature, and the bacteria had slow vital
functions. Since the other microcapsules were under greater influence of temperature change, this
had a significant effect on the decrease in their viability, as well as on the structure and
permeability of the capsules themselves, resulting a reduction in the protection of bacteria from
external influences.
195
Viability of B. japonicum 344 in Na-alginate coated with chitozan microcapsules at different
temperatures
Na-alginate coated with chitozan microcapsules showed greater decline in bacterial viability than
in Na-alginate microcapsules. As it is demonstrated in Fig 3., on first day of sampling there were
decreased bacterial numbers (8,33 log CFU/ml in every sample). The decrease in bacterial
number in samples coated with chitozan ranged in similar numbers. It was observed that wet
capsules stored at -20 °C had slightly bigger bacterial number than the rest of the samples. Final
number at -20 °C storage samples was 3,33log CFU/ml which is for 1 log greater than same
sample but without chitozan coating. Second greatest results gave wet chitozan coated
microcapsules stored at +7 °C. Decline between zero day of sampling (9,0 log CFU/ml) and last
day of sampling (2,33 log CFU/ml) was significant. The number of viable bacteria were reduced
for 74,11 %. Even more greater decline in bacterial numbers were noted in wet and lyophilized
microcapsules stored at room temperature. Their number of alive bacteria dropped from 9,0 log
CFU/ml to 1,33 log CFU/ml which is decline of 85,22 %. There are several reasons why chitozan
coated microcapsules affected on number of bacteria. One of the reasons for reduced bacterial
viability in capsules coated with chitozan is the deformation of the membrane capsule itself.
Second reason for capsule deformation is that chitozan does not enter deep into the alginate
structure but accumulates on the outside (Gaserod et al., 1998). Another reason is the competence
between the Ca2+ ions and the chitozan amino group. The Ca2+ immediately hardened, and
complex coacervation formation was made between chitosan and alginate. A porous gel structure
is formed, and chitozan molecules have come into contact with bacteria after a certain time
(Gaserod et al., 1998; Wittaya-arekul et al., 2006). Besides these reasons, chitosan has an
antimicrobial effect as it affects the cell membranes and damages the membrane function (Raafat
et al., 2008).
Fig 3. B. japonicum 344 viability during 30 days of storage in Na-alginate coated with chitozan microcapsules at
different temperatures and different storage conditions (log CFU/ml).
196
CONCLUSION
In conclusion, for yielding sufficient number of viable bacteria immobilized within sodium
alginate microcapsules the storage temperature should be low (e.g. -20°C) and constant. By
comparison, the consistent environment factors during storage time are also important in the
production of every available commercial inoculant. Supplementing Na-alginate microcapsules
with chitozan coating did not demonstrate to add protection to microcapsules itself, on the
contrary the number of bacteria was lower than single-coated Na-alginate capsules. This was
preliminary study, considering that encapsulation methods in agriculture is still not at the
industrial level. Additional investigations should be carried out, involving other species and
strains of soil-borne bacteria with further laboratory experiments and field trials, which should be
conducted for observations under natural conditions.
REFERENCES
BASHAN, Y. (1986): Alginate beads as synthetic inoculant carrieers for the slow release of
bacteria tat affect plant growth. App Environ Microbiol 51: 1089-1098
BASHAN Y. (1998): Inoculants of plant groeth-promoting bacteria for use in agriculture.
Biotechnol Adv 16: 729-770
BASHAN, Y., HERNANDEZ, J., P. LEYVA, L.A. BACILIO, M. (2002): Alginate microbeads
as inoculant carriers for plant growth-promoting bacteria, Biol Fertil Soils 35: 359-368
CASSIDY, M.B., LEE, H. TREVORS, J.T. (1996): Environmental application of immobilized
microbial cells: a review, J Ind Microbiol 16: 79-101
CATROUX, G., HARTMAN, A., AND REVELLIN, C. (2001): Trends in rhizobial inoculation
production and use. Plant and soil 230: 21-30.
CRAWFORD, N.M., KAHN, M., LEUSTEK, T., LONG, S.R. (2000): Nitrogen and sulfur. U:
Biochemistry and molecular biology of plants (Buchanan, B.B, Gruissern, W., Jones, R., Eds.).
American Society of Plant Physiologist, Rockville, 786-849p.
DEAKER, R., ROUGHLEY, R.J., KENNEDY, I.R. (2004): Legume seed inoculation technology
– a review, Soil Biol Biochem 36: 1275-1288
DENTON, M.D., PEARCE, D.J., BALLARD, R.A., HANNAH, M.C., MUTCH, L.A., NORNG,
S. (2009): A multi-site field evaluation of granular inoculants for legume nodulation. Soil Biol
Biochem 41: 2508–2516
DING, W. K., SHAH, N.P. (2009). An improve method of microencapsulation of probiotic
bacteria for their stabiltix in acidic and bile conditions during storage. Journal of Food Science 74
(2): 53-61
FALLIK, E., OKON, Y. (1996): Inoculants of Azospirillum brasilense: biomass production,
survival and growth promotion of Setaria italica and Zea mays. Soil Biol Biochem 28: 123-126
FAGES, J. (1992): An industrial view of Azzosporillium inoculants: formulation and application
technology. Symbiosis 13: 15-26
197
GASEROD, O., SMIDSROD, O., SKJAK-BRAK, G. (1998): Microcapsules ofalginate-
chitosan—I: a quantitative study of the interactionbetween alginate and chitosan. Biomaterials
19, 1815–1825
IVANOVA, E., TEUNOU, E. PONCELET, D. (2005): Alginate based macrocapsules as
inoculant carriers for the production of nitrogen fixing bio fertilizers. CI&CEQ 12(1): 31-39
JOHN, R.P., TYAGI, R.D., BRAR, S.K., SURAMPALLI, R.Y., PRÉVOST, D. (2011):
Bioencapsulation of microbial cells for targeted agricultural delivery. Critical Reviews in
Biotechnology 31 (3): 211–226.
KAREL, S.F., LIBICKI, S.B., ROBERTSON, C.R. (1985): The immobilization of whole cells:
engineering principles. Chemical Engineering Science 40 (8): 1321– 135p
PONCELET, D., DULIEU, C., JACQUOT, M. (2001): Description of the Support Material.
In:Wijffels, R.H. (Ed.), Immobilized Cells. Springer, Heidelberg, Berlin.
RAAFAT, D., VON BARGEN, K., HAAS, A., SAHL, H.G. (2008): Insights intothe mode of
action of chitosan as an antibacterial compound. Appl. Environ. Microbiol. 74, 3764–3773
RATHORE, S., DESAI, P.M., LIEW, C.V., CHAN, L.W., HENG, P.W.S. (2013):
Microencapsulation of microbial cells. Journal of Food Engineering 166: 369-381
THOMPSON, J.A. (1980): Production and quality control of legume inoculants. In: Methods for
evaluationg biological nitrogen fixation (Begerson, F.J., Ed.) Wiley, New york 489-533p.
ULUDAG, H., DE VOS, P., TRESCO, P.A. (2000): Technology of mammalian cell
encapsulation. Advanced Drug Delivery Reviews 42 (1–2), 29–64
WITTAYA-AREEKUL, S., KRUENATE, J., PRAHSARN, C. (2006): Preparation and in vitro
evaluation of mucoadhesive properties of alginate/chitosan microparrticles containing
prednisolone. Int J Pharm 312 (1-2): 113-118
YOUNG, C.C., REKHA, P.D., LAI, W.A., ARUN, A.B. (2006): Encapsulation of plant growth-
promoting bacteria in alginate beads enriched with humic acid. Biotechnol Bioeng 95:76–83
ZAEIM, D., SARABI-JAMAB, M., GHORANI, B., KADKHODAEE, R., TROMP, R. H.
(2015): Electrosprax assisted fabrication of hydrogel microcapsules by single- and double-stage
procedures for encapsulation of probiotics. Food and Bioproducts Processing 102: 250-259.
198
WIDESPREAD OF ASIAN FLY (DROSOPHILA SUZUKII, MATSUMURA) IN
DIFFERENT PLANTATIONS
Tamara Stanković
Mentor: dr. Aleksandra Popović
University of Novi Sad, Faculty of Agriculture, Department of plant and environmental
protection
Summary: The fruit fly Drosophila suzukii is a fruit crop pest and is a serious economic threat to
soft summer fruit. A polyphagous pest, it infest a wide range of fruit crops, included grape, as
well as an increasing number of wiled fruits. In Serbia, since its first occurrence in 2014
(Tosevski et al., 2014), D. suzukii has been causing damage in different crops. The damage
caused by D. suzukii larvae renders the fruit unmarketable (Bolda et al., 2010).This work
presents monitoring of D. suzukii in 2018 and it shows the widespread of this pest in the area of
regional centre Sremska Mitrovica. The presence of pests in traps was registered on 7 localities.
The increased number of individuals compared to 2017, widespread and favourable natural
conditions for development of D.suzukii on the territory of Srem indicate risks for production of
fruit and grape.
Key words: pest, Drosophila suzukii, fruit
INTRODUCTION
In june, 1916, larvae were found in pre-harvest cherries (Prunus avium) in Japan. The invaded
fruit were collected and the adult flies that appeared were confirmed as a species of Drosophila.
Later, in 1931, Dr. Shounen Matsumura described the species as Drosophila suzukii. On new
territories the species had spread by international trade. In Europe was recognized for the first
time in 2008 and in Serbia 2014 (Tosevski et al., 2014). It’s adaptability, large number of
generations and wide number of hosts enabled their survival and spreading on new territories.
Unlike other Drosophila, D.suzukii can cause damage on fruit in ripening phase. Drosophila
suzukii has two imago formes: summer and winter form. Male summer forms are brown-yellow
colored with black stripes on the abdomen and have red eyes. They are 2-3mm in lenght. The
main characteristics are a black-grey spot in the corner of the wings and short hairs on the front
feet. Females don’t have a spot on the wings, but have a large serrated ovipositor. Summer forms
are light colored and have black stripes on their back. The antennas are short. The main ankle is
winder, the upper ones are shortened by the ends. On the antennes are feather- like hairs. The oral
apparatus is specialized for sucking liquides. Compared to the body, the oral apparatus is long
and has inolucum of microorganisms for fruit degradation. Females lay eggs by ovipositor.
Larvae feeding in fruits can cause their damage. The development of an egg lasts 1-3 days, of a
larvae 3-13 days and of an adult 4-16 days (Kanzawa 1939 cit. Calabria et al. 2012). Adults live
3-9 weeks, and favourable natural conditions can cause the development of up to 13 generations
per year. During one oviposition term, a female lays maximum 3 eggs. Oviposition takes place in
the phase when the fruit changes it’s colour, when the fruit skin is softened and the sugar level is
199
higher. The female prefers juicy fruits with thin skin. Damage appears as an effect of feeding on
fruits, the fruit becomes soft, changes color and the places of stab can be susceptible for
secondary infections. Accommodation of D.suzukii for surviving winter takes place in fall when
it’s feeding is reduced and then transit to reproductive diapause. If the larvae were feeding
themselves and suddenly low temperatures appear, they die because of the „nucleus“ in their
intestines. Imagos search for a place to survive winter. Unlike other potentially invasive pests,
Drosophila suzukii is not an object of regulation in USA and Europe, for now it’s just monitoring
and it is on EPPO A2 quarantine list (EPPO 2013). The purpose of monitoring is to determine the
widespread od Asian fly in diffrent plantations in Srem and to determine flying dinamic during
vegetation season by the help of traps with apple vinegar basis.
Monitoring of widespread of Drosophila suzukii is implemented during 2018 in 5 vineyard, 3

raspberry, 2 peach and 2 blackberry plantations. Climate conditions and air temperature are very
important, so the data were taken from the Republic hidrometeorological institute for the region
of Sremska Mitrovica and because of this research the data was upgraded. The monitoring was
performed by the help of traps for which were used apple vinegar and wine solutions (40:60),
because it is more attractive than yeast. This solution was poured in plastic bottles which had a
small hole 3cm under the bottle cap, so the adults could go in it. A few drops of detergent were
added in apple cider vinegar (ACV), so the flies could fall to the bottom. The traps were set up: 1
trap per plot bigger than 5ha and 2 traps per plot smaller than 5ha. In vineyards and peach
plantations the traps were set up for the first time on, and in raspberry and blackberry plantations
July. Samples were collected once in a week untill October 2018. Vinegar was changed twice a
month. Most of the traps were set up in shadowy places. During the inspection of the catch, in
order to reduce the damage to the flies, vinegar solution was filtered through funnel or soft fabric,
and than destilated water was added to remove the excess. In this way refined catch is put on
white paper to decaut liquid , and than transffered into another bowl. Before recording, water was
added to separate the flies from each other.
The presence of Drosophila suzukii was registered for the first time in July in grape plantations in
all 5 localities. The total number of caught adults was 2200(Chart 1).
200
Chart 1. Number of adults of Drosophila suzukii in vineyards
In raspberry plantations the first individuals were also registered in July in 3 locations with total
number of 869. In peach plantations the first catch was at the end of July, the number of adults
were 748, while the first catch in blackberry plantations was registered at the begin of August,
with the number of 378 adults(Chart 2).
Chart 2. Number of adults of D. suzukii in plantations of blackberry, raspberry and peach
On the basis of these results it can be assumed that in plantations on the territory of Srem, the
number of pests was sporadical, increase was in ripening phase, and the further survival of the
population was enabled by the fruit they were in. According to research (dos Santos et al. 2017),
medium annual temperatures of 5°C to 20°C increase the survival of D.suzukii. Medium annual
temperature in Sremska Mitrovica is 13,3°C, which means that the terms were in favor of the
development of D. suzukii. Asplen et al. 2015 induce that the optimal temperature needed for
activity of Drosophila suzukii is 10-20°C, and the minimal needed for flying is 10°C. According
to Chart 3, it is obviously that the average monthly temperatures from March to November were
in the optimal range for activity. The minimum temperature for the flight was present during all
days in the months from July to November when it was 7°C.
201
Chart 3. Average monthly temeprature in Srem
The largest number of captured individuals was registered in the vineyard in the Divos 1267,
followed by the peach orchard in Molovin with 748 adults caught, while in raspberry plantations
the largest number was registered in Divos 400, and in the blackberry plantation at the locality of
Nocaj 352 individuals (Chart 4).
Chart 4. The largest number od D. suzukii
If the number and presence of Drosophila suzukii in Srem (44° 58’N 19° 36’E) are compared
with data from Zagreb (45°48’N 15°58’E), one can notice a large deviation in number in the
same platations of raspberries, peach and blackberries. During the research period, 847 adults
were captured in Zagreb, while 1545 individuals were caught in Srem at 3 locations, which
indicates that the climatic conditions in Srem, select variety, the surrounding vegetation
contributed to the greater settlement of Asian fly in this territory.
CONCLUSION
According to this results, based on monitoring of Drosophila suzukii in 12 plantations on the

territory of Srem in 2018, it can be concluded:
 Drosophila suzukii was present in all 12 plantations
 Totall number of caught adults in period of monitoring was 4195
 Climate conditions on the territory of Srem were in favor for the development of the pest,
medium annual temperature was 13,3°C.
202
 Compared to other monitorings on the territory of Srem it developed a large number of
generations thanks to favorable climate conditions, domestic and wild vegetation in the
surrounding area.
 The program of protection in Serbia should include all producers of fruits and grapes,
otherwise their plantations present potential danger for other plantations and localities in
which Drosophila suzukii is not present or it is present in a small number.
REFERENCES
CABI (2014) Invasive Species Compendium. Adults. Envirom. Entomol. 44(6):1619-26

BOLDA M, GOODHUE RE, ZALOM Spotted wing drosophila: potential economic impact of a
newly established pest. Agricultural and Resource Economics, 13(3):5-8.FG, 2010.
DOS SANTOS LA, MENDES MF, Krüger AP, BLAUTH ML, GOTTSCHLAK MS, GRACIA
FRM(2017) Global potential distribution of Drosophila suzukii (Diptera, Drosophilidae). PLoS
ONE 12(3): e0174318
EPPO (2013): Drosophila suzukii (Diptera: Drosophilidae). Spotted wing drosophila.
KANZAWA T, 1939. Studies on Drosophila suzukii Mats. 49pp
STEPHENS AR, ASPLEN MK, HUTCHINSON WD, VENETTE RC (2015): Cold
Hardninessof Winter – Acclimated Drosophila suzukii (Diptera: Drosophilidae)
TOŠEVSKI, I. MILENKOVIĆ S. KRSTIĆ O, KOSOVAC A., JAKOVLJEVIĆ M., MITROVIĆ
M.,CVRKOVIĆ T., JOVIĆ J.(2014): Drosophila suzukii, Diptera, Drosophilidae a new invasive
pest in Serbia, Zaštita bilja, Vol. 65 (1) No.289:03-139. Beograd.
203
TABLE OF CONTENT:
INDOOR ECOSYSTEM SERVICES: PLANTS IMPACT ON AIR QUALITY ............... 6
ASPECTS OF MORPHO-PHENOLOGICAL DEVELOPMENT AT Triticum aestivum

L. ........................................................................................................................................ 13
OPEN SPACES AND ELEMENTS ADAPTED TO THE LANDSCAPE OF

HORTOBÁGY NATIONAL PARK ................................................................................. 18
MORPHOLOGICAL DIFFERENCES OF FOREIGN GENOTYPES OF ....................... 24
MICROBIOME AND IMPORTANCE OF PGPR IN RHIZOSPHERE OF MAIZE (ZEA

MAYS L.) ............................................................................................................................ 31
GERMINATION AND PROTOCORM FORMATION OF OPHRYS SPHEGODES

MILL. – IN VITRO PROTOCOL FOR AN ENDANGERED ORCHID SPECIES .......... 37
ANALYSIS OF THE DEVELOPMENT OPPORTUNITIES OF AGRICULTURAL

LANDSCAPE WITH GREEN INFRASTRUCTURE NETWORK IN A HUNGARIAN
RURAL AREA .................................................................................................................. 45
IPARD – CHALLENGES AND PERSPECTIVES OF AGRICULTURAL HOLDINGS

IN SERBIA ........................................................................................................................ 51
REFERENCES ................................................................................................................... 59
HISTOMORPHOMETRIC AND HISTOLOGICAL STUDY OF THE EFFECT OF

FREE OMENTAL PEDICLE GRAFT ON NON UNION MODEL IN RABBIT RADIUS
............................................................................................................................................ 61
SYSTEMIC EFFECTS OF EPIDURAL ANESTHESIA WITH LIDOCAINE-

ADRENALINE IN DOGS ................................................................................................. 66
PANTARSAL ARTHRODESIS USING FIXIN HYBRID PLATE IN A FRENCH

BULLDOG ......................................................................................................................... 72
DIAGNOSIS OF HIP DYSPLASIA IN DOGS ACCORDING TO THE GUIDELINES

OF THE INTERNATIONAL CYNOLOGICAL ASSOCIATION (FCI) ......................... 79
SELECTION CRITERIA OF BREEDING BULLS FOR MILK PRODUCTION TRAITS

............................................................................................................................................ 85
204
EFFECTS OF NONSTEROIDAL ANTI-INFLAMMATORY DROGS ON BONE
HEALING .......................................................................................................................... 92
THE EFFECT OF CALCIUM CHLORIDE APPLICATION ON STORAGE CAPACITY

AND QUALITY OF KIWIFRUITS DURING COLD STORAGE .................................. 97
QUALITY OF RED WINES PRODUCED WITH DIFFERENT OENOLOGICAL

PRODUCTS ..................................................................................................................... 102
EFFECTS OF LOW LEVEL WCR EGG INFESTATION ON MAIZE PLANTS IN THE

FIELD*............................................................................................................................. 112
Elimination of iron from groundwater for irrigation purpose .......................................... 121
COMPARISON OF ANTIOXIDANT PROPERTIES AMONG TWO APPLE

CULTIVARS ................................................................................................................... 127
THE INFLUENCE OF MATURITY STAGE AND EXTRACTION SOLVENT ON

TOTAL PHENOLICS AND ANTIOXIDANT ACTIVITY OF STALK OF SWEET
CHERRY .......................................................................................................................... 134
tHE CHROMATOGRAPHIC ANALYSIS OF THE STAR ANISE ESSENTIAL OIL AS

THE POTENTIAL BIOPESTICIDE ............................................................................... 141
SENSITIVITY OF CERCOSPORA BETICOLA ISOLATES TO AZOXYSTROBIN ... 147
INTRODUCTION ............................................................................................................ 147
THE GENETIC INTERACTION BETWEEN SUNFLOWER LINS IN THE PROCESS

OF DEVELOPING OROBANCHE.CUMANA RESISTANCE HYBRIDS .................. 153
Onisan Grigore-Emilian ................................................................................................... 153
Mentor: Petrescu Irina ...................................................................................................... 153
The effect of sodium chloride on the morphological development of Zea mays L. hybrids
.......................................................................................................................................... 159
PRELIMINARY RESULTS OF DETECTION OF Xanthomonas arboricola pv. corylina

(Xac) ON HAZELNUT IN REPUBLIC OF SRPSKA .................................................... 165
205
PESTS INCIDENCE IN DIFFERENT BBCH STAGES IN SMALL GRAINS AGRO-
ECOSYSTEMS THROUGHOUT AGGRESSIVITY ASSESSMENT .......................... 171
EFFICACY ASSESSMENT OF DIFFERENT CONTROL METHODS OF WEEDS IN

SOYBEAN AGRO-ECOSYSTEM.................................................................................. 178
INFLUENCE OF SALINITY ON GROWTH AND DEVELOPMENT OF PANSY

(VIOLA X WITTROCKIANA) ....................................................................................... 185
EFFICIENCY OF IMMOBILIZED BRADYRHIZOBIUM JAPONICUM STRAIN USING

ENCAPSULATION METHOD ....................................................................................... 191
WIDESPREAD OF ASIAN FLY (DROSOPHILA SUZUKII, MATSUMURA) IN

DIFFERENT PLANTATIONS ........................................................................................ 199
206

Proceedings Book of 42nd Conference For Students PDF

Uploaded by

Copyright:

Available Formats

Proceedings Book of 42nd Conference For Students PDF

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Proceedings Book of 42nd Conference For Students PDF

Uploaded by

Copyright:

Available Formats

42nd Conference For Students Of

Agriculture And Veterinary Medicine

University of Novi Sad, Faculty of Agriculture

UNIVERSITY OF NOVI SAD

UNIVERSITY OF AGRICULTURAL SCIENCES AND

Calea Aradului 119, 300645 Timişoara, ROMANIA

Vice-dean for science and international cooperation

President of the organizing committee

Mentor: doc. dr Emina Mladenović

University of Novi Sad, Faculty of Agriculture, Department of Fruit Growing, Viticulture,

Picture 1. Sansevieria Picture 2: Extech Picture 3: Arduino Uno

RESULTS AND DISCUSSION

Mentor: Petrescu IRINA

Banat University of Agriculture Sciences and Veterinary Medicine `KING MICHAEL I

Key word: wheat , morpho-phenological, phases,

RESULTS AND DISCUSSIONS

1. Quantitative characters related to the height of the plant

2. Quantitative characters related to the leaf

Table 2 Quantitative font -related characters in the Alex variety

3. Quantitative characters related to the spice

Table 3 Quantitative characters related to spice in the Alex variety

before harvesting 31.22 ± 0.73 4.03 7.18 ± 0.45 11.03

1 . BILETEANU GH., AL. SALONTAI, C. VASILICA, V. BIRNAURE, I. BORCEAN , 1991 -

Fernando Arturo Mendez Garzón

Mentor: István Valánszki, PhD

The interactions of tourists and locals (users) with

RESULTS AND DISCUSSION

The implementation spaces are the fundamental

The cultural landscape of the Puszta represents

The proposal was designed based on the

COSTEA Marinela, ARDELEAN Abel

Mentor: SARAC Ioan

University of agriculture and veterinary medicine `KING MICHAEL I` of Romania,

MATERIAL. OTHER VARIETIES IN THE STUDY

RESULTS AND DISCUSSIONS

In the climatic conditions of the Timisoara Teaching and Experimental Center

VARIABILITY OF THE HEIGHT GROWTH OF Corylus avellana L.

V1.Tonda Gentile V2.Butler V3.Tonda Romana

Figure 1. Variability of height increase (cm) of Corylus seedlings avellana L

VARIABILITY OF THE INCREASE OF THE BUSH DIAMETER (CM) OF

V1.Tonda Gentile V2.Butler V3.Tonda Romana

VARIABILITY NUMBER OF BRANCHES / BUSHES JUVENILES FROM

VARIABILITY OF CHLOROPHYLL CONTENT OF Corylus avellana L.

V1.Tonda Gentile V2.Butler V3.Tonda Romana

Fig. 3. The variability of the chlorophyll content of Corylus varieties avellana L.

Sarac I., 2005, Genetics and the improvement of forests

Nikola HRICÁKOVÁ1, Renata CINKOCKI2

Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Sciences,

Analysis of bacterial communities based on nucleic acid isolation allows us to monitor

MATERIAL AND METHODS

RESULTS AND DISCUSSION

Coleman, D. C. – Crossley, D. A. (1996) Soil ecology.

Mentor: Mirjana Ljubojević, assistant professor

MATERIAL AND METHODS

Figure 1. Plant material: A) Mother plant; B) Viable seeds - red colored

Cultivation media composition and culture conditions

Data processing and statistical analysis

RESULTS AND DISCUSSION

Figure 4. Measurements Figure 5. Influence of Illumination and nutrition media 12 weeks