Chemical control of dry bubble disease induced by Verticillium fungicola [ Preuss ] Hassebr on white button mushroom , Agaricus bisporous

Plant Pathology Department, Parmanand Degree College Gajsinghpur Sri Ganganagar, 335024 Affiliated to Swami Keshawa Nand Rajasthan Agricultural University, Bikaner Rajasthan, India. Plant Pathology Department, CCSHAU, Hisar Haryana, India. Plant Pathology Department, Jodhpur Agricultural University, Rajasthan, India. Plant Pathology Department, Agricultural Research Station, Sri Ganganagar, Swami Keshawa Nand Rajasthan Agricultural University, Bikaner Rajasthan, India.


INTRODUCTION
White button mushroom is cultivated throughout the world, contributing about 40% of total world production of mushroom (Flegg, 1992).In India, white button mushroom is being cultivated in majority of the states both under seasonal and controlled conditions with an annual production of approximately 42,500 tons (Dandge, 2012).Haryana has become one of the leading states in white button mushroom production with 5312 tons/annum (Tiwari, 2004).Mushroom production is adversely affected by a large number of biotic and abiotic factors.Among the several biotic factors associated with reduction in yield of mushroom, fungal diseases significantly affect the mushroom production and yield.
Most common fungal diseases of white button mushroom are cobweb, dry/wet bubble, false truffle and green mould (Sharma, 1995).Of these diseases, dry bubble caused by Verticillium fungicola (Preuss) Hassebr is prevalent in all mushroom growing areas and has 25-50% incidence (Sharma, 1995).Dry bubble disease of white button mushroom causing brown spots was reported for the first time by Malthouse in 1901.He found a species of Verticillium associated with this disease.Two types of symptoms were observed.Initially, fungal growth appeared on the casing soil which later spread and turned grayish yellow.After that, light brown superficial spots appeared on the caps which finally coalesced to become large brown blotches.This disease is transmitted by contaminated compost, casing soil (Kumar et al., 2014), human beings and splash of water (Fekete, 1967;Cross and Jacobs, 1969).Mushrooms infected by V. fungicola shows typical thickening of stem, resulting in onion shaped fruiting bodies.However, the symptoms vary with the age of the mushroom and the stage of development at which the infection takes place.When mushrooms are infected by this fungus at an early stage, symptoms appear as small undifferentiated masses of tissue up to 2 cm diameter.Fruiting bodies are not properly formed and caps are partially differentiated.When infected at a later stage the stipes are distorted and have tilted caps.Infected mushroom show the presence of grey white mycelial growth and become discoloured and dry but do not rot.They show small pimple like outgrowth or brown grey spots (1-2 cm diameter) on the surface.Such spots often have a yellow or bluish grey halo around them.
In Haryana, the white button mushroom is being cultivated on compost prepared by long method in low cost mushroom houses under seasonal conditions.These factors coupled with poor sanitation and persistence of V. fungicola in soil pose a serious threat to the future of mushroom cultivation in Haryana.

Glassware and equipment
Glassware used in the present study were of Borosil.Polythene bags (30 x 45 cm), polypropylene (7.50 x 30 cm) bags and 500 ml empty glucose bottles were used for spawn and inoculum preparation.

Chemicals
Standard analytical grade chemicals were used in the present study.

Sterilization of glassware
Glasswares were sterilized at 180°C for 2 h in a hot air oven.

Maintenance of culture
Pure cultures of A. bisporus and V. fungicola were maintained on PDA at 20±1°C.

Preparation of compost
Two methods of composting viz., long method of composting (LMC) and short method of composting (SMC) were followed: Six types of composts were used: Wheat straw compost (LMC); Wheat straw compost with chicken manure (LMC); Wheat straw compost (SMC); Wheat straw compost with chicken manure (SMC); Brassica straw compost with chicken manure (LMC); Brassica straw compost with chicken manure (SMC).These composts were prepared for conducting the experiment.

Compost preparation
Six types of compost were used.The compost was prepared with or without chicken manure in both methods.Wheat and brassica straw were spread separately on a pucca floor and wetted thoroughly with clean water for 48 h to attain 70-75% moisture content.Wheat bran was dry mixed, with chemical fertilizer, moistened with water, covered with polythene sheets and kept overnight to facilitate adsorption of chemical fertilizer on the bran.The mixture was evenly spread on wet wheat and Brassica straw, mixed and stacked to make a compact rectangular pile.Seven turnings were given to the pile using the turning schedule of 0,6,10,13,16,19,22,25 and 28 days.At each turn, approximately 30 cm layer was separated from all the exposed surface of the pile and moistened, if necessary.The remaining pile was also dismantled and mixed well.The material was restacked in such a way that the outer portion of the previous pile was in the center of the new pile.Molasses was mixed at first turn, gypsum at the third.Two days after the last turn, the pile was dismantled and the contents were mixed thoroughly.The compost was checked for desirable characteristics, that is, dark brown colour, pH (7-8), absence of ammonia smell and appropriate moisture content (68-72%).The composition for LMC and SMC of the substrates are given in Table 1.

Spawning
Thorough spawning was done at 1% spawn before filling of compost in the polythene bags.In further studies, wheat straw compost with chicken manure prepared by LMC was used (Table 2).The compost was made separately.

Spawn run
After spawning, bags were covered with newspaper sheets sterilized with formalin (4%) and water was sprinkled to keep moist.Temperature was maintained 24 ± 2°C with relative humidity of 85-90%.After pin head initiation, temperature was lowered down to 16± 2°C and RH of 90%.Fresh air was circulated for 3-4 h daily during cropping.

Casing
Casing soil was prepared by mixing well decomposed (16-18 month old) farm yard manure and burnt rice husk (4:1 v/v).The casing mixture was disinfected with 4% formalin solution using 600 ml formalin (36%) diluted to 5 L for 100 kg of casing material.The formalin treated moistened casing material was kept covered with polythene sheets for at least 48 h followed by frequent turnings to evaporate formalin fumes.Before casing, the newspaper sheets were removed from the spawn impregnated compost and the surface was covered with disinfected casing material (4 cm) for uniform thickness.

Cropping
Adequate humidity (85-90%) was maintained inside the growing room by spraying water on the walls and the floor.Water was sprayed on the bags twice a day, very little or no ventilation was provided until the first appearance of pin heads.Thereafter, intermittent cross ventilation was given for a total 4-6 h per day.The mushrooms were harvested by gentle twisting of the fruit body.The depressions created in the casing layer were filled with fresh disinfected casing soil.The lower part of the pileus of harvested fruit bodies were trimmed off and yield was recorded.

Yield data and statistical analysis
The yield data was recorded for upto 45 days of cropping period.A daily record of the number of fruit bodies and their weight (g) per bag per treatment was maintained and the yield data was expressed as kg mushroom per 100 kg compost.The critical difference (CD at 5%) was calculated from the replicate data using factorial experiment and in common complete randomized design (CRD).

Isolation of pathogen
The diseased mushroom pileus showing typical symptoms of dry bubble disease were cleaned gently by wiping the outer surface with sterile cotton moistened with distilled sterilized water.Pieces of infected cut mushroom pileus were planted on PDA slants and incubated at 20±1°C.To suppress the bacterial contamination, the medium was amended with streptocyclin at a concentration of 50 ppm.
The subculturing was done periodically at regular intervals during the course of present investigations (Table 3).

Identification
Pathogen was identified as V. fungicola (Preuss) Hassebr on the basis of culture, colour, microscopic studies and type of sporulation with the help of mycologist in the Department of Plant Pathology, CCS HAU, Hisar.

Pathogenicity (Koch postulates)
The pathogenicity was proved by placing actively growing mycelia agar bit (5 mm diameter) of the V. fungicola on cut healthy white button mushroom (A.bisporus) fruit bodies incubated at 20±1°C
To evaluate the effectiveness of fungitoxicants in controlling dry bubble disease of white button mushroom, the fungitoxicants namely, Carbendazim.Dithane M-45, Dithane Z-78 and Thiophanate-methyl were used at 1, 10, 100 ppm concentrations in vitro.Treatment without any fungitoxicants served as control.For this purpose, double strength fungitoxicants were added to the double strength PDA media to get the desired concentrations.The PDA amended with test fungitoxicant was poured in Petri-plates (20 ml/plate) (Table 4).After solidification, the poisoned medium was seeded with 5 mm mycelial agar bit of actively growing V. fungicola and A bisporus separately.The three replications of each treatment were kept (Table 5 and 6).
Observations were recorded at regular intervals for radial growth of V. fungicola till whole plate (in control) was covered with mycelial growth of this pathogen (14 days) of incubation at 20±1°C.Growth inhibition (%) was calculated with the growth of the test fungus in control (devoid of fungitoxicant).Observations were recorded for radial growth of pathogen.
where C = Diameter of colony in the control; T = diameter of the colony in the treatment

Effect of different fungitoxicants on the development of dry bubble disease
The test fungitoxicants were mixed in compost to get desired concentration.In the case of Carbendazim and Thiophanate methyl, 400 mg each was dissolved in 4.0 L of water and sprayed on 20 kg of compost, spread over a clean polythene sheet.800 mg each of Dithane M-45 and Dithane Z-78 was dissolved in 4.0 L of water sprayed over the 20 kg compost.After mixing the compost thoroughly, spawning with M140 of A. bisporus (1%) and V. fungicola (0.3%) was done and filled in polythene bags weighing 5.0 kg each, four replication of each treatment were kept.In the control, water without fungitoxicant was sprayed before spawning.The yield of mushroom was recorded upto 45 days and compared with control treatment.The different concentrations of different fungicides were used so as to see comparable results and which was cheaper than the control of the disease.The spawn of A. bisporus (1%) is necessary for better results and for the inoculum of V. fungicola (0.3%), if percent inoculum taken is more, the more disease will occur (Table 7).

Isolation, purification and identification of dry bubble pathogen and pathogenicity
Isolation of pathogen was made on PDA from diseased sporophores suspected of having Verticillium infection.The pathogen cultures were further purified and incubited at 20±1°C.For morphological studies, pure culture was transferred in Petri dishes and incubated at desired temperature.The colony growth charactesrtics were recorded.
The colonies were white in appearance, under part of the plate was colorless to yellow and had scalloped C-T Percent inhibition = x 100 C  and McCauley (1987), Calonje et al. (2000), Khanna et al. (2003) and Justyna et al. (2011).So it was concluded that our pathogen was V. fungicola after proving the Koch's postulates.The pure culture was maintained and used in further experiment.

Effect of different fungitoxicants on Agaricus bisporus
Fungitoxicant have been reported to have some inhibition effect on the host (mushroom) though they are targeted at the pathogen.Thus, use of fungitoxicant may have bearing on the growth of mushroom being a fungus.Result of in vitro screening against A. bisporus revealed that 100 ppm concentration of Carbendazim inhibited growth of A. bisporus up to 47.75%, followed by Thiophanate -methyl (43.65%),Dithane Z-78 (40.89%) and Dithane M-45 (38.72%).The present results are in agreement with work done by Gandy (1985) who also reported that Carbendazim fungicides were less toxic to basidiomycetes than to other pathogen.Seth and Bharadwaj (1989) have shown that Benlate inhibited the growth of A. bisporus least followed by Bavistin during in vitro studies.
Similarly, the present studies support the work of Dhar and Kapoor (1990) and Navarro et al. (2011) who stated that use of Bavistin can control the fungal pathogens and competitors of white button mushroom and it also had less inhibitory action on mushroom mycelium.Thus, it may be inferred that to minimize damage to A. bisporus, extra care should be taken in the selection of fungicides for application to manage the disease.Bhalla (1998) reported that the inhibition percent of A. bisporus by Bavistin, Benlate, Sporogon, Dithane M-45 and Dithane Z-78 ranged from 18.18 to 100% at 50, 100, 200 and 500 ppm concentration.
Effect of different fungitoxicants on dry bubble disease Bhatt and Singh (2002) reported Sporogon (0.075%) to be effective against V. fungicola.Maximum yield and number of fruit bodies were obtained by using Bavistin.
Results of present study are in agreement with that which revealed that Carbendazim advanced spawn run by 6-7 days, pinhead initiation by 5 days resulting in 67.80 to 75.84% increase in yield.Other chemicals, Thiophanatemethyl Dithane Z-78 and Dithane M-45 also significantly increase the mushroom yield, shortened spawn run period and pinhead initiation as compared to the control.

Table 2 .
Compost substrate with chicken manure.

Table 3 .
Disease appearance on cut-mushrooms.
This table show pathogenicity test for dry bubble disease in white button mushroom caused by Verticillium fungicola.

Table 4 .
List of fungitoxicants and their active ingredient test against V. fungicola.

Table 5 .
Effect of different fungitoxicants on mycelial growth of V. fungicola in vitro.
and 85% humidity.The observations were recorded for disease appearance.Reisolated pathogen was compared with the original one.In the case of control, only agar bit was placed on the cut healthy fruit bodies.

Table 6 .
Effect of different fungitoxicants on mycelial growth of A. bisporus.

Table 7 .
Efficacy of different fungitoxicants in controlling dry bubble disease on Agaricus bisporus.