Morphological and Pathogenic Characteristics of the Fungus Cladobotryum dendroides , the Causal Agent of Cobweb Disease of the Cultivated Mushroom Agaricus bisporus in Serbia

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.

Cladobotryum species are soil-inhabiting cosmopolitan fungi found in all mushroom-growing countries worldwide (Van Zaayen and Van Andrichem, 1982;McKay et al., 1998McKay et al., , 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.

Isolates and growth conditions
The isolates of Cladobotryum spp.collected from diseased A. bisporus fruiting bodies in Serbia during 2003-2007 are shown in Table 1.
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 5 o C, 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 25 o C. 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   , 1997a, 1997b).

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 10 6 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 H 2 O were used as a negative control.Inoculated pilei were incubated at room temperature (22 ± 2 o C) for four days and the development of symptoms was observed.

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 10 6 conidia/ml) three days after casing.The bags were incubated at 25 o C during spawn-running of casing (for seven days) and then temperature was decreased to 18 o C (Grogan et al., 2000).Pathogen reisolation from the infected fruiting bodies of A. bisporus was performed on PDA in order to confirm pathogenicity.

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 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 H 2 O 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 became thicker, taking on pinikish hue, A. bisporus fruiting bodies turned dark brown from soft rot.There were no statistical differences among the studied iso-  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 25 o C. 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 25 o C when radial growth rate was in the range between 14 and 20 mm day -1 .No growth of the pathogen was recorded at 10 o C. Beyer and Kremser (2001) reported radial growth rate ranging from 15 to 20 mm day -1 at optimal temperature of 25 o C 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 18 o C. 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) 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) Slankamen of temperature on growth was studied by growing isolates on MEA at 10 o C, 13 o C, 18 o C, 20 o C, 25 o C, 28 o C and 30 o C 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 18 o C. The influence of different agar media: PDA, MEA, Czapek agar (CzA), modified Mushroom Dextrose Agar (mMDA) and Water Agar (WA) was examined at 18 o C. 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