Mushroom Biology and Mushroom Products. Sánchez et al. (eds). 2002UAEM. ISBN 968-878-105-3

COBWEB DISEASE OF AGARICUS BISPORUS: INCIDENCE, LOSSES AND EFFECTIVE MANAGEMENT

N. Bhatt and R.P. SinghDepartment of Plant Pathology. Mushroom Research Centre

G.B. Pant University of Agriculture and Technology, Pantnagar-263 145. (Uttaranchal) India

<nirmala_bhatt@hotmail.com>

ABSTRACT

Mushrooms are subject to attack by disease-causing organisms, including fungi, bacteria, viruses and nematodes. Some of them are capable of reducing the yield significantly or even result in crop failure depending upon their severity and stage of appearance. One of the mycoparasites, Hypomyces rosellus (the cause of cobweb disease) was observed in samples collected from seasonal mushroom farms of North Indian states. Symptoms of the cobweb disease appeared by the next day on the inoculated sporophores and inoculation in the casing resulted in the appearance of symptoms after 12 days. The pathogen covered the stipe and edges of the curved pileus, thus converting the fruit bodies into soft cottony balls. The mycelium of A. bisporus was overgrown by the mycelium of pathogen in-vitro and a 14.06 percent inhibition in growth was recorded. Temperatures of 250C and pH 6.5 were found optimal for the mycelial growth of the pathogen, both in compost and in casing. Inoculation of the pathogen in compost at spawning and casing resulted in 47.10-60.56 and 45.10-72.06 percent losses in yield respectively at 0.1-1.0 percent inoculum loads. The concentration of macronutrients and the pH of the casing decreased significantly with the advancement of the crop when the pathogen was inoculated in casing. Among the 18 fungicides screened, five were found promising. Of these, carbendazim and prochloraz manganese inhibited the growth of pathogen in-vitro at lower concentrations. Significantly higher yields were obtained by incorporating prochloroz manganese (0.075%) into the compost and spraying three times thereafter at 14 day intervals.

INTRODUCTION

The genus Cladobotryum syn. Dactylium, introduced into mycological literature by Nees (1816, 1817) was identified to be the imperfect stage of Hypomyces rosellus (Gams and Hoozemans 1970, Sinden 1971, Fletcher and Atkinson 1977). In India it was first recorded in Himachal Pradesh at Chail and Shimla (Seth 1977) and later in Solan and Kasauli with a natural incidence ranging from 8.17-25.63 percent in 1986 (Seth and Dar 1989). Symptoms of the disease appear as fluffy growth of the fungus at the surface of casing, and symptoms and severity vary with the time of infestation of the casing soil and the type of casing material used. The pathogen H. rosellus is capable of growing at temperatures ranging from 10-330C and pHs ranging from 3-8 (Dar and Seth 1992). Management of this fungal diseases of A. bisporus poses problems because both the host and pathogen are fungi. Consequently, there is a need to select fungicides which have least effect on the growth of the mushroom. The present studies of incidence, losses and effective management of cobweb disease caused by the fungal pathogen Hypomyces rosellus were conducted bearing these considerations in mind.

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MATERIALS AND METHODS

Various commercial farms cultivating A. bisporus were intensively surveyed during crop season from November to March 1999-2000 to locate/isolate the pathogen H. rosellus. Incidence was calculated on the basis of samples examined and the presence of the pathogen in the samples.

Mycelial cultures of H. rosellus were grown from infected fruitings of A. bisporus and casing soil disinfected in 0.01 percent mercuric chloride solution for 30 seconds. The medium was supplemented with oxytetracycline (50 ppm) to suppress bacterial growth. Spores were harvested by flooding 6-day-old culture on malt extract agar with sterile distilled water and lightly rubbing the culture surface with a sterile loop. The concentration of resulting suspension was adjusted to 2x107

spores/ml. The inhibitory effect of the pathogen on A. bisporus was tested by keeping a 5 mm. disk of each at a distance of 25 mm. on petri plates containing malt extract agar medium.

Compost was sterilized in polypropylene bags at 650C (3 p.s.i.) for 90 minutes. Then 3g grain spawn of A. bisporus and H. rosellus were placed separately at the bottom of sterilized glass tubes of 180 mm. in length and 4 mm. in diameter. Twenty grams of sterilized compost were filled into each tube under aseptic conditions. Similarly, the casing mixture of FYM + spent compost (3:1) was autoclaved. Then the sterilized glass tubes were filled with 3g spawn of A. bisporus and pathogen separately and covered by placing 40g of sterilized casing in each tube under aseptic conditions. Growth rates at different pHs were studied adjusting the initial pH of compost and casing mixture using CaCO3 and CaSO4.2H2O before sterilization. Experiments were replicated thrice and incubated for mycelial growth, which was measured at 24 h intervals for 10 days.

The loss in yield was assessed by inoculating the pathogen into the compost and casing separately by weight at the rate of 0.10, 0.50, 0.75 and 1.00 %. Grain spawn of A. bisporus strain X-13 was mixed into the compost by weight at the rate of 0.75 %. Ten kg compost were filled into each bag and treatments were replicated thrice. Standard cultivation practices were followed. Uninoculated but spawned compost bags were kept as a control.

Eighteen different systemic and non-systemic fungicides were evaluated at four different concentrations to observe their inhibitory effects on the mycelial growth of A. bisporus using the food poison technique of Falk (1907). Percent inhibition compared to the control was calculated by applying the following formula (Vincent 1947):

I = C-Tx100C

Where I = Percent Inhibition; C = Growth in control specimens; and T = Growth in treated specimens

In the bioassay test, the five fungicides which displayed the least adverse effects on the growth of A. bisporus were further evaluated to determine their inhibitory effect against the pathogen H. rosellus. The fungicides tested were: prochloraz manganese, carbendazim, ridomil, captaf and carbendazim+formalin at four different concentrations. All the treatments were replicated three times and incubated for 6-10 days at a temperature of 24±10C.

This trial incorporated two different doses of fungicides in the casing. The prepared doses of inocula (0.50 and 0.75 %) were mixed into the casing. Then the casing was applied into the bags with compost fully colonized by A. bisporus strain X-13. In both the trials three fungicidal sprays were

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applied at 14-day intervals. Controls with inoculum and without fungicides (Control I) and without inoculum and fungicides (Control II) were kept for comparison of yields.

RESULTS AND DISCUSSION

The incidence of H. rosellus varied from 0-17.85 percent, lower than the 8.17-25.63% reported by Seth and Dar in 1989. The pathogen isolated from infected fruiting and casing soil on potato dextrose agar was multiplied on wheat grains and inoculated in the casing as well as in the fruiting bodies on the mushroom beds. Symptoms of the disease appeared by next day on the inoculated sporophores. Inoculation on casing resulted in symptoms after 12 days. In both cases, younger mushrooms were more susceptible than mature ones. On the casing, the pathogen appeared as a white fluffy growth (Figure 1A). It completely engulfed the young developing mushrooms after creeping over the surface of casing. Patches 2.5-35-cm. in diameter consisting of a felty layer 4 to 8 mm thick were observed. The attacked mushrooms were trapped under this web, which decomposed shortly, leaving cottony balls behind (Figure 1B). When the infected mushrooms were incubated at 250C under normal conditions, enormous spores appeared on the mycelial tissue of the pathogen. The mycelium of the fungus was initially white, later became light yellow in colour. The hyphae were hyaline, septate, and prostrate, with 3-4 pointed and oppositely-placed branches. The conidiophores were erect, simple, and branched in many whorls. Conidia were single, elongated, pointed at the base, two-celled, slightly constricted at the septa and measured 15-32 x 8-14 µ (Figure 1C).

Contrary to the observations of Olivier and Guillaumes (1976) who found that mushroom mycelium showed antagonism to Hypomyces sp, a 14.06 % inhibition in the growth of A. bisporus mycelium was recorded (Table 1, Figure 2).

Data presented in Table 2 reveals that at 50C, A. bisporus failed to grow. The optimal temperature for the mycelial growth of both the fungi was 250C. The growth of the pathogen was greater in the casing, and the growth of A. bisporus was greater in the compost. The optimal pHs for the growth of the pathogen and A. bisporus were 6.5 and 7.0, respectively (Table 3). H. rosellus and A. bisporus failed to grow at pH 8 and at pH 5.50, respectively. However, Dar and Seth (1992) reported a pH range of 3-8 and temperatures of 21-240C for the growth of pathogen.

Inoculation of compost and casing with different concentrations of pathogen showed that losses in yield were higher when the pathogen was inoculated in casing (Table 4). Maximum loss in yield, (76.2 %), was recorded at 1.0 percent inoculum load (inoculum in casing) A 61.15 % loss in yield occurred when the inoculum was mixed with compost at spawning. Losses in yield recorded by earlier workers (Seth and Dar 1989) were considerably lower (21.95 – 48.95 %) than those recorded in this study. Environmental factors during experimentation and the strain of the pathogen used may be responsible.

In order to identify the fungicides that could be safely used to control the pathogen in A. bisporus beds, eighteen different fungicides (systemic and non-systemic) were tested at four different concentrations. Captaf, carbendazim, prochloraz manganese (at 25 ppm), ridomil (200 ppm) and formalin (250 ppm) had little inhibitory effect on the growth of A. bisporus, so they were selected for further studies with regard to their effect on H. rosellus. It has been reported earlier that benomyl and MBC did not have any adverse effects on the growth of mushroom mycelium even at concentrations as high as 500 ppm (Jandaik et al. 1978). It has also been reported that spraying carbendazim, thiophanate methyl in-vitro and in-vivo at 50 and 500 ppm concentrations had no adverse effects on the mushroom mycelium. The spawn-run in treated beds was in fact reported to

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be faster, more whitish and luxuriant compared to growth in a control. The fungicides were found to be slightly inhibitory to the mycelial growth at 1000 ppm (Thapa and Raina 1989).

Five funigicides (carbendazim, prochloraz manganese, captaf, ridomil and formalin) selected on the basis of their harmlessness to A. bisporus were screened for effectiveness against the pathogen at different concentrations (Figure 3). Carbendazim and prochloraz manganese (10 ppm) and a combination of carbendazim and formalin (4+4 ppm) caused 100% inhibition of the growth of the pathogen. Other fungicides inhibited the growth of H. rosellus at concentrations above 20 ppm.

Table 1: Inhibitory effect of the pathogen Hypomyces rosellus on AgaricusAgaricus bisporus in-vitro.bisporus in-vitro.

Treatments Radial growth (mm) Percent Inhibition

H. rosellus 18.33 14.06

A. bisporus 21.33 0.00 CD at 5% 1.67

Table 2: The effects of different temperatures on the growth of Agaricus bisporus and the pathogen

Hypomyces rosellus in compost and casing. in compost and casing.Temperature

0C

Growth in mm

Compost Casing

H. rosellus A. bisporus H. rosellus A. bisporus

5 6.33 0.00 9.67 0.00

10 15.00 6.34 18.00 4.34

15 19.00 17.00 25.00 12.67

20 42.00 51.00 51.67 47.00

25 98.34 95.67 122.67 84.00

30 70.67 37.00 100.00 28.67

35 7.34 0.00 9.34 0.00 CD at 5% 1.67 1.97 2.50 1.47

The results obtained under in-vitro trials revealed that carbendazim, prochlaraz manganese, ridomil,

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Mushroom Biology and Mushroom Products. Sánchez et al. (eds). 2002UAEM. ISBN 968-878-105-3

captaf and formalin were comparatively safe in the sense that they did not adversely affect the growth of A. bisporus to any significant extent. These fungicides were sprayed on the mushroom beds at 14 day intervals. The results obtained are presented in Table 5. A significant decrease in

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yield was obtained with increased inoculum loads. The yield obtained after spraying with prochloraz manganese and carbendazim (0.075%) was on a par with Control II at a 0.50 % inoculum load. Yields recorded with carbendazim and carbendazim + formalin were on par at both concentrations and inoculum loads. Application of prochloraz manganese at 1.5-3 g/m2 on 9th day from casing controlled the pathogen with no marked phytotoxity (Zaayen 1982, Fletcher 1983, Fletcher et al.1983).

Table 3: The effect of pH on the mycelial growth of the pathogen Hypomyces rosellus and Agaricus bisporus in compost and casing.

pH Growth in mm

Compost Casing

H. rosellus A. bisporus H. rosellus A. bisporus

5.50 2.00 0.00 8.33 0.00

6.00 10.34 39.00 15.33 10.00

6.50 98.67 61.00 121.67 26.67

7.00 87.67 96.00 114.00 85.67

7.50 69.67 83.00 97.33 81.00

8.00 0.00 4.00 0.00 28.00 CD at 5% 2.21 1.25 1.67 2.78

Table 4: Assessment of losses in yield of Agaricus bisporus from the pathogen Hypomyces rosellus

in mushroom beds.in mushroom beds.Pathogen/Inoculum Inoculum

load (%)Av. Yield (kg. / qtl. Compost)

Inoculum mixed in compost at spawning

Inoculum mixed in casing at casing

Wt. Loss (%) Wt. Loss (%)H. rosellus 0.10 9.19 46.13 9.09 46.72

0.50 7.10 58.38 7.34 56.970.75 6.40 62.48 6.01 64.771.00 6.56 61.15 4.06 76.20

Check ---- 17.06 ---- 17.06 ----CD at 5% for

Inoculum 0.43Yield 1.54Inoculum x Yield 2.17

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Table 5: Effect of different fungicides on the yield of Agaricus bisporus at 0.50 and 0.75%

inoculum load of the pathogen Hypomyces rosellus . .Fungicide Dose (%)

Yield in kg/qtl. CompostInoculum loads (%)

0.50 0.75Prochloraz manganese 0.025 13.34 9.34

0.050 14.08 10.450.075 16.38 14.50

captaf 0.025 11.13 8.260.050 10.88 13.580.075 13.05 14.16

carbendazim 0.025 13.06 9.910.050 13.38 11.000.075 16.70 15.70

carbendazim + formalin 0.01+0.05 12.16 12.460.015+0.10 14.25 13.530.025+0.20 15.38 14.38

ridomil 0.075 8.46 10.300.10 8.74 8.710.15 10.70 11.67

Control I -- 7.40 5.38Control II -- 17.08 17.08CD at 5%

Fungicide 0.60Inoculum 1.25Fungicide x Inoculum 2.51

Control I: With inoculum and without fungicideControl II: Without inoculum and fungicide

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Figure 1. Cobweb symptoms. Conidial attachment and Conidia.

Figure 2. Interaction of mycelium of Agaricus bisporus and the pathogen Hypomyces rosellus.

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Figure 3. Laboratory screening of fungicides against the pathogen Hypomyces rosellus.

(A) 1. Bavistatin(a) 1 ppm(b) 4 ppm(c) 7 ppm(d) 10 ppm(ch) Control

B 2. Sporgon(e) 1 ppm(f) 4 ppm(g) 7 ppm(h) 10 ppm(ch) control

(B) 1. Captaf(a) 5 ppm(b) 10 ppm(c) 15 ppm(d) 20 ppm(ch) Control

2. Ridomil(e) 50 ppm(f) 100 ppm(g) 150 ppm(h) 200 ppm(ch) Control

3 Bavistatin + formalin(i) 1+1 ppm(j) 2+2 ppm(k) 3+3 ppm(l) 4+4 ppm(ch) Control

ACKNOWLEDGEMENT

The senior author is thankful to the administration of the Experimental Station for providing necessary facilities and assistance during the period of this investigation.

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