Pestic. fitomed. (Beograd), 23 (2008) 175-181
Pestic. Phytomed. (Belgrade), 23 (2008) 175-181
UDC: 632.4:635.82
Scientific paper * Naučni rad
Morphological and Pathogenic
Characteristics of the Fungus
Cladobotryum dendroides, the Causal
Agent of Cobweb Disease of the
Cultivated Mushroom Agaricus bisporus
in Serbia
Ivana Potočnik, Emil Rekanović, Svetlana Milijašević, Biljana Todorović and
Miloš Stepanović
Institute of Pesticides and Environmental Protection, Banatska 31b, 11080 Belgrade, Serbia
(ivanapotocnik@yahoo.com)
SUMMARY
Twenty isolates were isolated from diseased fruiting bodies of Agaricus bisporus collected from Serbian mushroom farms during 2003-2007. The isolates formed white, cottony,
aerial colonies on agar media. With age, conidia and colonies turned yellow and redish.
Pathogenicity of these isolates was confirmed by inoculation of harvested basidiomes of
A. bisporus and by casing inoculation. Symptoms similar to natural infection were recorded.
Based on pathogenicity tests and morphological characteristics, the isolates were identified as Cladobotryum dendroides (Bulliard : Fries) W. Gams & Hoozemans.
Keywords: Cladobotryum dendroides; Agaricus bisporus; Morphological characteristics
INTRODUCTION
A decade ago, Verticillium fungicola and Mycogone
preniciosa, the causing agents of bubble diseases, were
the most important pathogens of cultivated mushroom
(Agaricus bisporus (L)) in Serbia (Potočnik, 2006).
Today, cobweb disease is one of the most serious diseases in Serbian mushroom farms affecting both quality
and yield (Potočnik et al., 2007). Cobweb disease of A.
bisporus is caused by three Cladobotryum species: C. dendroides (Bulliard : Fries) W. Gams & Hoozemans (teleomorph Hypomyces rosellus (Albertini & Schweinitz : Fries)
Tulasne and C. Tulasne), C. mycophilum (Oudemans)
W. Gams & Hoozemans (teleomorph Hypomyces odoratus G. R. W. Arnold) and C. varium Nees : Fries (teleomorph Hypomyces aurantius (Persoon) Tulasne) (Eicker
and Van Greuning, 1991). All three species cause more
or less similar symptoms: cottony fluffy, white/greyish
175
Ivana Potočnik i saradnici
colonies on mushroom casing, rapid colonization of casing surface and covering of host basidiomata by mycelia,
and its decay. With time, mycelium becomes yellowish
or redish/pink in colour (McKay et al., 1998). The red
colouration is due to the pigment aurofusarin (Rogerson
and Samuels, 1993). C. varium does not exhibit the typical pinkish red coloration of the mycelium as C. dendroides and C. mycophilum. C. dendroides produces phialide extensions/rachises and conidia with conspicuous
basal hilum. C. mycophilum does not share these characteristics and its colonies have a typical camphor odor.
Cladobotryum species are soil-inhabiting cosmopolitan
fungi found in all mushroom-growing countries worldwide (Van Zaayen and Van Andrichem, 1982; McKay
et al., 1998, 1999). They also occur on other mushroom
species growing in uninhabited regions (Rogers and
Samuels, 1989, 1993, 1994).
Cladobotryum spp. produce verticillately or irregularly branched conidiophores. Conidiophore carriers
are branched into three to four phyalides. The conidiophores are hyaline, initially single-celled and later have 1
to 3 septae. The spores are approximately 21-30 x 9-10.5
µm and have a symmetrically placed basal scar where
they had previously joined the phialide. Mycelia produce dark microsclerotia (Hughes, 1978). Ordinarily,
Cladobotryum spp. spores can survive for a maximum
of seven days in sterile water. The survival rate of microsclerotia is considerably higher. Saved in sterile water, 100% of microsclerotia still have germinating power
after four months. It is noted that even when the casing
layer is strongly infested by spores, symptoms usually appear during the last flushes. When the casing layer is infested by mycelium of the pathogen, disease symptoms
are observed just before the first flush, when pins begin
to develop. Very often, the disease begins developing on
mushroom stipes that have been left after harvesting, or
on dead A. bisporus fruit bodies. Cobweb spores are easily dislodged by air and are carried to considerable distances by air (Adie and Grogan, 2000). Flies, people and
equipment are also vectors of the pathogen. The pathogen
thrives under warm moist conditions and grows rapidly
under ideal mushroom growing conditions. Higher casing moistures and lower evaporation rates provide conditions more conducive to disease development.
The aims of this study were to isolate and identify the
causal agent of cobweb disease of the cultivated mushroom A. bisporus in Serbia, and examine pathogen variations as evidenced by the morphology of its colonies
under different growth conditions and their pathogenic characteristics.
176
MATERIAL AND METHODS
Isolates and growth conditions
The isolates of Cladobotryum spp. collected from diseased A. bisporus fruiting bodies in Serbia during 20032007 are shown in Table 1.
Table 1. List of isolates of Cladobotryum spp. and their
origin
Tabela 1. Lista izolata Cladobotryum spp. i njihovo poreklo
Oznaka izolata
Isolate code
SP1C4
P3C1
Ba1C1
B1C1
Ku1C1
NSl1C1
OB1C2
OB1C3
P 7C1
Res1C1
VG3C2
Beč1C1
Jak1C1
Kal1C1
NSl 2C1
Beč2C1
Veg2C1
VG2C2
NSl3C1
NSl4C1
Poreklo izolata
Origin of isolate
Smederevska
Palanka
Požarevac
Beograd –
Banjica
Beograd – Savski
Venac
Kurjače
Novi Slankamen
Ovčar Banja
Ovčar Banja
Požarevac
Resnik
Vračev Gaj
Bečej
Jakovo
Kaluđerica
Novi Slankamen
Bečej
Veliko Gradište
Vračev Gaj
Novi Slankamen
Novi Slankamen
Godina skupljanja
Year of collection
2003
2003
2004
2004
2004
2004
2004
2004
2004
2004
2004
2006
2006
2006
2006
2007
2007
2007
2007
2007
Isolation was done by taking small pieces (2 x 2 x 5
mm) of fruiting bodies with disease symptoms, immersing them in a 1% sodium hypochlorite solution for 1
min, and placing onto Potato Dextrose Agar (PDA). The
isolates were kept on PDA, at 5oC, in the culture collection of the Institute of Pesticides and Environmental
Protection, Belgrade. Colony morphology was studied
after three days of cultivation on Malt Extract Agar
(MEA) at 25oC. Conidium size, the number of septa
per conidium, the presence or absence of phialide extension/rachis, and the conspicuous basal hilum on
the conidia were studied. Chlamidospore and microsclerotium production was also noted. The influence
Pestic. fitomed. (Beograd), 23 (2008) 175-181
of temperature on growth was studied by growing isolates on MEA at 10oC, 13oC, 18oC, 20oC, 25oC, 28oC
and 30oC after three days. Optimal pH for pathogen
growth was studied on Potato Dextrose Agar (PDA) by
adjusting the pH on a scale of 5-9 at 18oC. The influence of different agar media: PDA, MEA, Czapek agar
(CzA), modified Mushroom Dextrose Agar (mMDA)
and Water Agar (WA) was examined at 18oC. Each
plate was inoculated with an inverted mycelium agar
disc (10 mm), taken from the edge of four-day-old cultures of Cladobotryum spp. isolates placed centrally onto the agar media. Colony diameter was measured after three days of cultivation. Three replicates per each
treatment and isolate were submitted to statistical analyses. Data were analysed separately for each trial using
ANOVA and the means were separated by Duncan‘s
multiple range test (EPPO, 1997a, 1997b).
RESULTS AND DISCUSSION
Diseased fruiting bodies of A. bisporus with symptoms resembling cobweb disease were observed on 13
Serbian mushroom farms. Early symptoms were round,
fleshy, yellowish brown lesions on A. bisporus caps
(Figure 1). Late symptoms progressed when the parasitic fungus formed white cobweb-like circular colonies
on dead or damaged pinheads, spread on the surface
of the casing and covered entirely A. bisporus fruiting
bodies (Figure 2). With age, the fluffy mycelia became
thicker and granular, taking on pinkish hue. A. bisporus
Pathogenicity test I
Pathogenicity assay was performed on harvested basidiomes of A. bisporus by a modified method of Collopy
at al. (2001). Approximately 1 ml of spore suspensions
containing 3 x 106 conidia mL-1 were prepared out of
four-day-old cultures of all tested Cladobotryum spp.
isolates. Pilei were converted and inoculated at a site
of previously removed stipes. Pilei treated with 1 ml of
sterile H2O were used as a negative control. Inoculated
pilei were incubated at room temperature (22 ± 2oC)
for four days and the development of symptoms was
observed.
Figure 1. Early symptoms of cobweb disease on naturallyinfected Agaricus bisporus caps by fungus Cladobotryum
dendroides
Slika 1. Rani simptomi paučinaste plesni na šeširima
Agaricus bisporus nakon prirodne zaraze gljivom
Cladobotryum dendroides
Pathogenicity test II
Spawn-run compost (A. bisporus Italspown F 56),
produced by Uča & Co., Vranovo, Serbia, was used for
the pathogenicity test. Compost bags were cased with a
40-50 mm layer of black peat/lime casing („Makadam”
Co., Belgrade), which was artificially inoculated with
the studied Cladobotryum spp. isolates. Casing inoculation was done by spore suspension spraying (approximately 106 conidia/ml) three days after casing. The
bags were incubated at 25oC during spawn-running of
casing (for seven days) and then temperature was decreased to 18oC (Grogan et al., 2000). Pathogen reisolation from the infected fruiting bodies of A. bisporus was
performed on PDA in order to confirm pathogenicity.
Figure 2. Progressed symptoms of cobweb disease on
naturally-infected Agaricus bisporus fruiting bodies by
fungus Cladobotryum dendroides
Slika 2. Kasni simptomi paučinaste plesni na plodonosnim
telima Agaricus bisporus nakon prirodne zaraze gljivom
Cladobotryum dendroides
177
Ivana Potočnik i saradnici
caps turned dark brown and eventually shrunk due to
soft rot. The described symptoms fit those caused by
Cladobotryum spp.
Pathogenicity assay on mushroom pilei showed that
each of the twenty isolates had high virulence level for
A. bisporus. The symptoms were not produced on pilei
treated with sterile H2O that was used as a negative control. All isolates induced severe disease symptoms on A.
bisporus pilei. The growth of pathogen mycelia was recorded two days after inoculation. White cobweb mycelium extended beyond the inoculation site. Three days
after inoculation, the sporocarps were completely covered with white cottony mycelium and profuse sporulation was noted, resembling the symptoms of natural
infection. The pilei were completely rotten, soft and decayed on the fourth day of incubation (Figure 3). There
were no significant differences in the levels of symptom
development among the different isolates.
The first symptoms were noticed twelve days after artificial inoculation of casing layer with the investigated
Cladobotryum spp. isolates. The white fluffy mycelium
first appeared on the casing layer and covered the fruiting bodies of A. bisporus. Colony was initiated as small,
circular patches of infection on casing soil. The diameter of infection was usually no larger than 3 to 4 centimetres. Infection spreaded from dead pinheads and
stalks. The mycelium quickly overwhelmed A. bisporus
fruiting bodies. The infected mushrooms were browncoloured and decayed.
Cobweb mycelium was initially white or grayish
(Figure 4). Later, the mycelium and infected mushrooms assumed reddish colour. Colonies growing
on the casing were circular and overwhelmed mushrooms, causing rapid decay. As the cobweb mycelia be-
Figure 3. Left – Agaricus bisporus sporocarp three days after
artificial infection by fungus Cladobotryum dendroides,
isolate B1C1; right – negative control
Slika 3. Levo – Izgled sporokarpa Agaricus bisporus tri dana
nakon veštačke zaraze gljivom Cladobotryum dendroides,
izolat B1C1; desno – negativna kontrola
178
Table 2. Conidial size of investigated Cladobotryum
dendroides isolates
Tabela 2. Veličina konidija ispitivanih izolata Cladobotryum
dendroides
Isolate Conidial length μm Conidial width μm
Izolat Dužina konidije μm Širina konidije μm
SP1C4
18.99 a
10.33 a
(12.3-26.06)
(7.38-11.07)
P3C1
19.99 a
9.53 a
(12.3-26.06)
(7.38-11.07)
Ba1C1
22.24 a
9.05 a
(12.3-26.06)
(7.38-11.07)
B1C1
17.83 a
11.07 a
(12.3-26.06)
(6.15-11.07)
Ku1C1
19.37 a
9.34 a
(12.3-26.06)
(7.38-11.07)
NSl1C1
20.29 a
10.46 a
(12.3-26.06)
(7.38-11.07)
OB1C1
22.45 a
8.61 a
(12.3-26.06)
(7.38-11.07)
OB1C2
20.27 a
9.25 a
(12.3-26.06)
(7.38-11.07)
OB1C3
21.22 a
9.25 a
(12.3-26.06)
(7.38-11.07)
P 7C1
20.60 a
8.92 a
(12.3-26.06)
(7.38-11.07)
Res1C1
19.97 a
9.15 a
(12.3-26.06)
(7.38-11.07)
VG3C2
17.53 a
9.84 a
(12.3-26.06)
(7.38-11.07)
Beč1C1
20.91 a
8.91 a
(12.3-26.06)
(6.15-11.07)
Jak1C1
19.68 a
8.61 a
(12.3-26.06)
(7.38-11.07)
Kal1C1
18.76 a
9.53 a
(12.3-26.06)
(7.38-11.07)
NSl 2C1
19.68 a
9.84 a
(12.3-26.06)
(6.15-11.07)
Beč2C1
21.22 a
8.92 a
(12.3-26.06)
(7.38-11.07)
Veg2C1
19.98 a
9.84 a
(12.3-26.06)
(7.38-11.07)
VG2C2
19.37 a
8.61 a
(12.3-26.06)
(7.38-11.07)
NSl3C1
20.30 a
9.25 a
(12.3-26.06)
(7.38-11.07)
*mean values in columns followed by the same letter do not differ
significantly, p=0.05
*ista slova u koloni označavaju da nema statistički značajne razlike,
p=0,05
came thicker, taking on pinikish hue, A. bisporus fruiting bodies turned dark brown from soft rot. There
were no statistical differences among the studied iso-
Pestic. fitomed. (Beograd), 23 (2008) 175-181
Figure 4. Fruiting bodies of Agaricus bisporus artificiallyinfected by fungus Cladobotryum dendroides, isolate SP1C4
Slika 4. Inokulisana plodonosna tela Agaricus bisporus
izolatom SP1C4 gljive Cladobotryum dendroides
lates. Pathogenicity of all investigated isolates was confirmed, on which occasion symptoms had attributes of
cobweb disease. The results were in accordance with
those recorded by Bhatt and Singh (1992) and Beyer
and Kremser (2001).
The isolates formed white, cottony, aerial mycelium
on MEA at 25oC. The mycelia produced spores four
days after inoculation and changed the colour from
white to yellow (Figure 5). After nine days the colour
of colonies turned pink, and after 12 days red. The maximum mycelial growth of the Serbian Cladobotryum
spp. isolates was noted on MEA at 25oC when radial
growth rate was in the range between 14 and 20 mm
day-1. No growth of the pathogen was recorded at 10oC.
Beyer and Kremser (2001) reported radial growth rate
ranging from 15 to 20 mm day-1 at optimal temperature of 25oC on MEA. Among the media evaluated, the
best growth of the investigated Serbian Cladobotryum
spp. isolates was recorded on PDA (44.20 mm), followed by CzA (43.18 mm), mMDA (38.48 mm),
MEA (33.60 mm) and WA (30.38 mm) three days after inoculation at 18oC. Consistent with observations
of Dhar and Seth (1992) and Bhatt and Singh (1992),
the optimal pH for pathogen growth was 7.0. The
hyphae were hyaline, septate and prostrate, with 3-4
pointed and oppositely placed branches. The conidiophores were erect, hyaline, simple, arising from aerial mycelium. They were branching verticillately, terminating in groups of phialides that tapered toward
the apex. Conidia were hyaline, oblong, and had one to
three septa, with centrally or laterally placed conspiciuous basal hilum (Figure 6). Secondary extension (ra-
chis) was evident on the phialides. Their dimensions
were 6.15 – 9.38 - 11.07 μm x 12.30 – 19.96 - 27.06
μm. Similar observations were reported by Bhatt and
Singh (1992), Rogerson and Samuels (1993) and McKay
et al. (1998). Chlamidospores and microsclerotia were
present. Chlamydospores and microsclerotia are regularly produced both by C. dendroides and C. mycophilum, but conidia with conspicuous basal hilum and
a secondary extension on the phialides are typical of C.
dendroides (McKay et al., 1998). According to the investigated morphological and pathogenic characteristics,
the microfungal isolates from screened Serbian A. bisporus farms were identified as C. dendroides (Bulliard :
Fries) W. Gams & Hoozemans.
Figure 5. Colony of fungus Cladobotryum dendroides,
isolate SP1C4
Slika 5. Izgled kolonije gljive Cladobotryum dendroides,
izolat SP1C4
Figure 6. Conidiophores of fungus Cladobotryum
dendroides, isolate VG2C2, terminating in groups of
phialides (a); conidia had one to three septa (b)
Slika 6. IIzgled konidiofore i konidija gljive Cladobotryum
dendroides, izolat VG2C2: fijalide u grupama na završecima
konidiofora (a); konidije sa jednom do tri septe (b)
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Ivana Potočnik i saradnici
ACKOWLEDGEMENT
This study is part of Project TR 20036, which is financially supported by the Ministry of Science and
Technological Development of Serbia.
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Pestic. fitomed. (Beograd), 23 (2008) 175-181
Morfološke i patogene
karakteristike gljive Cladobotryum
dendroides, prouzrokovača
paučinaste plesni šampinjona
Agaricus bisporus u Srbiji
REZIME
Dvadeset mikrofungalnih izolata je dobijeno iz obolelih plodonosnih tela Agaricus bisporus prikupljenih iz gajilišta u Srbiji u periodu od 2003. do 2007. godine. Izolati su obrazovali bele, vazdušne kolonije na krompir-dekstroznoj podlozi. Nakon nekoliko dana kolonije su poprimile žutu i ružičastu boju. Veštačkim inokulacijama ubranih plodonosnih tela A.
bisporus i pokrivke za gajenje šampinjona, pojavili su se simptomi paučinaste plesni. Izolati
su identifikovani na osnovu morfoloških i patogenih osobina kao Cladobotryum dendroides
(Bulliard : Fries) W. Gams & Hoozemans.
Ključne reči: Cladobotryum dendroides; Agaricus bisporus; morfološke karakteristike
181