Mass multiplication of C Globosum for farm use against plant pathogens

M Debbarma / P Dutta *

The application of fungal biocontrol agents (BCAs) is becoming an increasingly important alternative to chemicals in protecting crops against weeds, insects and fungal pathogens in both agriculture and forestry. The population dynamics of biocontrol agents in the field need to be well understood for their successful exploitation (Lo et al., 1996).

Out of the different fungal biocontrol agents, Chaeomium-globosum (Fam, Chaetomiaceae) is so far the most common and cosmopolitan mesophilic fungi especially on plant remains, seeds, compost paper and other cellulosic substrates (Domsch et al., 2007), primarily resides on plants, soil, straw, and dung. But considered as a common soil colonizer and cellulose producer with ability to degrade cellulosic and other organic materials and it is one of the common species growing sapro- phytically in the rhizosphere and phyllosphere.

It has been reported to be a potential bio-control agent and suppresses the growth of bacteria and fungi through antagonistic mechanisms which include competition for space and nutrients (Vannaci and Harman, 1987), mycoparasitism (Mandal et al., 1999) and metabolite production (antibiosis) such as chaetomin, chaeto-globosin, cochliodinol, chaetosin and prenisatin (Brewer et al., 1970; Brewer et al., 1972; Brewer and Taylor, 1978). There are many studies with promising results on using endophyte Chaetomium spp. as a biocontrol agent.

Their asymptomatic colonization supports plant tolerance to mental toxicity Heavy metals such as copper, suppress plant growth and disrupt metabolic processes, e.g. photosynthesis. A plant i.e., Ginkgo biloba harbors this fungus endophytes to suppress bacterial pathogens. Even inoculation of ascospores reduces bacterial disease symptoms such as wilting, apple scabs, and seed blight in treated plants.

Enhancing plant stress tolerance and microbial defense, renders C.globosum application beneficial for agricultural use. This successful use of biological control measure particularly reduce the chemical usage and improved agro ecosystem for sustainable agriculture and has maintained ecological balance.

IMPORTANCE

The specific strain of C.globosum Kunze has been found to control many plant pathogens, for example C.globosum was shown to be antagonistic to Fusarium spp. and Helminthosporium spp. (Tveit and Moore, 1954) and was found antagonistic to Alterna- riabrassicicola (Vannacci and Harman, 1987), and reduced the inoculum of Botrytis cinerea (Kohl et al., 1995).

Strains of C.globo-sum have been screened and found to control other economically important plant pathogens like Phytopthora-palmivora, P.parasitica, and Colletotrichumgloeosporioides. A novel cytotoxin named chaetomugilin was obtained from C.globosum isolates originating from Ginkgo biloba. C.globosum is effective against seed rot and damping off caused by several seed-and soil-borne plant pathogens like Pythiu-multimum, Alternaria- raphani, A.brassicicola and Fusarium spp.

C.globosum is also antagonistic to the rice blast pathogen (Pyriculariaoryzae) (Tyler, 2001). C.globosum is also known to produce various metabolites such as chaetomin, chaetoglobosin and Butylated-hydroxy-toluene (Di Pietro et al., 1992; Biswas et al., 2012) having antifungal properties against phytopathogenic fungi.

C.globosum has been found as the best antagonistic microorganism against onion white rot, caused by Sclerotiumcepivorum. The efficacy of C.globosum as a biocontrol agent against the late blight pathogen P.infestans has been reported in potato plants. C.globosum Cg-6 was formulated as a liquid and applied as a tuber, soil and foliar treatment either individually or in combination against Phytophthora infection in potato plants.

Among different treatments, combined application of C.globosum as a tuber treatment @ 1 ml kg-1 of tubers, as a soil application @ 1 ml kg-1 of Farm Yard Manure (FYM) and foliar spray @ 0.7% resulted in significantly less late blight infection (72%) compared to untreated control (100%) under field conditions.

The application of C.globosum resulted in greater tuber yield by reducing late blight infection in two field trials when compared to untreated controls. Three sprays of the C.globosum based bioformulation proved effective in controlling the Phytophthora blight disease upto 30% and increased the yield up to 10% (Anonymous, 2011).

STEPS INVOLVED IN FORMULATION OF C.GLOBOSUM

Preparation of Culture Media: C.globosum can be mass multiplied in potato dextrose broth medium.
Preparation of Potato Dextrose Broth
Materials Required
Readymade PDB (HIMEDIA): 24 g, Distilled water: 1000 ml, Beaker, Measuring cylinder, Conical flasks, Non-absorbent cotton, brown paper, Rubber band etc.

Procedure

o Suspend PDB (24 g) media in 1000 ml distilled water
o Boil it until the medium gets completely dissolved
o Dispense about 250 ml of medium into each of the four 500 ml conical flasks
o Plug the mouth of the flasks with non-absorbent cotton
o Sterilize the medium by autoclaving at 15 lbs pressure (121oC) for 15 minutes
o Mix it well before distributing

ISOLATION OF C.GLOBOSUM

Serial dilution technique is used for isolation of C.globosum. For serial dilution technique, suspend one gram of soil sample in 9 ml of sterile water, stir in a vortex for 3 min. Dilute the mixture serially achieve dilutions of 10-1 to 10-6. Replicate these dilutions three times separately and use for analysis.

Place 1 ml of aliquot (1 ml) from each dilution in petridishes containing PDA medium and incubate at 28+1oC for 5 days. The pure cultures will be maintained on potato dextrose agar medium in slants for further studies and stored in refrigerator at 4oC.

Mass Multiplication of C.Globosum
Materials Required
Pure culture of C.globosum, Inoculating needles, Alcohol, Spirit lamp, Non absorbent cotton, Brown paper, Thread

Procedure

o Inoculate the PDB with mycelial disc of 5 mm size cut out from 3-4 days old culture of C.globosum.
o Concentration of C.globosum should be 1 x 108cfu ml-1of water
o Incubate the inoculated flasks at 28+1oC for 7 days
o Count the spore concentration and colony forming unit (cfu) ml-1 of water after 7 and 14 days of inoculation

C.globosum formulated in the form of powder and pellets for the biological control of plant diseases is registered as Ketomium ®. Survival rate is better in the biopellets (77%) compared to that of the biopowder formulation (57%) following three years of storage.

Development of Ketomium® formulations and their shelf-life formulations of mycofungicides have gradually developed since 1992, and mycofungicides have now been formulated into both pellet and powder preparations (Soytong and Soytong, 1997). The steps involved are:

1. Grow C.globosum on potato dextrose agar (PDA) for three weeks at room temperature (27-30oC).
2. Make a spore suspension and then add to alginate mixture and drop through a pipette into a solution of 0.1 M calcium gluconate.
3. Dry the pellets overnight before packaging. Grind the pellets to make biopowder before packaging.
4. Determine the viable spore populations by dilution plate assays before pellet formation, 24 h after, and every two months for a period of one year.
5. During this period store the pellets at room temperature (27-30o) in screw-capped jars.
6. To test viability, dissolve the pellets in water with a mixture of 8 x 102 (M)KH2PO4 for dilution plating assays.
7. Assess the viable spore populations of the biopowder formulation in the same way.

METHOD OF APPLICATION

Con-Blight is a biofungicide preparation containing spores and mycelial fragments of the naturally occurring antagonistic fungus, Chaetomium- globosum. It is available in liquid @ 1 x 109 ml-1) and powder @ 2 x 106 g-1) formulation. Therefore, it can be applied in following ways:

1. SEED/TUBER TREATMENT

o Con-Blight @ 3 l or 5 kg is to be mixed with the required quantity of water (infused with spreaders and stickers) sufficient enough for treating the potato tuber/seeds needed for planting one hectare area.
o Then the seeds/tubers can be treated either in a seed treater mechanically or by dipping in the seeds manually in Con-Blight solutions/slurry for 30 minutes before sowing.
o Seed treatment of bio-phos can be done by sticking solution (jaggary solution @ 125 g 1-1 water) on seeds.
o Sprinkle 30 g of inoculants in the seeds and mix thoroughly.
o Air dries the seeds in shade after treatment and then use for sowing.

2. Surface Drench/Soil Application

o Surface drench: Plough and surface drench the main field with Con-Blight (C.globosum) solution prepared by mixing Con-Blight @ 5 l or 10 kg in 500 l of water. Surface drenching of soil with Con-Blight can be done either before ploughing or at the time of last ploughing.
o Soil application: Alternatively, Con-Blight @ 5 1 or 10 kg ha-1 is to be mixed with 10 tons of organic fertilizer. The Con-Blight blended organic fertilizer is to be kept for 7 days for curing before applying in the soil.
o Dilution: 5 1 or 10 kg in 500 l of water for soil application. Products available: Ketomium®, Con- Blight

PRECAUTION MEASURES

o Proper protective clothes, hand gloves and masks should be used while applying in the field.

CONCLUSION

C.globosum can adapt to harsh microclimate conditions in infested soils and have the ability to survive during dry periods and to colonize the organic substrates rapidly in the soils. C.globosum can be applied as a broad-spectrum mycofungicide in the field condition. It has been shown that this biological product can be applied to the field-soils infested with the test pathogens.

However, further field trials and observations need to be carried out in order to establish a better integrated approach in using these fungal biocontrol agents. For field use, the effects of soil pH, content of organic matter, soil aeration, moisture and chemical residue plays and important role for effective results.

In different soils however, the ecology and climate may possibly result in no or poor biological control. Thus, we need to establish the biological diversity of soil microorganisms, the ecology of microbial antagonists, and the effects of chemical residues in the soil.

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CAU, Imphal.
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