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

CASING SOIL BACTERIA AS BIOCONTROL AGENTS AGAINST THE MYCOPARASITIC FUNGI OF AGARICUS BISPORUS

N. Bhatt and R.P. Singh. Department of Plant Pathology, Mushroom Research Centre

G. B. Pant University of Agriculture and Technology, Pantnagar-263 145 (Uttaranchal) India<nirmala_bhatt@hotmail.com>

ABSTRACT

Various proportions of organic matter of plant origin, farm animal manure and soil are used as casing materials in India. These mixture contain a variety of microbiota. For this study, a 3 :1 casing mixture of Farm Yard Manure (FYM) and two-year-old spent compost was prepared and analyzed for its microbial content. Bacteria, especially pseudomonads, are a significant presence in casing. Many of them produce substances which improve the growth of mushrooms and also reduce the growth and parasitic activities of mycoparasitic fungi. The interaction of these isolates with the mycoparasites of the button mushroom was investigated. Of the sixteen bacterial isolates, five were selected on the basis of their harmlessness to Agaricus bisporus and designated as BI (Bacterial Isolates) I, II, III, IV and V. All of these isolates were effective against Verticillium fungicola and Fusarium moniliformae, and BI I, II and V were found to be effective against Mycogone perniciosa and Sepedonium chrysosporium. BI III controlled Trichoderma harzianum. The isolates selected against pathogens in vitro were further inoculated in the casing to assess their effectiveness in the mushroom beds. The following bacterial isolates were found to reduce pathogens in the mushroom beds as indicated, and thus favored an increase in mushroom yield : BI V (against F. moniliformae and Verticillium fungicola,) BI I (against S. chrysosporium and Mycogone perniciosa) and BI III (against T. harzianum).

INTRODUCTION

Use of bacterial flora for management of plant diseases and yield improvement started in the early part of the 20th century (Cook and Baker 1983). A number of bacterial species have been tested as biocontrol agents (Weller 1988). Mechanisms by which bacteria exhibit biological control include antibiosis, competition, parasitism and production of plant growth promoting compounds (Klopper 1992). Jhune et al. (1990) have studied biological control of Mycogone perniciosa, which parasitizes Agaricus bisporus. Some of the bacteria isolated from biofertilizers have exhibited a strong antagonism against common competitor moulds (Ahlawat and Rai 1997). Experiments were conducted to explore and exploit the potential of antagonistic bacteria naturally occurring in casing mixtures for controlling pathogenic fungi of A. bisporus.

MATERIALS AND METHODS 

Isolation and identification of pathogens

Pathogens (Mycogone perniciosa, Verticillium fungicola, Trichoderma harzianum, Fusarium moniliformae and Sepedonium chrysosporium) were isolated from compost/casing/fruit bodies showing symptoms of disease and using peptone dextrose rose bengal agar medium (Martin 1950). The cultures of the above pathogens were purified and then their identities were confirmed by comparing their morphological characteristics with the literature (Fletcher et al. 1986, Sharma

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1995). Subsequently purified cultures were maintained on potato-dextrose- agar (PDA) or compost decoction agar (CDA) slants and further multiplied on PDA and CDA media.

Isolation of bacterial biocontrol agents 

Employing the serial dilution plate technique (Waksman 1922), total bacterial colonies were isolated from casing mixtures on soil extract medium (Allen 1957) and modified King’s B medium (King et al. 1954, Sands and Rovira 1970), respectively.

Interaction between bacterial isolates and A. bisporus was studied using a method developed by Weller et al. (1985) with slight modification. Five mm disks from a 21-day-old culture of A. bisporus were placed at the centre of petri plates containing compost decoction agar (CDA) medium and then incubated at 22±20C for 20 days. Then a bacterial suspension containing 106 cfu ml-1 was streaked one cm from the growing A. bisporus colony. The plates were incubated for another seven days. Each treatment was replicated three times with a suitable control and observations were made of growth enhancement as well as inhibition.

Screening of the antagonists 

The antagonism between selected bacterial isolates and the pathogenic fungi were tested following the method suggested by Morton and Stroube (1955). Twenty ml of sterilized and melted PDA was poured into 90 mm sterilized petri plates. After solidification, the plates were inoculated with the tested fungus using a five mm diameter disk of young culture. Then a five mm diameter filter paper disk dipped in a bacterial suspension of each individual isolate was placed at a distance of ten mm from the periphery of the inoculated plates. The plates were incubated at 25 0C for ten days and observations were recorded. Each treatment was replicated three times.

Interaction of isolates and pathogens in mushroom beds 

Five selected isolates were cultured in nutrient broth. Four hundred ml of each isolate containing 106 cfu ml-1 were thoroughly mixed with four kg casing mixture. Inocula of the pathogens, prepared on wheat grains, were mixed into the casing (0.75% of casing weight). Then four cm of casing was applied onto the compost of fully colonized bags. For comparison, there were two controls. In one control, only a pathogen was mixed in casing (Control I); in the other (Control II), neither a pathogen nor an antagonist was used. All the treatments were replicated three times.

RESULTS AND DISCUSSION

Biological control using bacteria holds great promise for controlling soil borne diseases caused by fungi (Weller 1988). Like field crops, mushrooms suffer parasitization by several fungal pathogens. Since bacteria constitute a sizable part of microbial populations of casing microflora and many of them produce substances which improve plant growth and also reduce parasitic activities (Byer and Sikora 1990), their biocontrol potential is worthy of investigation.

Out of sixteen bacteria isolated from casing, five were selected on the basis of their harmlessness to A. bisporus in in vitro tests in dual cultures (Figure 1A, B). These isolates were tested against the pathogens, The data recorded are presented in Table 1 and Figure 2. Out of these five isolates, all the isolates were effective against F. moniliformae. These isolates reduced the growth of this pathogen by about 40-60 percent. (Figure 2A). BI I, II and V were effective against the pathogen M. perniciosa and 50-60 percent reduction in growth was recorded (Figure 2D). T. harzianum, itself

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is a strong antagonist against soil-borne pathogens of plants (Weller and Cook 1986), was effectively reduced by BI I (Figure 2E). The remaining isolates were ineffective and overrun by the pathogen  itself . V. fungicola was significantly controlled by all the isolates (Figure 2B). Radial growth of S. chrysosporium was effectively reduced by BI I, II and V (Figure 2C). Unidentified microorganisms in the casing soil suppressed the development of M. perniciosa (Han et al. 1974). From more than a thousand bacterial isolates from rhizopheres and rhizoplanes of various plants and mushrooms, seven isolates inhibited microconidia of F. oxysporum (Isaka and Okamoto 1989). Of 343 Bis, only 12 showed antimicrobial activity and five of them were selected for antibiotic activity on casing soil with M. perniciosa (Jhune et al. 1990).

Figure 1. Interactions (dual culture) between A. bisporus and bacterial isolatesA. (i) BI-I

(ii) BI-II(iii) BI-III

B. (i) BI-IV(i) BI-V

Interaction of isolates and pathogens in beds

To determine the effectiveness of the BIs against various pathogens, the bacterial strains found effective against the particular pathogen in vitro were selected to test them for A. bisporus yield. The data in Table 2 show that all the bacterial isolates significantly enhanced the yield in case of F. moniliformae and V. fungicola compared to Control I (with pathogen and without bacteria).

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However, BI V was found to be the best in controlling F. moniliformae and V. fungicola in terms of mushroom yield. The BIs enhanced the yield significantly compared to Control I, but the yield recorded was very poor compared to Control II. The BI I was found to be more effective than BI, II and BI, V. A higher yield (30.45 kg) was recorded in case of T. harzianum when BI III was used against it. Of the three isolates (BI I, BI II and BI V) tested for control the S. chrysosporium in mushroom beds, BI I proved best in terms of mushroom yield.

Results clearly reveal that some microflora naturally existing in casing can be used for management of different fungal diseases of A. bisporus. Earlier studies by Baker (1987), Jhune et al. (1990), Kloepper (1992) and Ahlawat and Rai (1997) support the usefulness of bacterial flora in control of pathogens affecting the yield of A. bisporus.

Table 1 : Biocontrol potential of selected bacterial isolates against pathogenic fungi.

Bacterial Isolates

Fusarium moniliformae

Mycogone perniciosa

Trichoderma harzianum

Verticillium fungicola

Sepedonium chrysosporium

(1) (2) (1) (2) (1) (2) (1) (2) (1) (2)

BI-I 40.00 39.75 35.34 50.13 89.00 0.00 51.00 29.89 46.00 37.75

BI-II 45.33 35.79 43.67 37.80 89.67 0.00 51.67 30.70 41.00 43.03

BI-III 50.33 32.79 88.33 0.00 47.00 38.79 54.00 28.70 89.67 0.00

BI-IV 53.67 30.76 88.29 0.00 89.00 0.00 56.67 28.00 89.67 0.00

BI-V 36.34 51.83 36.00 50.75 89.34 0.00 44.00 34.70 54.67 31.75

Check 89.67 0.00 88.34 0.00 89.00 0.00 89.34 0.00 89.67 0.00

(1) Radial growth of the fungus in mm(2) Inhibition zone in mm

CD at 5% For isolate (a) 0.87 For pathogenic fungus 0.89

For radial growth and inhibition zone 0.40 (axb) 2.25

(bxc) 1.31 (axc) 1.31

(axbxc) 3.50

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Table 2 : Interaction of Bacterial Isolates and pathogens in mushroom beds.

Bactrial Isolates

Average yield of Agaricus bisporus (Kg/qlt. Compost)

Fusarium moniliformae

Mycogone perniciosa

Trichoderma harzianum

Verticillium fungicola

Sepedonium chrysosporium

BI – I 10.25 7.50 --- 10.08 12.58

BI - II 9.00 6.17 --- 9.25 9.50

BI – III 10.92 --- 12.58 10.17 ---

BI – IV 9.58 --- --- 8.59 ---

BI – V 11.84 5.08 --- 10.58 10.25

Check – I

7.44 0.00 8.75 6.88 7.65

Check - II

16.50 16.50 16.50 16.50 16.50

CD at 5% 0.902 870 1.042 1.106 1.12

BI = Bacterial Isolate Check I : With inoculum and without bacteria

Check II : Without inoculum and bacteria

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Figure 2. Biocontrol potential of bacterial isolates against pathogens.

(A) Against F. moniliforme(a) BI-I (b) BI-II (c) BI-III (d) BI-IV (e) BI-V (ch) control(B) Against V. fungicola(a) BI-I (b) BI-II (c) BI-III (d) BI-IV (e) BI-V (ch) control(C)Against S. chrysosporium(a) BI-I (b) BI-II (c) BI-V (ch) control(D)Against M. perniciosa(a) BI-I (b) BI-II (c) BI-V (ch) control(E)Against T. harzianum(a) BI-I (b) BI-II (c) BI-III (d) BI-IV (e) BI-V (ch) control

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ACKNOWLEDGEMENT

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

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