Journal of Biology, Agriculture and Healthcare www.iiste.

org
ISSN 2224-3208 (Paper) ISSN 2225-093X (Online)
Vol.4, No.25, 2014

Isolation and Identification of Fungal Pathogens Associated with

Cold Storage Type of (Coffee Arabica.) Seed, at Jimma
Agricultural Research Center, Western Ethiopia
Alemu Nega
Department of Horticulture and Plant Science, Jimma University, P. O. Box 307, Jimma, Ethiopia
Corresponding Author: Alemu Nega. Email Address: alemunega531@gmail.com

Abstract
Coffee is the most important commercial crop in the national economy of Ethiopia. Coffee seed are subject to
various operations of contamination by microorganisms during growth (while the beans are on trees), after
harvesting (when the beans are de-hulled, washed and stored) and during storing. The aim of this research is to
isolate and identify the fungal pathogens associated with cold storage type of coffee (coffee arabica L.) seed.
Different fungi were associated with coffee seed under cold storage condition. These different fungal species
was isolated and identified both in blotter and agar plate method. In blotter test method the identified fungi were;
Aspergillus sp.; Pencillium sp.; Fusarium sp., and another some unidentified species were isolated and identified
at genesis level from the two month coffee seeds storage. Among these, Aspergillus spp. had the highest
(49.375%) frequency of occurrence, followed by Penicillium spp. (11.875%), Fusarium spp. (5.625%) and
unidentified species (0.626%). In addition, in blotter test method the obtained result indicated that the infection
mean percentage in blotting methods W0V74-1 (without parchment) followed by W0V74-110
(without parchment) were highly infected mean percentage 80% and 97.5% respectively. However the lowest
fungal infestation was noted on both W1V74-1 and W1V74-110 (45 %) with parchment of coffee seed was verified.
The results of germination test obtained in blotter plate method showed that the germination mean percentage of
W0V74-110 (without parchment) were highly germinated with mean percentage of (98%). However the lowest
germination mean percentage was noted on W1V74-1 (27.5%) with parchment of coffee seed. In agar plate method
also the identified fungi were; Fusarium sp. Aspergillus sp. Pencillium sp. and another some unidentified
species. Among these, Aspergillus spp. had the highest (31.25%) frequency of occurrence, followed by
Penicillium spp. (10.625%) and Fusarium spp. (11.875%) and unidentified
spp. (9.375%). Moreover, in agar plate method the maximum coffee seeds mean infection percentage were recor
ded 82.5% and the mean minimum infection percentage were (52.5%) in the treatment W1V74_110 (with parchmen
t) and W0V74_110 (without parchment) respectively. From total four treatments (62.5%) maximum fungal contam
inations were recorded. The result of present study, storage fungi chiefly comprise several "group species" of the
genera Aspergillus spp, Penicillium spp, Fusarium spp. and another unidentified species. The species identified
in this study are among the most common species of fungi present in storage environments at high moisture.
They can tolerate growth in different substrates and environmental conditions, and their complete elimination is
difficult. However, the use of good hygiene practices and using optimum moisture of coffee seeds in storage
management and can minimize mycroflora association of coffee seeds. Although the present study was carried
out in one location in Jimma University College of Agriculture and Veterinary Medicine (JUCAVM)/Jimma
Agricultural Research Center for three month in 2013 and, it has clearly indicated that different fungi were
associated with coffee seed under storage condition, especially Aspergillus species. In general, further research is
needed to identify all recovered fungal pathogens and evaluation of promising treatments for use in integrated
disease management strategy to manage not only fungal but also other coffee seed diseases and also further
investigation of storage temperature, relative humidity, periods of storage and storage types in wide range across
the location that suitable for good supply of health coffee seeds.
Keywords: Coffee, seed, cold storage, mycoflora.

1. INTRODUCTION
Coffee belongs to the family Rubiaceae, which is widely distributed throughout the tropical region. Although
there are many species of coffee, the only two commercially important ones are (Coffea arabica) and (Coffea
robusta) (Pieters and Vander-Graff, 1980). Both species can grow best on deep, free- draining, loamy soils, with
a good water holding capacity and a slightly acid soil (PH 5-6) (CTA, 1999; Kimani et al., 2002; Lewis Ivey et
al., 2003) and soil fertility is important for good production. Coffee is the most important commercial crop in the
national economy of Ethiopia, contributing 60% of its foreign exchange earnings and nearly 25% of Ethiopian
population depends, directly or indirectly on coffee for a livelihood by involving in the production, processing,
and marketing of coffee as the major contribution to the development of the rural and the national economy
(CTA, 1999; Paulos Dubale and Demil Teketay, 2000). Currently, Ethiopia is a leading arabica coffee producer
in Africa, ranking the fifth largest Arabica coffee producer and tenth in coffee export worldwide. Its total coffee

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Journal of Biology, Agriculture and Healthcare www.iiste.org
ISSN 2224-3208 (Paper) ISSN 2225-093X (Online)
Vol.4, No.25, 2014

production and export respectively increased by 107% and 226% for the crop year 2009/10 and 2010/11 (ICO,
2011). In Ethiopia, coffee had been and still contributes to the Lion’s share in its national economy being the
leading source of foreign exchange earnings. Besides, the livelihood of a quarter of the Ethiopian population
depends directly or indirectly on the different processes of production and marketing along the coffee value-
chain (Girma et al., 2008). Even though coffee is the second largest commodity after oil in the World Bank
ranking (Smith, 1985), its production suffers from many constraints, including diseases, pests and weeds,
resulting in substantial losses. According to Oerke et al., (1994), losses in coffee production are estimated at
40% of the attainable harvest worldwide, broken down into 14.8% due to diseases, 14.9% due to insect pests and
10.3% due to weeds. To these important losses should be added post-harvest losses, which also affect very
significantly the total yield obtained by the poor coffee farmers of the developing countries. Post-harvest
problems of coffee adversely affect the quality of coffee beans because of fungal contamination and production
of mycotoxins. Moreover, coffee quality is evaluated by key factors including the selection of the Coffea
arabica variety, climatic conditions during growth, processing method and storage conditions. The main aim of
coffee processing is removal of the pulp, mucilage, parchment and silver skin surrounding the coffee beans,
which leaves the so called 'green' coffee beans. In Brazil and Ethiopia and for robusta coffees generally, the dry
or natural method of fermentation is usually used. During the natural processing, coffee fruits are spread on the
ground (earth, concrete or tarmac) in layers approximately 10 cm thick, heaped at night and re-spread each
morning. During the course of 10-25 days of sun drying, the natural microbial fermentation that occurs can
influence the final quality of the product (Silva et al., 2000). Microbial contamination can occur in the cherries
and during harvesting, fermentation, drying and storage coffee beans (Silva et al., 2000). Bacteria, yeasts and
filamentous fungi have been already reported in the pulp and beans of coffee processed in Brazil, India, Hawaii,
Congo, Argentina, Colombia, Costa Rica, Ethiopia and Mexico (Avallone et al., 2001)). Filamentous fungi
predominate at the end of the processing and during storage, and may affect the quality and safety of the final
product due to production of mycotoxins (Batista et al., 2003). Several studies have reported the occurrence of
toxin-producing fungi and ochratoxin in green coffee beans (Batista et al., 2003). Hence, the current study was
meant to isolate and identify the fungal pathogens associated with cold storage type of coffee (coffee arabica L.)
seed. Therefore, the objective of this study was conducted to isolate and identify the fungal pathogens associated
with cold storage type of coffee seed.

3. MATERIALS AND METHODS

3.1. Study Area and Period
The field study was conducted in Jimma zone Ethiopia which is found at about 345 km from Addis Ababa in
South west and lies between 36° 10´ E longitude and 7° 40´ N latitude. The two varieties of coffee arabica such as
variety one (V74_1) and variety two (V74_110) coffee seeds sample from cold storage type stored at the 100%
purity, 70% relative humidity, 12% moisture content and temperature (8-100c) were collected from Jimma
Agricultural Research Center (JARC) February 2005 and the experiment were carried out for three months
(February, March and April) in 2013 at Jimma University College of Agriculture and Veterinary medicine
(JUCAVM) plant pathology laboratory. Jimma which is a zone for coffee production and supplier to the other
towns. This area experiences annual average rainfall of 1000 mm for 8 to 10 months. The zone has an elevation
ranging from 880 to 3360 masl. The temperature of Jimma zone varies from 8-28°C. The average annual
temperature is 20°C [15] (Haile A. and Tolemariam T, 2008).
3.2. Blotter Tests Methods
The blotter method was used to isolate the fungal pathogens associated with the coffee seeds were as to
determine the health seeds after the storage periods. The samples were tested according to (ISTA, 1981). A total
of 340 coffee seeds of the two varieties (V74_1) and (V74_110) and the total treatment combination was 4 (2x2), in
each four replicates, were tested from each sample. The sample with and without parchment and disinfection
then seeds were plated directly on top of three layers of well-soaked blotter paper. Ten seeds were plated on per
plastic Petri-dish of 9cm diameter by surface disinfection. The plated coffee seeds were incubated at 22-25°C for
7 days under alternate cycles of 12h daylight and darkness. After incubation each coffee beans were observed
under different magnifications in the stereomicroscope for fungal growth. Pathogenic fungi developing on coffee
seeds were isolated. Seed borne pathogen and the micro flora associated with seeds were cultured on Potato
dextrose agar media and identified to geneses level following the descriptions of (Singh et al., 1991 and Samson
et al., 1995).The infection level (%) is determining as the ratio of infected seeds over the total number of coffee
seed tested.
3.3. Agar Plate Method
For agar plate method the two varieties of cold storage types of coffee seeds were equal placed aseptically on
PDA in 9cm Petri plate 10 seeds per plate 40 seeds per 4 treatment with four replication. In comparable set the
seeds with and without parchments and surface sterilization with 5% sodium hypochlorite for three minutes
washed in sterilized distilled water before plate on the PDA medium and the plated coffee seeds were incubated

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Journal of Biology, Agriculture and Healthcare www.iiste.org
ISSN 2224-3208 (Paper) ISSN 2225-093X (Online)
Vol.4, No.25, 2014

for 7-10 days at 25oc under 12hr alternate cycles of light and darkness. At the end of the incubation period, fungi
growing out from the seeds on the agar medium were examined and identified. Identification was done by
preparation of slide mounts of spores or other bodies in a drop of water and examine under a compound
microscope for shape, size and color colony characters and morphology of sporulation structures (Mathur and
Kongsdal, 2003).
3.4. Germination Test
With and without parchment of coffee seeds were collected and socked for 48 hours for imbibitions. Carefully
saturated the absorbent material for each of ten (10) days each day check that absorbent material remains moist
record the number of germinated seeds.
Germination (%) = Number of seeds germinated x 100
Number of seeds on petri plate
3.5. Statistical analysis
Microsoft Excel software programs were used in the calculations of treatment means and summary tables
presented wherever required.

4. RESULT AND DISCUSSION

4.1. Detection and identification of seed borne fungi of coffee seed by blotter plate method
This study showed that coffee seeds could be attacked by several economically important post harvest fungal
pathogens under storage condition. A total of four post harvest fungal disease (mould fungi) of different genera
were identified. High number of fungal mycoflora was also associated with coffee bean seeds (Table 1 and 2). In
blotting methods coffee seed are subjected to contamination and consequent colonization by microorganism
during different phases of development, harvesting, preparation, transport and storage were tested. In this study
the results showed that four fungal species including Aspergillus sp.; Pencillium sp.; Fusarium sp., and another
some unidentified species were isolated and identified at genesis level from the two month coffee seeds storage
(Table 1). The results showed that the occurrence of these fungal species was somewhat heterogeneous and
Aspergillus spp. had the highest (49.375%) frequency of occurrence, followed by Penicillium spp. (11.875%),
Fusarium spp. (5.625%) and unidentified species (0.626%). Photographic images of these isolates have been
depicted below. Among these isolates species of the main toxigenic fungal genera (Aspergillus, Penicillium and
Fusarium) are natural coffee contaminants and are present from the field to the warehouse (Bokhari et al., 2002).
These toxic genera consist of some species which produce Aflatoxin (AFB1), Ochratoxin (ATO), P. variadile, P.
verrucosum, P. viridcatum, P. chrysognum, P. commune, P. palis and P. cyclopium (Frysvad et al., 1990).
Furthermore, the climatic conditions of some areas of the countries like Ethiopia, where temperature and relative
humidity are high, as well as the poor conditions and long duration of coffee storing could promote fungi,
Aspergillus Fusarium and Penicillium, in particular, to invade coffee beans during storage (Bokhari et al., 2002).
Table 1. Fungal pathogens associated with stored coffee seeds at JARC (seed health test by blotting techniques)
at jimma in 2014.
Fungal flora observed and its Percentage of occurrence
TRT Aspergillus spp. Penicillium spp. Fusarium spp. UN
W0V74_1 37.5 20 22.5 0
W1V74_1 27.5 17.5 0 2.5
W0V74_110 97.5 0 0 0
W1V74_110 35 10 0 0
Mean 49.375 11.875 5.625 0.626
TRT = Treatment, W0V74_1=Variety one wituout parchement, W1V74_1 = Variety one with parchement, W0V74_110
= Variety two wituout parchement, and W1V74_110 = Variety two with parchment.

4.2. Detection and identification of seed borne fungi of coffee seed by agar plate method
Coffee beans are subject to various operations of contamination by microorganisms during growth (while the
beans are on trees), after harvesting (when the beans are de-hulled, washed and stored) and during storing. In
Agar plate methods also coffee seed are subjected to contamination and consequent colonization by
microorganism during different phases of development, harvesting, preparation, transport and storage. In this
study the results showed four fungal species including Fusarium sp. Aspergillus sp. Pencillium sp. and another
some unidentified spp. were isolated and identified at genesis level from the two month coffee seeds storage
(Table 2). The results showed that the occurrence of these fungal species was somewhat heterogeneous.
Aspergillus spp. had the highest (31.25%) frequency of occurrence, followed by Penicillium spp. (10.625%) and
Fusarium spp. (11.875%) and unidentified spp. (9.375%). Bokhari (2007a) reported that coffee seeds were
highly contaminated with toxigenic fungal isolates and toxins especially ochratoxin A. The occurrence of fungal
contamination and mycotoxin production started at the beginning of harvest due to high moisture content of the
seeds and increased during transport, storage, or marketing. Within the last decade, significant advances have

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Journal of Biology, Agriculture and Healthcare www.iiste.org
ISSN 2224-3208 (Paper) ISSN 2225-093X (Online)
Vol.4, No.25, 2014

been made in mycotoxin detection methods and control strategies as well as in studying the effect of
environmental factors on toxin synthesis (Patterson, 1984; Nakajima et al., 1997).
Table 2. Fungal pathogens associated with stored coffee seeds at JARC (seed health test by agar plat techniques)
at jimma in 2014.
Fungal flora observed and its Percentage of occurrence
TRT Aspergillus spp. Penicillium spp. Fusarium spp. UN
W0V74_1 35 7.5 0 10
W1V74_1 30 15 0 5
W0V74_110 45 7.5 5 0
W1V74_110 20 7.5 42.5 2.5
Mean 32.5 9.375 11.875 4.375
TRT = Treatment, W0V74_1=Variety one wituout parchement, W1V74_1 = Variety one with parchement, W0V74_110
= Variety two wituout parchement, and W1V74_110 = Variety two with parchment.

4.3. Detection of seed infection percentage in both blotter and agar plate method
The result showed that an infection mean percentage in blotting methods Variety one (W0V74-1) without
parchment followed by Variety two (W0V74-110) without parchment were highly infected mean percentage of
80% and 97.5% respectively. However, the lowest fungal infestation was noted on both with parchment of
Variety one (W1V74-1) and Variety two (W1V74-110) 45 % of coffee seed. In agar plate methods the result showed
that the maximum coffee seeds mean infection percentage were recorded 82.5% and the mean minimum
infection percentage were 52.5% in the
treatment Variety two (W1V74_110 ) with parchment and Variety two (W0V74_110 ) without parchment respectively.
From total four treatments 62.5% maximum fungal contaminations were recorded. Detection of major seed
borne pathogens using the standard blotter method gave the higher amount of seed infection as compared to the
agar plate method (Figure 2 and 3). This might be due to the availability of more free moisture on the moistened
blotter papers than the agar plates. The coffee bean seed coat absorbs more water from the wet blotters and
enables the fungi to easily grow and sporulate. Whereas on the agar plates, the coffee bean seeds remain dry and
sporulation of the fungus was reduced as compared to the standard blotter method. Blotter method is the easiest,
efficient and economical detection technique for Fusarium sp. Aspergillus sp. Pencillium sp. and another some
unidentified species from naturally infected coffee bean seeds under storage condition.
120

100

80
% of Infection

60

40

20

0
W0V74_1 W1V74_1 W0V74_110 W1V74_110
Treatment
Figure 1.The infection percentage of coffee seeds by blotter plating method.

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ISSN 2224-3208 (Paper) ISSN 2225-093X (Online)
Vol.4, No.25, 2014

100
80
% of Infection

60
40
20
0
W0V74_1 W1V74_1 W0V74_110 W1V74_110
Treatment

Figure 2.The infection percentage of coffee seeds by agar plating method.

4.4. Germination test

Most crop seed is stored for some period of time. During this time, seed may deteriorate considerably. Good
storage conditions can slow the rate of deterioration, but seed germination and vigor cannot be improved,
regardless of the storage facilities. Many costly storage problems actually begin during field exposure,
harvesting, and conditioning of the seed. Excessive harvesting delays, mechanical injuries, and improper drying
techniques, followed by poor storage conditions, can lead to rapid deterioration of seed germination and vigor.
Fast drying decreases the vigor of seeds independent from where they dry. In cold chamber it is possible to store
regular seeds or slowly dried seeds for up to nine months. In a regular warehouse environment the vigor is
affected independently from the way they are dried. The result showed that the germination mean percentage of
Variety two (W0V74-110) without parchment were highly germinated with mean percentage of 98%. However the
lowest germination mean percentage was noted on Variety one (W1V74-1) (27.5%) with parchment of coffee bean
seed were recorded in blotter plate method (Fig. 3).
Wooton (1970) conducted a study to determine the best storage conditions for parchment coffee for a period up
to one year. He found that conventional storage of coffee in sisal bags stacked on wooden pallets, exposed coffee
to storage pests and possible spoilage depending on moisture content. Drying to a low moisture content and
hermetic sealing of coffee bag was found to be a desirable and necessary condition for storage.
120
% of Germination

100 Mean
80
60
40
20
0
W0V74_1 W1V74_1 W0V74_110 W1V74_110
Treatment

Figure 3.The germination percentage of coffee bean seeds.

5. CONCLUSION
Coffee is the most important commercial crop in the national economy of Ethiopia. Coffee beans are subject to
various operations of contamination by microorganisms during growth (while the beans are on trees), after
harvesting (when the beans are de-hulled, washed and stored) and during storing. The loss of seed viability, what
makes it difficult to keep the high physiological quality, is one the greatest problems faced by the coffee seed
producers during seed storage. For all storage systems, good store management aims to maintain the coffee
within the ‘safe’ range of moisture content over the required duration of storage, to protect the product from
damage by insects or other pests, and to prevent cross contamination or new contamination from other sources. It
also facilitates identification and handling of coffee lots. Different fungi were associated with coffee seed under

24
Journal of Biology, Agriculture and Healthcare www.iiste.org
ISSN 2224-3208 (Paper) ISSN 2225-093X (Online)
Vol.4, No.25, 2014

cold storage condition. These different fungal species was isolated and identified both in blotter and agar plate
method. In blotter test method the identified fungi were; Aspergillus sp.; Pencillium sp.; Fusarium sp., and
another some unidentified species were isolated and identified at genesis level from the two month coffee seeds
storage. Among these, Aspergillus spp. had the highest (49.375%) frequency of occurrence, followed by
Penicillium spp. (11.875%), Fusarium spp. (5.625%) and unidentified species (0.626%). In addition, in blotter
test method the obtained result indicated that the infection mean percentage in blotting methods W0V74-1
(without parchment) followed by W0V74-110 (without parchment) were highly infected mean percentage 80% and
97.5% respectively. However the lowest fungal infestation was noted on both W1V74-1 and W1V74-110 45 % (with
parchment) of coffee bean seed were verified. The results of germination test obtained in blotter plate method
showed that the germination mean percentage of W0V74-110 (without parchment) were highly germinated with
mean percentage of (98%). However the lowest germination mean percentage was noted on W1V74-1 (27.5%)
with parchment of coffee seed. In agar plate method also the identified fungi were; Fusarium sp. Aspergillus sp.
Pencillium sp. and another some unidentified species. Among these, Aspergillus spp. had the highest (31.25%)
frequency of occurrence, followed by Penicillium spp. (10.625%) and Fusarium spp. (11.875%) and unidentified
spp. (9.375%). Moreover, in agar plate method the maximum coffee seeds mean infection percentage were
recorded 82.5% and the mean minimum infection
percentage were 52.5% in the treatment W1V74_110 (with parchment) and W0V74_110 (without parchment) respecti
vely. From total four treatments 62.5% maximum fungal contaminations were recorded. The result of present
study, storage fungi chiefly comprise several "group species" of the genera Aspergillus spp, Penicillium spp,
Fusarium spp. and another unidentified species. The species identified in this study are among the most common
species of fungi present in storage environments at high moisture. They can tolerate growth in different
substrates and environmental conditions, and their complete elimination is difficult. However, the use of good
hygiene practices and using optimum moisture of coffee seeds in storage management and can minimize
mycroflora association of coffee seeds.
Although the present study was carried out in one location in Jimma University College of Agriculture and
Veterinary Medicine (JUCAVM)/Jimma Agricultural Reserch Center (JARC) for three month in 2014 and, it has
clearly indicated that different fungi were associated with coffee bean seed under storage condition, especially
Aspergillus spp. In general, further research is needed to identify all recovered fungal pathogens and evaluation
of promising treatments for use in integrated disease management strategy to manage not only fungal but also
other coffee bean seed diseases and also further investigation of storage temperature, relative humidity, periods
of storage and storage types in wide range across the location that suitable for good supply of health coffee
seeds.

5. Acknowledgements
The author is thankful to the department of horticulture and plant science of Jimma University, for allowing the
conveniences to conduct the experiment.

6. REFERENCES
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Oduor, G., Phiri, N., Hakiza, G. J., Abebe, M. Asiimwe, T., Kilambo, D. L., Kalonji- Mbuyi, A., Pinard, F.,
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Silva, C.F.; Schwan, R.F.; Dias, E.S.; Wheals, A.E., 2000. Microbial diversity during maturation and natural
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26
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