See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/330263176

Factors Promoting Pycnidia Production of Didymella Bryoniae, the causal of

Gummy Stem Blight in Cucurbits

Preprint · January 2017

DOI: 10.13140/RG.2.2.34731.28963

CITATIONS READS

0 138

3 authors, including:

Amal Khalil
Agricultural Research Center, Egypt
5 PUBLICATIONS   10 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Efficiency of eugenol oil nanoemulsion against Banana bunchy top virus and contamination with fungi in plant tissue culture. View project

Didymella bryoniae View project

All content following this page was uploaded by Amal Khalil on 09 January 2019.

The user has requested enhancement of the downloaded file.

11 Egypt. J. Phytopathol., Vol. 45, No. 1, pp. 173-187 (2017)

Factors Promoting Pycnidia Production of

Didymella Bryoniae, the causal of Gummy
Stem Blight in Cucurbits
A.A. El-Wakil, Amal A. Khalil and I.H. El -Abbasi
Seed Pathol. Res. Dept., Plant Pathol. Res. Inst., ARC, Egypt

ummy stem blight in Cucurbitacea caused by the pathogen

G Didymella bryoniae under high humid conditions was previously
recorded in many countries. In Egypt, the disease was observed for the
first time on cantaloupe grown under sprinkling irrigation system in El-
Bostan (Beheira governorate). Percentage of diseased plants ranged from
10 to 15% in the surveyed fields. Many isolation trials were carried out
without success in obtaining conidial structures; only undistinguished
mycelium was obtained. Among 9 natural media tested, V-8 Agar
medium (V-8A) was the fastest in producing the distinguished pycnidia
and non-septate pycnidiospores, followed by Bean Dextrose Agar
medium (BDA) for number of produced pycnidia. Three concentrations
(full strength, ½ and ¼ concs.) of V-8A, Cucumber Dextrose Agar
(CDA) and Potato Dextrose Agar (PDA) were also tested, where ¼ conc.
of V-8A and CDA produced pycnidia after 7 days only. Near Ultra Violet
light boosted pycnidia production when compared with two other
lightening systems. In case of incubation materials tested, Pyrex glass
plates affected pycnidia production positively. Among several kinds of
plant debris evaluated for pycnidia promotion, coriander debris was the
best while debris of squash and cucumber, each alone, were the least.
Moreover, plant debris affected the shape and number of pycnidia
produced. Some bioagents promoted pycnidia production. Trichoderma
harzianum was the best. The fungus started in producing pycnidia after
the 4th day when the surface of mycelial culture was damaged, while no
pycnidia were produced if wounding was made before media inoculation.
Keywords: Didymella bryoniae, gummy stem blight, pycnidia
production and T. harzianum.

Didymella bryoniae (Auersw.) Rehm, the anamorph of Mycospharella melonis,

the causal fungus of gummy stem blight of cucurbits is one of the most important
diseases of cucurbits causing considerable damage in many countries (Crüger and
Schneider, 1964; Fletcher and Preece, 1966; Kagiwata, 1967; Schenck, 1968;
Figueiredo et al., 1970; Arny and Rower, 1991; Young et al., 2010; Mason et al.,
2011; Keinath, 2013 and Basim et al., 2016).
The pathogen commonly attacks members of Cucurbitaceae especially
cucumber (Cucumis sativus L.), muskmelon (Cucumis melo L.), pumpkin
(Cucurbita pepo L.) watermelon (Citrullus vulgaris Shard.) and cantaloupe
(Cucumis melo var. cantalupensis). The disease produces a variety of symptoms on
the above ground parts of all cucurbits. In early infection, seedlings die quickly
after infection. On older plants, leaf spots, stem canker, vine wilt and black fruit rot
174 A.A. EL-WAKIL et al.

appeared. The fungus is known to be seed-borne on cucumber and watermelon

(Chupp and Sherf, 1960 and Richardson, 1979). In Egypt, Ragab et al. (1986)
mentioned that disease symptoms were observed in watermelon fields, but they did
not mention anything about the causal pathogen. El-Wakil and Khalil (2016)
observed typical symptoms of gummy stem blight on cantaloupe plants grown
under sprinkling irrigation at El-Bostan. Isolation, purification and identification
were carried out from the collected diseased plants.
The objective of this study is an attempt to solve the difficulty in obtaining
fungal pycnidia by employing some physiological factors promoting mycelia
growth and pycnidia production by D. bryoniae.

Materials and Methods

1. Collecting infected samples:

Diseased cantaloupe plants showing stem cankers with characteristic red to
brown gummy exudates and diseased fruits showing spots of greasy green,
brownish and/or brown colors, were obtained from some cantaloupe fields in El-
Bostan, Beheira governorate in 2015.
2. Isolation, Purification and Identification:
Infected samples of cantaloupe plants were carefully washed in running tap
water to get rid of sand and soil particles. Infected samples were cut into small
pieces and soaked in fresh 2% sodium hypochlorite solution for 3 minutes. These
pieces were washed four times in sterilized water and dried between two sterilized
filter papers. Then samples were transferred into Petri dishes containing Potato
Dextrose Agar medium (PDA) and incubated in the dark at 20 ºC for seven days.
The fungal growth was examined using stereo- and compound microscopes.
Cultural purification was done using hyphal tip technique (Riker and Riker, 1936).
Pure cultures were kept in PDA slants and reserved for further studies. To obtain
the pycnidial stage of the isolated fungus, discs of the fungal growth were plated on
V-8 medium (Young et al., 2010) and incubated at 25oC in dark. After 10 days,
plates were examined for pycnidia formation. Identification of the fungal pycnidia
was carried out according to Lee et al. (1984).
3. Impact of some factors on D. bryoniae sporulation:
3.1. Effect of media:
Nine different media were used to determine fungal reaction in relation to
variable nutrition sources, i.e. Sugar Free Carnation-leaves Agar (SFCA), Yeast
Extract Agar (YEA), Corn Meal Dextrose Agar (CMDA), V-8 Agar (V-8A), half
concentration V-8 Agar (0.5 V-8A), Oat Meal Agar (OMA), Cucumber Dextrose
Agar (CDA), Bean Dextrose Agar (BDA) and Potato Dextrose Agar (PDA).
Mycelial discs (5 mm) were used to inoculate the above mentioned media in Petri
plates. Eight replicates were employed and the average was obtained. Plates were
incubated for one month under room conditions and results were obtained on the
10th, 15th and the 30th day.

Egypt. J. Phytopathol., Vol. 45, No. 1 (2017)

 FACTORS PROMOTING PYCNIDIA PRODUCTION….. 175

Four habit characters of fungal growth were evaluated; mycelial color after 7
days of incubation, number of pycnidia in area unit (cm2), location of pycnidia on
medium surface and days to first pycnidium formation.
3.2. Effect of media concentration on mycelial growth and pycnidia production:
The effect of quarter, half and standard concentration of three different media,
i.e. PDA, CDA and V-8 juice was studied through this experiment. Mycelial discs
of 5 mm diameter were cut from the growing margins of fresh fungal culture and
placed in the center of 9-cm Pyrex Petri plates. Four replicates were carried out for
each treatment. After 7 days of incubation at 22±2 oC under alternating cycle of
12/12 NUV/darkness, results of diameter of radial mycelial growth (mm), mycelial
density, pycnidia production and pycnidia location were recorded.
3.3. Effect of light sources on mycelia growth and pycnidia production:
This experiment was designed to study the effect of different light sources on
growth habits of D. bryoniae. Discs from 7-days old fungal culture were cut using
flamed cork-borer and transferred onto PDA in Pyrex Petri plates, then exposed to
one of the following light regimes; alternating cycle of Near Ultra Violet light
(NUV)/complete darkness (12/12 hrs.), continuous day light (CDL) or complete
darkness (CD). Plates were incubated at 22 ± 2 oC for 14 days in quadruplicates for
each of the designed lighting regime. Near Ultra Violet was provided by two Philips
black light lamps TL 40 w/08, while CDL was supplied by two cool white
fluorescent Philips TLF 40 w 143 deluxe tubes, hanged 50 cm above the dishes.
Impact of lighting regimes on mycelial color, radial growth and days to first
pycnidium formation was evaluated.
3.4. Effect of container material on mycelial growth and pycnidia production:
Permeability to light and material clarity play an important role in introducing
light from surrounding climate to in-container limited climate which could affect
growth characters of any inoculated organism. Pyrex glass dishes, standard plastic
dishes (perspex) and commercial glass dishes were used as the most commonly
used plates locally and internationally to study their effects on some major growth
habits of D. bryoniae. The same light sources mentioned previously were used.
Mycelial color, radial growth and number of days to the first pycnidium formation
were measured. Four replicates of each treatment were incubated and the averages
were presented.
3.5. Effect of plant debris on pycnidia production:
Didymella bryoniae has a wide host range within Cucurbitaceae. The pathogen
can survive on organic debris from previously infected cucurbits or on wild or
volunteer cucurbits. This experiment was conducted to uncover if the fungus can
survive on plant debris of cucurbit crops only or on other plant debris as well.
Various kinds of plant debris representing 5 different field crops and 4 vegetable
crops were used in this experiment. Dead dried main and secondary stems and
branches of barley (Hordeum vulgaris L.), chickpea (Cicer arietinum L.), alfalfa
(Medicago sativa L.), flax (Linum usitatissinum L.), rice ( Oryzae sativa L.), bean
(Phaseolus vulgaris L.), coriander (Cardimum sativum L.), squash (Cucurbita pepo
L.) and cucumber (Cucumis sativus L.) were dried and autoclaved. Stems and
Egypt. J. Phytopathol., Vol. 45, No. 1 (2017)
176 A.A. EL-WAKIL et al.

branches were then cut into 5 cm segments and put on water agar in Pyrex plates
and 4 parts in each plate. Each treatment was replicated four times.
Discs from 7-days old fungal culture were aseptically transferred onto the water
agar in Petri plates close to plant debris segments and incubated at 22±2 oC for eight
days under alternating cycle of 12/12, NUV/darkness. Density of mycelium and
pycnidia formation were recorded after 4 and 8 days.
4.1 Effect of some antagonists on pycnidia production by D. bryoniae in vitro:
Four well-known bioagents were used as induction factors of D. bryoniae for
pycnidia production. One isolate of each of Trichoderma harzianum, T. viride
(Martinez et al., 2013) and Penicillium aurantiogrism were freshly isolated from
native soil, purified, identified and maintained on PDA slants while Bacillus subtilis
was obtained from Bacterial Diseases Res. Dept., Plant Pathology Res. Institute,
ARC. Each treatment (individual bioagent) consisted of four replicates. Discs (5-
mm diameter) of D. bryoniae culture and each of the bioagents were grown in two
opposite-side positions in 9-cm Pyrex dishes containing PDA medium and
incubated at 22±2oC for eight days under alternating cycle of 12/12, NUV/darkness.
Control treatment was carried out using both discs of D. bryoniae only. Inhibition
zones and number of aligned pycnidia were estimated. This experiment was ended
when mycelial growth from the two discs met in the control treatment.
4.2 Effect of wounding mycelial mat on pycnidia production of D. bryoniae:
This experiment was planned to understand cultural behavior of D. bryoniae in
vitro. Eight PDA Pyrex plates of 9-cm diameter were divided into two groups, 4
plates each. Random punctures of 5-mm were made in each plate medium of the
first group using flamed cork-borer, then inoculation with 5-mm disc of D. bryoniae
at the center. Plates of the second group were first inoculated and punctured later.
Plates of both groups were incubated at 22±2oC for eight days under alternating
cycle of 12/12, NUV/darkness. On the eighth day, plates of the second group were
fully covered with very light growth of mycelium. Random punctures of 5-mm
were made in each plate medium of the second group using flamed cork-borer, then
resumed incubation for one more week with daily observation for both groups.

Results and Discussion

Detection of D. bryoniae:

Diseased cantaloupe plants showing stem cankers with characteristic red to

brown gummy exudates and diseased fruits showing spots of greasy green,
brownish and/or brown colors were collected from El-Bostan (Fig. 1a). At later
stages, the pathogen produces typical symptoms on the radicle with numerous
pycnidia.

Didymella bryoniae has been shown to cause fruit rot of cucurbits (Waint, 1945;
Kagiwata, 1967; Figueiredo et al., 1970; Cardoso et al., 1974 and Sitterly and
Keinath, 1996). During the maturation of the seeds and seed harvesting there is
Egypt. J. Phytopathol., Vol. 45, No. 1 (2017)
 FACTORS PROMOTING PYCNIDIA PRODUCTION….. 177

ample opportunity for the spores of this pathogen to spread, germinate and invade
seeds. The seed can easily be inoculated artificially with spores of the fungus
(Rankin, 1954 and Brown and Preece, 1968).

In the current investigation, most of the conidia in these pycnidia were

nonseptate (Fig. 1b), some uniseptate and few biseptate as compared with slightly
smaller, generally nonseptate, spores in pycnidia were produced (Lee et al., 1984).
The results obtained in this investigation indicated that there was a great chance
of introducing the disease to nursery beds. The significance of seed-borne infection
by D. bryoniae lies large scale development of the disease, but also in the
introduction of inocula to previously uninfested areas. For this reason, the presence
of D. bryoniae on/or in the seed may provide an unsuspected and potentially
dangerous source of infection.

Factors affecting pycnidia production by D. bryoniae:

1. Effect of media:

Among media tested in Table 1, three of them gave white mycelial growth, i.e.
CDA, BDA and PDA media. While SFCA, YEA, CMDA, V-8A, 0.5V-8A and
OMA gave off-white color ranging between dull and pinkish white colors.
Regarding the number of pycnidia appeared in square cm, the 0.5V-8A showed the
highest number with an average of 17 pycnidia/cm2 followed by BDA (14
pycnidia/cm2) and V-8A (8 pycnidia/cm2). Average number of pycnidia formed on
other media was lesser than 5 pycnidia/cm2.
Only V-8A (full and half- concentrations) were the earliest to show pycnidia
formation after 10 days only. However, location of formed pycnidia differed in the
two concentrations tested. In case of the full concentration, pycnidia localized
around the center, scattered and/or upper surface, while in case of half-
concentration, locations of pycnidia developed were toward the edge and/or upper
surface only. On the other media tested, pycnidium formation was delayed to 15 –
25 days. These results are in agreement with those reported by Young et al. (2010)
who stated that pycnidia location varied according to the medium. Alam et al.
(2001) mentioned that the pycnidia of Botryodiplodia theobromae were often found
partially embedded in the medium, they were visible from the reverse side of Petri
dish. This clearly indicated that the different media; with different nutritive
components, affected mycelial color, number and location of pycnidial formation.

Egypt. J. Phytopathol., Vol. 45, No. 1 (2017)

178 A.A. EL-WAKIL et al.

Table 1. Physiological impacts of different media on some habit characters of

D. bryoniae the cause of gummy stem blight on cucumber plants
Habit Character
Days to first
Media Av. number of **Pycnidia
Mycelial color pycnidium
pycnidia/cm2 location
formation
SFCA White beige 2 1&4 15
YEA Pinkish white 4 1&5 20
CMDA White beige 3 5&7 20
V-8A Dull white 8 1&3&4 10
0.5 V-8A Dull white 17 2&4 10
OMA Pinkish white 2 6 20
CDA White 2 1&4 20
BDA White 14 7 20
PDA White 2 3&4 25
** Pycnidia location:1 = around center, 2 = edge, 3 = scattered, 4 = upper surface,
5 = reverse surface, 6 = beneath mycelium and 7 = embedded

2. Effect of media concentration on mycelial growth and pycnidia production:

It is clear from the data presented in Table 2 that richest media concentrations
did not boost the fungus to produce pycnidia, meantime, least concentrations of
media used revealed maximum pycnidial units where V-8 (¼ cons.) and CDA (¼
cons.) gave numerous pycnidia when compared to PDA (¼ cons.) which ranked the
least. This is in agreement with Keinath (2013) who mentioned that one-quarter-
strength potato dextrose agar produced pycnidia and conidia typical of D. bryoniae
3 days after culturing on the surrounding agar. On the contrary, mycelium was very
dense on V-8 medium, dense on CDA and 0.5 V-8A media, while other treatments
gave light mycelial densities. Figure 2 illustrates variable effects of V-8 medium
concentrations on pycnidia production.

Regarding mycelial radial growth, only V-8 and PDA gave distinguished
records while others were more or less similar. Data related to location of pycnidia
produced on media surface were not markedly affected by media concentration.
Alam et al. (2001) noticed negative correlation and highly significant effect on
formation of pycnidia and reduction of colony diameter when glucose was
increased in PDA.

Egypt. J. Phytopathol., Vol. 45, No. 1 (2017)

 FACTORS PROMOTING PYCNIDIA PRODUCTION….. 179

a b
Fig. 1: a = symptoms collected from El-Bostan, Beheira governorate;
b = nonseptate pycnidiospores released from pycnidium (1000x)

Fig. 2. D. bryniae grown on three different V-8 concentrations revealed the maximum
number of pycnidia on V-8 (¼ conc.)

Egypt. J. Phytopathol., Vol. 45, No. 1 (2017)

180 A.A. EL-WAKIL et al.

Table 2. Effect of media concentration on mycelial growth and pycnidia

production after 7 days of incubation at 22±2 ºC under alternating cycle of
NUV/darkness (12/12hrs.)
Habit character
Media Mycelial
Mycelial ** Pycnidia Pycnidia
concentration radial
Density production location
growth
PDA 2.5 Light None None
PDA (½ cons.) 1.5 Light None None
PDA (¼ cons.) 1.2 Light + Around the disc
CDA 1.2 Dense None None
CDA (½ cons.) 1.3 Light ++ Around the disc
CDA (¼ cons.) 1.2 Light +++ Around the disc
V-8 2.6 Very dense None None
V-8 (½ cons.) 1.6 Dense + Around the disc
3mm away from
V-8 (¼ cons.) 1.5 Light +++
the disc
** + = 1 to 10 pycnidia ++ = 11 to 20 pycnidia +++= More than 20 pycnidia

3. Effect of light sources on mycelia growth and pycnidia production:

Data in Table 3 show variable effects of the three lighting regimes applied on
mycelial color where NUV gave the most clear and distinguished color of pink,
while CW and CD regimes gave immature colors, beige and white, respectively.
Regarding number of days till pycnidium formation, NUV was the only regime that
boosted the fungus toward pycnidium formation. It seemed that NUV and CW had
similar effects on linear growth of the mycelium. Overall, NUV proved its necessity
for coloring the mycelial pad and pycnidia formation and missing such an important
tool of incubation process, results obtained might be misleading. Current results are
in harmony with those obtained by Neergaard (1979) who stated that using
monochromatic radiation represented by NUV region of spectrum, 3200 – 4000 Å
is very efficient in inducing sporulation. He added that black light fluorescent
which emits light mainly at wavelengths near 3650 Å, and cool white daylight
fluorescent which emits some NUV light had become standard equipment in seed
health testing. Moreover, Leach (1967) found that pigmentation of fungi was
greatly influenced by the presence or absence of light. Irradiation such as by NUV
usually stimulates strong pigmentation.

Egypt. J. Phytopathol., Vol. 45, No. 1 (2017)

 FACTORS PROMOTING PYCNIDIA PRODUCTION….. 181

Table 3. Effect of three light sources on some habit characters of D. bryoniae

incubated in Pyrex plates for 14 days at 22±2oC
Habit character

Mycelial Av. of radial Days to first pycnidium

Plate material
color growth (cm) formation

Pyrex glass Pink 7.0 14

Standard plastic Pink 5.6 14
Commercial glass Beige 5.8 None
NUV = near ultra violet. CDL = continuous day light CD = complete darkness

4. Effect of container material on mycelia growth and pycnidia production:

Obviously, data presented in Table 4 clarify that Pyrex glass was superior to the
other two culturing containers with respect of linear growth (7 cm in 7 days).
Surprisingly, the standard plastic plates (perspex) revealed the lowest average of
mycelium radial growth of 5.6 cm. Neergaard (1979) mentioned that plastic
containers transmit light with the wave lengths stimulating sporulation of fungi and
had been recommended by the sixth International Workshop on Seed Pathology in
preference to glass containers except Pyrex. Commercial glass material did not
promote the fungus to form pycnidium for as long as 14 days of incubation. Most
probably, using plates made of commercial glass material was one of the main
reasons that delayed the pycnidia recovery in D. bryoniae cultures since the disease
symptoms were noticed in Egypt on watermelon starting from 1985 (Ragab et al.,
1986) as it did not allow the transmission of wavelengths needed for sporulation.
Recently, El-Wakil and Khalil (2016), completed the picture by obtaining,
identifying and recording D. bryoniae the causal pathogen of the gummy stem
blight on cantaloupe for the first time in Egypt.

Table 4. Effect of plate material permeability on some morphological habit

characters of D. bryoniae when incubated under NUV light for 7
days at 25+2ºC

Habit character
Light sources Mycelial Av. radial Days to first pycnidium
color growth (cm) formation
NUV Pink 6.7 14
CDL Beige 6.7 None
CD White 7.1 None

Egypt. J. Phytopathol., Vol. 45, No. 1 (2017)

182 A.A. EL-WAKIL et al.

5. Effect of plant debris on pycnidia production:

Nutrition is considered one of the most important factors affecting growth

behavior of any organism. The pathogen in the current investigation seemed to
produce surviving elements, the pycnidia under certain nutritional conditions.
Moreover, shape of formed pycnidia differed according to crop species depending
on the supporting plant species Shahidul et al. (2001).
Data presented in Table 5 reveal that short incubation period of 4 days was not
enough for D. bryoniae to express sufficient mycelial growth and/or pycnidia
production. Where most of the used plant debris did not show any mycelial growth
while, alfalfa, squash and cucumber gave light dense mycelial growth. The plant
debris of chickpea, flax and cucumber did not help the fungus to produce pycnidia
within the period of 4 days. In the current investigation, it was clearly noticed the
development of dense pycnidia formed on the node of plant debris while less
pycnidia were formed on internode. Beck et al. (1983) emphasized that pycnidia
development abundantly on nodes could be referred to structural and anatomical
factors of nodes cells and tissues; mainly thickness and/or compactness which
might lead subsequently to lesser notorious contents in nodes more than internodes
(Fig. 3).

Coriander plant debris boosted D. bryoniae to produce pycnidia at the maximum

number in this experiment, being < 20 and < 30 after 4 and 8 days of incubation,
respectively. These results could be explained by coriander plant debris components
were the poorest source of nutrition and did not satisfy the fungal needs to grow. On
the contrary, squash plant debris was the only nutritional material enhanced the
mycelial growth to the level "very dense" after 8 days of incubation. The expression
of D. bryoniae pycnidial production against flax plant debris (> 10) was the least at
the end of incubation period.

Table 5. Effect of 9 plant debris materials on mycelial density and number of

pycnidia production after 4 and 8 days of incubation at 22±2 oC
under alternating cycle of NUV/darkness
Habit character
4 days 8 days
Debris Mycelium Number of Mycelium Number of
density pycnidia/cm2 density pycnidia/cm2
Barley - > 10 - 10-20
Chickpea - - Light 10-20
Alfalfa Light 10-20 Light 10-20
Flax - - Light > 10
Rice - 10-20 - 10-20
Bean - 10-20 - 10-20
Coriander - < 20 Light < 30
Squash Light > 10 Very dense 10-20
Cucumber Light - Light 20-30

Egypt. J. Phytopathol., Vol. 45, No. 1 (2017)

 FACTORS PROMOTING PYCNIDIA PRODUCTION….. 183

6. Impact of antagonism on pycnidia production:

Data presented in Table 6 show that antagonism between the fungus D. bryoniae
and the bioagents tested may act as an induction factor for pycnidia production. It
was also clear that there was no relation between the developed inhibition zone and
pycnidia produced. The bioagent T. harzianum was superior in boosting D.
bryoniae to produce the maximum number of pycnidia (> 10 pycnidia/cm 2)
Martinez et al. (2013). Trichoderma viride and Bacillus subtilis gave almost equal
and least number produced (3-5 pycnidia/cm2), while the bioagent Penicillium
aurantiogrisum moderately induced the pathogen to produce 6-10 pycnidia/cm2.
Aries et al. (1997) stated that exudates of P. aurantiogrisum might have promoted
the fungus to produce pycnidia, while metabolites produced by T. harzianum and
T. viride negatively affected the number of pycnidia.

Table 6. Effect of 4 bioagents on pycnidia production of D. bryoniae in vitro

Treatment Inhibition zone (cm) Number of pycnidia (cm2)

Control Zero --
T. harzianum 1.2 > 10
T. viride 1.0 3-5
P. aurantiogrisum 1.75 6-10
B. subtilis 2.0 3-5

7. Effect of wounding mycelial mat:

Plates of the first group did not show any pycnidia production of D. bryoniae.
Figure 4 shows plates of the second group where 6-15 pycnidia on the average were
developed on edge of each puncture after 4 – 7 days of wounding. Although James
et al. (1991) reported that wounding of mycelium did not significantly increase
conidial production. Results of the current investigation are in agreement with other
researchers who proved the opposite. Campbell et al. (2003) obtained similar
results related to number of conidia produced by a number of fungi. They
mentioned that wounding and exposing culture and medium after 7 days to
temperature cycle of 23/19ºC (light/darkness) increased conidia production by
800% or more than the unwounded. They stated that the inhibition of vegetative
development through wounding commonly enhances sporulation. They also cited
that sporulation of Pyrenophora tritici-repentis is routinely enhanced by removal of
aerial mycelium, while wounding of mycelium enhanced sporulation of
Pyrenophora graminea and Alternaria species.

Egypt. J. Phytopathol., Vol. 45, No. 1 (2017)

184 A.A. EL-WAKIL et al.

a b

c d

Fig. 3. a, b and c = different shapes of D. bryoniae pycnidia formed on

different plant debris materials, d= dense pycnidia formed on the
node of plant debris while less pycnidia were formed on the
internode.

Fig 4. Pycnidia formed on cuttings edges made after the mycelium grew
on PDA medium 4-7 days after cutting.

References

Alam, M.S; Begum, M.S.; Sarker, M.L.; Islam, M.R. and Alam, M.S. 2001. Effect
of temperature, light and media on growth, sporulation, formation of pigments
and pycnidia of Botryodiplodia theobromae. Pakistan. J. Biol. Sci.,
4(10):1224-1227.
Aries, S.M.S.; Machado, J.D.A.C. and Arias, E.R.A. 1997. Evaluation of the
antagonistic property of Penicillium. FitoPathologia Brasilerra, 22(3): 437-440.

Egypt. J. Phytopathol., Vol. 45, No. 1 (2017)

 FACTORS PROMOTING PYCNIDIA PRODUCTION….. 185

Arny, C.J. and Rower, C. 1991. Effect of temperature and duration of surface
wetness on spores production and infection of cucumbers by Didymella
bryoniae. Phytopathology, 81: 206-209.
Basim, E.; Basim, H.; Abdulhai, M.; Baki, D. and Oztrürk, N. 2016. Identification
and characterization of Didymella bryoniae causing gummy stem blight disease
of watermelon (Citrullus lanatus) in Turkey. Crop Protec., 90:150-165.
Beck, C.B.; Schmid, R. and Rothwell, G.W. 1983. Stellar morphology and the
primary vascular system of seed plants. Bot. Rev., 48(4): 691-815.
Brown, M.E. and Preece, T.F. 1968. Examination of cucumber seed for
Mycosphaerella melonis. Plant Pathol., 17:116-118.
Campbell, M.A.; Medd, R.W. and Brown, J.B. 2003. Optimizing conditions for
growth and sporulation of Pyrenophora semeniperda. Plant Pathol.,
52:448-454.
Cardoso, R.M.G.; Figueiredo, M.B.; Palazzo, D. and Matinez, J.A. 1974.
Epidemiology of fruit rot (Mycosphaerella melonis) on Italian squash
(Cucurbita pepo). Arquivos do Instituto Biológico, 41(1): 35-37.
Chupp, C. and Sherf, A.F. 1960. Vegetable Diseases and Their Control. New York.
693p.
Crüger, G. and Schneider, R. 1964. On the occurrence of Mycosphaerella melonis
on glasshouse cucumber. Nacher Bl. dt. Pflschutzdienst, Stuttgart. 16:82-85.
(Rev. appl. Mycol. 43, 3366).
El-Wakil, A.A. and Khalil, Amal A. 2016. First report of Didymella bryoniae, the
causal fungus of gummy stem blight on cantaloupe (Cucumis melo var.
cantalupensis) in Egypt. Egypt. J. Phytopathol., 44(1): 229-230.
Figueiredo, M.B.; Cardoso, M.G. and Arrude, H.V. 1970. Blossom excision as a
method of controlling fruit rot infection caused by Mycosphaerella melonis in
Italian squash (Cucurbita pepo). Archivos do Instituto Biologico., 37: 285-292.
Fletcher, J.T. and Preece, T.F. 1966. Mycosphaerella stem rot of cucumbers in the
Lae Valley. Ann. Appl. Biol., 58: 428-430.
James, T.D.W.; Summerell, B.A. and Burgess, L.W. 1991. Production of
pseudothecia and conidia by Pyrenophora tritici-repentis in relation to nutrients
and substrate. Austral. Plant Pathol., 20: 92-96.
Kagiwata, T. 1967. Brown heart rot of cucumber caused by Mycosphaerella
melonis and its control. Bull. Agric. Exp. Stn., Kanagawa, 105: 40-49.
Keinath, A.P. 2013. Diagnostic guide for gummy stem blight and black rot on
cucurbits. Online, Plant Management Network, Plant Health Progress
doi:10.1094/PHP-2013-1024-01-DG.
Egypt. J. Phytopathol., Vol. 45, No. 1 (2017)
186 A.A. EL-WAKIL et al.

Leach, C.M. 1967. The Light Factor in Detection and Identification of Seed-borne
Fungi. Proc. Int. Seed Test. Ass., 32: 565-589.
Lee, D.H; Mathur, S.B. and Neergaard, P. 1984. Detection and location of seed-
borne inoculum of Didymella bryoniae and its transmission in seedlings of
cucumber and pumpkin. J. Phytopathol., 109: 301-308.
Martinez, B.; Perez, J.; Infante, D.; Duarte, Y. and Moreno, M. 2013. Antagonism
of Trichoderma spp. isolates against Didymella bryoniae (Fuckel) Rehm. Revista
de Protecciʹon Vegetal., 28(3):192-198.
Mason, N; Mathews, L.P.; Nicholas, S.D. and Joshua, H.F. 2011. Management of
gummy stem blight (Black rot) on Cucurbits in Florida. UF/IFAS Exten.,
Gainesville, Fl 32611, 280-288.
Neergaard, P. 1979. Seed Pathology. Revised Edition, Volumes I & II, The
Macmillan Press LTD, 1190p.
Ragab, M.M.; Fahim, M.M.; Abdo, Y.A. and Salama, E.A. 1986. Plant Pathology.
4th Ed., 676 p (In Arabic).
Rankin, H.W. 1954. Effectiveness of seed treatment for controlling anthracnose and
gummy stem blight of watermelon. Phytopathology, 44: 657-680.
Richardson, M.J. 1979. An Annotated List of Seed-Borne Diseases. 3rd Ed., Int.
Seed Test. Ass., Zürich, Switzerland. 320p.
Riker, A.J. and Riker R.S. 1936. Introduction to Research on Plant Diseases: A
Guide to the Principles and Practice for Studying Various Plant-Disease
Problems. St. Louis, John S. Swift Co., Inc.
Schenck, N.C. 1968. Epidemiology of gummy stem blight (Mycosphaerella
citrullina) on watermelon. Ascospore incidence and disease development.
Phytopathology, 58: 1420-1422.
Shahidul, A.M.; Begum, M.F.; Sakar, M.A.; Islam, M.R. and Alam, M.S. 2001.
Effect of temperature, light and media on growth, sporulation, formation of
pigments and pycnidia of Botryodiplodia theobromae. Pakistan. J. Biol. Sci.,
4(10): 1224-1227.
Sitterly, W.R. and Keinath, A.P. 1996. Gummy Stem Blight. In: Compendium of
Cucurbits Diseases. Am. Phytopathol. Soc., St. Paul, MN.
Waint, J.S. 1945. Mycosphaerella black rot of cucurbits. J. Agric. Res., 71:193-213.
Choi, I.Y., Choi, J.N., Choi, D.C., Sharma, P.K., & Lee, W.H. 2010. Identification
and characterization of the causal organism of gummy stem blight in the
muskmelon (Cucumis melo L.). Mycobiology, 38(3): 166-170.

(Received 12/04/2017;
in revised form 22/05/2017)

Egypt. J. Phytopathol., Vol. 45, No. 1 (2017)

 ‫‪FACTORS PROMOTING PYCNIDIA PRODUCTION…..‬‬ ‫‪187‬‬

‫العوامل المحفزة إلنتاج بيكنيديات الفطر دايدميال‬

‫بريوناى مسبب مرض لفحة الساق الصمغية فى‬
‫الفصيلة القرعية‬
‫عبد الفتاح عبد الحميد الوكيل‪ ،‬أمل عبد الوهاب‬
‫خليل‪ ،‬إبراهيم حافظ العباسي‬
‫قسم بحوث أمراض البذور ‪ -‬معهد بحوث أمراض النباتات ‪-‬‬
‫مركزالبحوث الزراعية – مصر‬

‫سجل مرض لفحة الساق الصمغية المتسبب عن الفطر ‪Didymella‬‬

‫‪ bryoniae‬فى كثير من بلدان العالم حيث يصيب أغلب محاصيل القرعيات‬
‫خاصة تحت ظروف الرطوبة العالية‪ .‬وفى مصر ورغم تسجيل األعراض‬
‫النموذجية للمرض إال أنه لم يتم عزل وتسجيل الفطر المسبب لسنوات عديدة حتى‬
‫تم عزله ألول مرة فى ‪ 6102‬من نبات الكنتالوب تحت ظروف الرى بالرش فى‬
‫منطقة البستان‪ .‬حيث ظهرت االعراض على هيئة تقرحات ذات لون بنى محمر‬
‫مصحوبة بإفرازات صمغية بينما كانت األعراض على الثمار عبارة عن بقع ذات‬
‫لون أخضر المع فى البداية ثم تتحول الى اللون البنى مؤخرا‪ .‬تمت محاوالت‬
‫العزل من تلك االعراض على بيئة ‪ PDA‬إال أنه لم يتم عزل أى تراكيب جرثومية‬
‫حيث ظهرت نموات هيفية فقط غير مميزة للفطر‪ ،‬ومن ثم كان حتميا توسيع نطاق‬
‫الدراسة لتشمل بعض العوامل التى قد تشجع إنتاج البكنيديات ومنها اختبار ‪9‬‬
‫بيئات طبيعية كمصادر تغذوية مختلفة فكانت بيئة ‪ V-8‬أكثر البيئات تحفيزاً على‬
‫تكوين األوعية البكنيدية وجراثيمها غير المقسمة المميزة للفطر كما كانت أفضلها‬
‫فى سرعة تكوين البكنيديات ثم بيئة ‪ CDA‬فى عدد البكنيديات المتكونة‪ .‬وشملت‬
‫الدراسة تأثير ثالث تركيزات لثالثة بيئات طبيعية هى ‪ PDA‬و ‪ CDA‬و ‪V-8‬‬
‫(تركيز كامل و ½ و¼)‪ ،‬فكان أفضلهم بيئتى ‪ V-8‬و ‪ CDA‬بتركيز ¼ فى إنتاج‬
‫البيكنيديات بعد ‪ 7‬أيام فقط مقارنة بالبيئات والتركيزات األخرى‪ .‬وعند اختبار‬
‫تأثير نوع اإلضاءة فكان استخدام ‪ NUV‬مع ظالم التام فى دورة تبادلية ‪06/06‬‬
‫له دورا واضحا فى تكوين البكنيديات المميزة للفطر‪ .‬كما كان اختبار مادة أوعية‬
‫التحضين له تأثير أيجابى عند استخدام آطباق ال‪ Pyrex‬على تكوين البكنيديات‪.‬‬
‫كما كان لتنمية الفطر على بقايا ‪ 9‬أنواع نباتية جافة تأثير كبير على تكوين عدد‬
‫أكبر من البكنيديات محسوبا ً فى ‪ 0‬سم‪ ،6‬فكانت أفضلهم البقايا الجافة لسوق وأفرع‬
‫نبات الكزبرة‪ ،‬فى حين كان أقلهم تحفيزا إلنتاج البكنيديات هى السوق واألفرع‬
‫الجافة لنبات الكتان بينما كانت بقايا الكوسة والخيار متوسطة التحفيز على تكوين‬
‫البكنيديات‪ .‬وكان لنوع البقايا النباتية المستخدمة تأثير متباين على شكل وعدد‬
‫البكنيديات الناتجة‪ .‬وقد اندفع الفطر إلى تكوين البكنيديات اعتبارا من اليوم الرابع‬
‫بعد إحداث الجروح فى النموات الميسليومية فى حين لم تتكون البكنيديات إذا تم‬
‫التجريح قبل تلقيح األطباق بالفطر‪.‬‬

‫)‪Egypt. J. Phytopathol., Vol. 45, No. 1 (2017‬‬

‫‪View publication stats‬‬