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Original Article

Providing inoculum for Didymella bryoniae studies: the effect

of light spectrum and storing at low temperature
Fornecendo inóculo para estudos de Didymella bryoniae: o efeito do espectro de luz e
armazenamento em baixa temperatura

M. C. S. Virtuosoa* , E. H. C. Silvab , E. M. Silvaa , T. S. Valentec,d , P. F. Vargase , L. T. Braze  and R. C. Panizzie 

a
Universidade Estadual Paulista – UNESP, Faculdade de Ciências Agrárias e Veterinárias, Programa de Pós-graduação em Agronomia (Genética e
Melhoramento de Plantas), Jaboticabal, SP, Brasil
b
Instituto Taquaritinguense de Ensino Superior “Dr. Aristides de Carvalho Schlobach” – ITES, Departamento de Agronomia, Taquaritinga, SP,
Brasil
University of Alberta – UAlberta, Department of Agricultural, Food and Nutritional Science, Edmonton, Canada
c

d
Universidade Estadual Paulista – UNESP, Faculdade de Ciências Agrárias e Veterinárias, Departamento de Zootecnia, Jaboticabal, SP, Brasil
e
Universidade Estadual Paulista – UNESP, Faculdade de Ciências Agrárias e Veterinárias, Departamento de Ciências da Produção Agrícola,
Jaboticabal, SP, Brasil

Abstract
The in vitro sporulation of Didymella bryoniae is of great importance for studies that require pure inoculum and in
large quantities. Thus, the objectives of this study were to identify the best condition for D. bryoniae sporulation
combining different light spectra (UV-A or UV-B light, white light, and continuous dark), with distinct culture media
(PDA, V8, ML, and PDAB) and, to evaluate fungus’ survivability stored at -20°C over time. The fungus samples were
only able to sporulate when subjected to the UV-B light treatment, regardless of the culture medium. The highest
appearance of spores conidium type was observed in the PDAB medium, and the lowest production occurred in
the ML medium. Reproductive structures, such as perithecia and pycnidia, were observed in all culture media.
However, there was considerable variation in the amount of each structure between the different culture media.
The ML and V8 media showed a greater number of perithecia and the PDA and PDAB media presented a greater
proportion of pycnidia compared to perithecia. The storage duration at -20°C did not affect mycelial growth or
mycelial growth rate. In conclusion, the UV-B light is essential for D. bryoniae in vitro sporulation. Moreover,
the culture medium composition influences the type of fungal structure produced, as well as spores’ size and
quantity. Freezing at -20°C is an efficient technique that can be used to store D. bryoniae for at least five months
without loss of viability.
Keywords: fungi preservation, gummy stem blight, sporulation, UV-B light.

Resumo
A esporulação de Didymella bryoniae in vitro é de grande importância para estudos que requerem inóculo puro e em
grandes quantidades. Assim, os objetivos deste estudo foram identificar a melhor condição para esporulação de D.
bryoniae combinando diferentes espectros de luz (luz UV-A ou UV-B, luz branca e escuro contínuo) com distintos
meios de cultura (PDA, V8, ML e PDAB) e, avaliar a sobrevivência do fungo armazenado a -20°C ao longo do tempo.
As amostras de fungo só esporularam quando submetidas ao tratamento com luz UV-B, independentemente do
meio de cultura. Maior aparecimento de esporos do tipo conídio foi observado no meio PDAB, e a menor produção
ocorreu no meio ML. Estruturas reprodutivas, como peritécios e picnídeos, foram observadas em todos os meios de
cultura. No entanto, houve uma variação considerável na quantidade de cada estrutura entre os diferentes meios de
cultura. Os meios ML e V8 apresentaram maior número de peritécios e os meios PDA e PDAB apresentaram maior
proporção de picnídeos em relação aos peritécios. A duração do armazenamento a -20°C não afetou o crescimento
micelial ou a taxa de crescimento micelial. Em conclusão, a luz UV-B é essencial para a esporulação de D. bryoniae
in vitro. Além disso, a composição do meio de cultura influencia o tipo de estrutura fúngica produzida, bem como
o tamanho e a quantidade dos esporos. O congelamento a -20°C é uma técnica eficiente que pode ser usada para
armazenar D. bryoniae por pelo menos cinco meses sem perda de viabilidade
Palavras-chave: Preservação de fungos, crestamento gomoso do caule, esporulação, Luz UV-B.

*e-mail: marcos.virtuoso@unesp.br
Received: June 18, 2021 – Accepted: September 30, 2021
This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

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Virtuoso, M.C.S. et al.

1. Introduction at -20°C for the storage of D. bryoniae isolates. However,

these authors did not carry out systematic evaluations
The Didymella bryoniae (Auersw.) Rehm, synonym
to verify the preservation and survival of the fungus over
Stagonosporopsis cucurbitacearum (Fr.) [anamorph Phoma
time, nor of its structures. Thus, little is known about the
cucurbitacearum (Fr.) Sacc (=Ascochyta cucumis Fautrey &
survival capacity of D. bryoniae stored at low temperatures.
Roum)] (Mao et al., 2020), is an ascomycete that causes
Based on the above considerations, there is a need
the gummy stem blight (GSB) in several cucurbits.
to establish the ideal condition for in vitro D. bryoniae
When present in cucurbits cultures this fungus might
sporulation, as well as to evaluate the survival capacity
negatively impact yield with direct consequent economic
of this fungus when stored at low temperature. Thus,
revenue decreases for growers (Liu et al., 2017). Given
the objectives of the present study were to establish the
the importance of D. bryoniae, studies involving in vitro
best condition for D. bryoniae sporulation combining
cultivation and sporulation are relevant, especially when
different light spectra and culture media, and to evaluate
a large amount of pure inoculum is needed. For example,
the fungus’ ability to survive over time when submitted
screening for resistant plants requires a high number of
to the -20°C storage.
spores to guarantee the intensity of the disease. Moreover,
GSB-related studies often require the combination of
inoculations in a greenhouse and subsequent field tests
(Gusmini et al., 2003). 2. Material and Methods
To date, several studies evaluating the best condition The present study was carried out at São Paulo State
for D. bryoniae sporulation in vitro have been reported, University, School of Agricultural and Veterinarian Sciences,
testing different culture medium (CM) combinations Jaboticabal Campus (21°15′19″S, 48°19′21″ O), São Paulo -
(Gusmini et al., 2003; Hassan et al., 2018; Leão et al., Brazil. Melon plants (Cucumis melo L.) with symptoms of
2012; Nga et al., 2010; Skarshaug, 1981; Somai et al., GSB, such as the presence of viscous exudate and fruiting
2002), ultraviolet (UV) light (Kwon et al., 1997; Lee, 1982; bodies of the fungus, were used to obtain the D. bryoniae
Tsay et al., 1990; Tsutsumi and Silva, 2004; Wolukau et al., isolate. The pycnidia/perithecium were directly removed
2007; Zhang et al., 2017), and temperature and pH from lesions on the stem was performed with the aid of
(Bhat et al., 2009). However, despite the vast literature on a magnifying glass. These structures were placed in Petri
the subject, there is still no consensus among researchers on dishes containing PDA (potato-dextrose-agar) medium,
the main factors influencing in vitro D. bryoniae sporulation, subsequently closed with PVC film, and incubated in
with frequent disagreement between studies.
growth chambers, with a constant temperature of 24°C
According to Keinath et al. (1995) and Wiant (1945),
and a 12-hour photoperiod for 4 consecutive days. After
some isolates do not sporulate in any CM. On the other
obtaining the fungal culture, a subculture was carried out
hand, Leão et al. (2012) observed that the CM can influence
to purify the fungus, transferring 5 mm diameter discs
D. bryoniae sporulation. In addition, Choi et al. (2010) and
with mycelium to new plates and conditioning them for
Kwon et al. (1997) concluded that the presence of UV light
10 days, forming the stock cultures.
is important to promote the sporulation of this fungus.
The study was conducted in two stages to assess
Despite the direct effect that UV light may have on D.
the ability of in vitro sporulation and survival after
bryoniae sporulation, many studies do not classify the type
freezing. In the first phase of the study, a completely
of UV used, hampering the use of a similar methodology.
randomized design was used in a 4 x 4 factorial scheme
Such information is important because UV lights can be
with 10 repetitions, the factors being represented by the
classified according to the emitted wavelength, being UV-A
different culture media and types of light. In the second
(315–400 nm), UV-B (280–315 nm), or UV-C (100–280 nm)
phase, the growth capacity was evaluated after different
(Braga et al., 2015).
periods of freezing at -20°C.
In addition to the CM and the type of light, the
storage time is also important for the development of
2.1. Culture medium and light spectrum
research with D. bryoniae. Properly stored samples serve
as quick-access natural resources banks, which can be To evaluate the sporulation capacity of D. bryoniae, four
used for the development of studies on biodiversity, culture media were used, namely: i) potato-dextrose-agar
systematics, phytopathology, and related areas, such as (PDA), ii) concentrated vegetable juice (V8, commercial
plant breeding for resistance to diseases (Aparecido et al., product), iii) melon leaf extract (ML), and iv) potato-
2018; Hawksworth and Colwell 1992; Sakr, 2019). The most dextrose-agar + melon branches (PDAB). In addition,
used methods for fungi preservation are submersion in four light spectra were used, being: i) white fluorescent
sterile water, mineral oil, or glycerol, use of filter paper light 440-740 nm/10W, ii) UV-A white fluorescent light,
discs, wood, soil, and silica gel (Al-Bedak et al., 2019). Also, 350-400 nm/8W, iii) UV-B white fluorescent light,
cryopreservation, lyophilization, and preservation under 280-360 nm/8W, and iv) control group in continuous dark.
liquid nitrogen can be employed (Ayala-Zermeño et al., PDA and PDAB culture media were prepared with 200g
2017). of potatoes chopped into small cubes and placed in 1.0 L
Freezing at -20°C has also been shown to be a safe of deionized water and cooked at 100°C for 20 minutes.
method for storing various microorganisms and, unlike To the potato extract, 20 g of dextrose and 20 g of agar
cryopreservation and preservation under liquid nitrogen, were added, completing the volume with deionized water
which requires more sophisticated equipment, storing at to 1.0 L. For the preparation of the V8 medium, 20 g of
-20°C is simple and of low cost (Tortora et al., 2011). To the agar was added to 200 mL of concentrated vegetable
best of our knowledge, only Grube et al. (2011) used freezing juice, then the volume was adjusted to 1.0 L, and finally,

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 Providing inoculum for Didymella bryoniae studies

3.2 g of CaCO3 was added to raise the pH to 7.0. For the ML the colony perimeter, discounting 10.0 mm corresponding
medium, 200g of melon leaves were washed in running to the initial inoculum of the fungus, for five consecutive
water and then processed in a blender with 500mL of days. The data were used to calculate the average mycelial
deionized water, then sieved twice in non-woven fabrics growth at five days and to obtain the mycelial growth rate
(TNT) and then added with 20g of agar and 20g of dextrose, index (MGRI), using the method proposed by Maia et al.
completing the process. volume to 1.0 L. The pH of all (2011), as follow (Equation 1):
culture media was assessed and, when necessary, adjusted
to standardize at 7.0. ∑ ( D − Da )
MGRI = (1)
After preparation, all culture media were sterilized N
by autoclaving for 20 minutes at 121ºC, then 200 mg
of antibiotic (streptomycin) was added. Subsequently, where D is the current average diameter of the colony,
5 mL of each medium was poured into test tubes (14.5 x Da is the average diameter of the colony of the previous
2.5 cm). In this step, three pieces of 5 cm melon branches day, and N is the number of days of evaluation.
were added to the PDAB medium, previously disinfected
in 1% bleach, and dried on filter paper. Finally, the tubes 2.3. Statistical analysis
were covered with cotton, sealed with aluminum foil, and The spore concentration data were transformed to
again autoclaved. root(x) and subjected to analysis of variance and comparison
At the beginning of the experiment, 5 mm diameter of means by the Tukey test (Tukey, 1953). The data
discs with mycelium of the fungus were removed from relating to AG and MGRI were submitted to analysis of
the stock plates and transferred to the center of each
variance. The analyzes were performed using software R
test tube. Subsequently, the tubes were incubated in a
(R Development Core Team, 2018).
growth chamber with a constant temperature of 24°C and
subjected to a photoperiod of 16 hours of light, except for
the continuous dark treatment, for 20 consecutive days.
During this photoperiod, all tubes were placed 20 cm away 3. Results
from the lamps allowing maximum possible radiation. There was no significant interaction between the
The spore concentration was determined for culture medium and the type of light used to promote
all treatments after 20 days of incubation, using a in vitro sporulation of D. bryoniae. The fungus samples
hemacytometer. For the solution preparation, the culture
were only able to sporulate when subjected to the UV-B
medium was removed from the tube and placed in a
light treatment, regardless of the culture medium.
crucible with 20 mL of sterile water, then macerated to
The other light conditions did not cause any stimulus to
release the spores. After maceration, filtration in voile
sporulation, or even traces of reproductive structures.
tissue was carried out to remove remnants of the culture
As a result, comparisons using the mean test were only
medium and mycelium, followed by the addition of 0.2%
Tween 20. The results were expressed as number of performed between different culture media under UV-B
spores per mL. In addition, the mycelium staining, type of light treatment and, a significant difference was observed
sexual structures (perithecia and pycnidia), and the spore for the number of spores among the four culture medium
morphology were visually evaluated with the aid of an evaluated (P<0.001). The highest appearance of spores
optical microscope with an attached camera (Olympus conidium type was observed in the PDAB medium, and the
cellSens standard 2.3). The morphological classification lowest production occurred in the ML medium (Figure 1).
of spores followed the methodology proposed by Chiu
and Walker (1949).

2.2. Storing at -20°C

To verify fungal survivability after different periods
of storing at -20°C, a completely randomized design
was employed, considering the storage time (in months)
as treatment, with five repetitions. Following the
aforementioned methodology, PDAB tubes were prepared
and then conditioned in the growth chamber under UV-B
light to sporulate. After the fungus had sporulated, tubes
were taken to a freezer (-20°C) and stored for up to five
consecutive months.
A tube of PDAB medium was removed from the freezer
every 30 days of storage to obtain D. bryoniae fungal
structures. These samples were transferred to five Petri
dishes containing PDA medium, which were sealed with
plastic film and incubated in a growth chamber at a Figure 1. Average conidia concentration of Didymella bryoniae
constant temperature of 24ºC, with a 12-hour photoperiod produced in different culture media under UV-B light. PDA = potato-
and white fluorescent light regime. dextrose-agar; PDAB = potato-dextrose-agar + melon branches;
In this phase, daily evaluations of mycelial growth were V8 = concentrated vegetable juice (commercial); ML = melon
performed, measuring two diametrically opposite points of leaf extract.

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Virtuoso, M.C.S. et al.

Reproductive structures, such as perithecia and pycnidia, structures. When submitted to growth in UV-B light, the
were observed in all culture media. However, there was mycelium showed a darker color (dark gray) compared
considerable variation in the amount of each structure to all other evaluated light conditions. Samples subjected
between the different culture media used as shown in to continuous dark, or UV-A light presented white color,
Table 1. The ML and V8 media showed a greater number and those under fluorescent light were greyish-white.
of perithecia in relation to the pycnidia. This variation In addition, it was observed that the mycelium volume
might have influenced the final spore count since, despite is smaller when subjected to UV-B light but with a larger
a large number of structures the ascospores are difficult number of perithecia and pycnidia (Figure 2).
to visualize on the hemacytometer, resulting in reduced Regarding morphological aspects evaluated, the
total spores’ count. In contrast, the PDA and PDAB media appearance of septate and non-septate conidia was
presented a greater proportion of pycnidia compared observed in all culture media under UV-B light as shown
to perithecia, resulting in a higher conidia appearance in Figure 3A. However, an important size variation was
(Figure 1). It worth mentioning the importance of PDAB observed in which the largest microconidium production
medium to promote pycnidia production since most of the occurred in the V8 medium, with structure size ranging
formed structures were present in the melon branches. from 3 to 5 µm. In the other culture media, there were a
The visual analysis employed for mycelium higher proportion of macroconidia, ranging from 6 to 10 µm
characterization indicated that the light type is an important in length according to the classification proposed by Chiu
factor affecting both the appearance and color of the fungus’ and Walker (1949). Moreover, in all assessed media the
asci’s size ranged from 35 to 50 µm and the ascospores
from 6.5 to 12 µm in length (Figure 3B).
Table 1. Spores of Didymella bryoniae produced in different media As greater D. bryoniae sporulation was observed in
under UV-B light. samples submitted to PDAB medium and under UV-B light,
this treatment was used to produce biological material for
Culture mediaa Perithecia Pycnidia further freezing. At this stage of the experiment, the fungus
PDAB ++ ++++ survivability was evaluated after different storage periods
at -20°C. However, there was no significant difference
PDA ++ +++
(P>0.05) between the freezing times on mycelial growth
V8 +++ ++ and the mycelial growth rate index (Figure 4). This result
ML ++++ + indicates that when subjected to a combination of PDAB
medium with UV-B light, the fungus can survive, and
+ = scarce; ++ = moderate; +++ = good; ++++ = abundant. aPDAB = potato-
dextrose-agar + melon branches; PDA = potato-dextrose-agar; biological functions are preserved after freezing at -20°C
V8 = concentrated vegetable juice (commercial); ML = melon leaf extract. for up to five months.

Figure 2. Characteristics of Didymella bryoniae reproductive structures in vitro. (A) PDAB medium = potato-dextrose-agar + melon
branches, (B) on the branch; and (C) on the medium.

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 Providing inoculum for Didymella bryoniae studies

Figure 3. (A) Conidia of the imperfect form of Didymella bryoniae; (B) Asci and ascospores of the perfect form of Didymella bryoniae
(100x amplification, scale = 10µm).

Figure 4. Average values of mycelial growth (A) and mycelial growth rate index (B) for the Didymella bryoniae fungus stored over five
consecutive months at -20° C.

4. Discussion During preliminary tests (unpublished data), it was possible

to stimulate the formation of reproductive structures
The results obtained in our study showed that regardless
when using a 12-hour photoperiod with tubes placed
of the culture medium, the in vitro sporulation of Didymella
25 cm away from the lights, however, sporogenesis or
bryoniae isolate occurs only in the presence of ultraviolet spore maturation did not occur. Chiu and Walker (1949)
radiation, more specifically, the light spectrum in the also observed the formation of reproductive structures
wavelength between 280-360 nm (peaking at 315nm). without the presence of spores. These authors report that
According to Sepúlveda et al. (2009), some photoreceptors the absence of sporulation on the material used occurred
are only activated when stimulated by light with specific due to specific nutritional requirements by the different
wavelengths. In addition, other authors reported that in isolates of D. bryoniae. Considering that in the natural
several species the light has a regulatory effect on the environment the fungus receives appropriate quality
development and behavior of fungi (Corrochano, 2007; and amount of light to sporulate, these factors (number
Pulz and Massola Jr., 2009). of light hours, intensity, and spectrum) are key elements
The quality and intensity of light are also important that need to be considered under laboratory conditions
aspects to promote in vitro sporulation of D. bryoniae. to promote satisfactory sporulation of this fungi.

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Virtuoso, M.C.S. et al.

During the first phase of the experiment, the appearance was not possible to systematically assess survivability
of reproductive structures occurred both on the surface after five months and, thus, further studies are required
and submerged in the culture medium, which is why to improve our knowledge on this behalf.
maceration was defined as the best method for obtaining Another important aspect that should be highlighted
the spores produced. Similar behavior was observed by is the fact that the freezing was carried out using the
Leach (1962) when working with the sporulation of the PDAB medium, which biological material contains a high
fungus Ascochyta pisi. During preliminary evaluations, it concentration of fungal spores. According to Meryman
was observed that the sporulation induction occurred in (1966) and Sakr (2019) spores, sclerotia, and dormant
young mycelia only, soon after their exposure to UV-B mycelium are the most resistant fungal structures to
light. On the other hand, there was no induction when freezing because their cells have less moisture content.
mature mycelia were placed under UV-B light (personal On the other hand, cellular structures with a higher
communication). Leach (1962) also reported that young moisture content tend to be more susceptible to damage
mycelium is more sensitive to UV light radiation acting caused by low temperatures.
as a receptor compared to mature mycelium. To the best of our knowledge, to date, no studies have
The fungal structure type formed varied among the been developed to identify the main factors related to in
four media used (Table 1). The ML medium showed higher vitro sporulation of D. bryoniae neither its ability to survive
production of perithecia than pycnidia, indicating that after freezing. Our results showed that it is possible to
there is a higher occurrence of sexual reproduction in this stimulate the sporulation of this fungus in vitro and that
medium caused by the greater availability of essential approachable techniques can be used for the storage and
substances for fungus development. The greater appearance conservation of biological material.
of perithecia might be explained by the fact that sexual In conclusion, this is the first study identifying
reproduction has higher physiological requirements key elements of light and culture medium affecting
and energy costs compared with asexual reproduction the Didymella bryoniae in vitro sporulation and the
(Chamberlain and Ingram, 1997). Conversely, in other media consequences of freezing during five consecutive months.
(PDA, PDAB, and V8) there is less availability of nutritional The spectrum emitted by UV-B light is the main factor
resources with consequently increased occurrence of promoting in vitro sporulation. Moreover, the culture
asexual reproduction. These results partially corroborate medium composition influences the type of fungal structure
Lee (1982), who obtained different sexual structures of D. produced, as well as spores’ size and quantity. Freezing at
bryoniae when submitted to PDA, V8, and plant extracts.
-20°C is an efficient technique that can be used to store D.
However, this author attributed the observed differences
bryoniae for at least five months without loss of viability.
to the origin of their isolates. In practical terms, the PDAB
medium is more suitable to be used when screening
to identify resistant plants, since the pycnidiospores
Acknowledgements
produced are more easily visualized and quantified on
a hemacytometer. On the other hand, the ML medium is This study was financed in part by the Coordenação
mainly indicated to study genetic aspects of this fungus. de Aperfeiçoamento de Pessoal de Nível Superior - Brasil
In addition to the influence on the development (CAPES) - Finance Code 001, which had no role in study
of reproductive structures, the medium composition design, data collection and analysis, decision to publish, or
also proved to be a determining factor for the size of preparation of the manuscript. The study was part of the
pycnidiospores. In the V8 medium, there was a higher doctoral thesis of the first author, prepared for the Graduate
proportion of microconidia compared to all other media. Program in Genetics and Plant Breeding at Universidade
In this case, the carbon source may be responsible for the Estadual Paulista (UNESP), Faculdade de Ciências Agrárias
variation observed in spore size, given that the V8 medium e Veterinárias, Jaboticabal, SP, Brasil.
is the only that dextrose was not added. Similar results
were observed by Chiu and Walker (1949) and Lee (1982)
who associated the conidia size to the type of medium
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