occurrence, biocontrol potential against fungal Biocontrol technologies have gained

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1 The Journal of Plant Protection Sciences, 2(2) : 1-8, 2010 Mass production, formulation, quality control and delivery of Trichoderma for plant disease management B. Ramanujam, R. D. Prasad, S. Sriram and R. Rangeswaran National Bureau of Agriculturally Important Insects, HA Farm post, P.B.No. 2491, Bellary Road, Bangalore Directorate of Oil Seeds Research, Rajendranagar, Hyderabad. bonamramanujam58@gmail.com A B S T R A C T Trichoderma has gained maximum attention as biocontrol agent due to the fact that it is effective against a large number of soil-borne plant pathogenic fungi, suppressive effects on some root nematodes without adversely affecting beneficial microbes like Rhizobium and capable of promoting growth of certain crops. There are two major methods of inoculum production of Trichoderma spp. viz., solid state fermentation and liquid state fermentation. In solid fermentation, the fungus is grown on various cereal grains, agricultural wastes and byproducts. The solid state production is highly labour intensive and fit for cottage industry. These products are used mainly for direct soil application in nurseries/main fields to suppress the soil-borne inoculum. In liquid state fermentation, Trichoderma is grown in inexpensive media like molasses and yeast medium in deep tanks on a commercial scale. Biomass from the liquid fermentation can be made into different formulations like, dusts, granules, pellets, wettable powders. Trichoderma formulations can be applied to the seed either by dry seed treatment or by seed biopriming for control of several soil-borne diseases of some field crops. Similarly, seedlings of horticultural crops and rice are treated by dipping the roots in Trichoderma suspensions before planting. Granular or pellets preparations and Trichoderma enriched FYM have been used for soil application directly and have provided effective control of diseases both nurseries and field conditions. To ensure that the products of Trichoderma do not affect the environment, human beings and other living organisms adversely and to prevent the sale of poor quality products to the farmers, the Central Insecticide Board of Government of India has made registration of microbial pesticides mandatory before commercial production/import/sale. Guidelines and data requirements for registration of microbial pesticides have been provided in the annexure of Insecticide Act. Quality control parameters set by CIB are inadequate for knowing potentiality of a bioagent. Apart from the counts of live propagules in the formulation, bioefficacy also should be taken as a quality parameter to ensure availability of better products to farmers. Keywords : Trichoderma, mass production, formulation quality control, registration delivery, shelf-life, disease management. Introduction occurrence, biocontrol potential against fungal Biocontrol technologies have gained and nematode diseases as well as host defense momentum in disease control of crop plants in inducing ability (Haraman & Kubicek 1998). recent times as these technologies not only Bioagents like, T. viride, T harzianum and minimize or replace the usage of harmful T.virens are being successfully used for the chemical pesticides but also found to be cheaper control of some of the dreaded diseases like, foot and efficient in certain disease control rot of black pepper, root rots of pulses, damping programmes. Successful use of fungal off, collar rots and Fusarium wilts of biocontrol agents like Trichoderma spp. for the horticultural crops. control of soil borne diseases caused by Mass Production Technology for Trichoderma pathogens like, Rhizoctonia, Sclerotium, spp. Fusarium, Pythium, and Phytophthora in The major aspects of successful biological several crops have been reported (Cook & Baker control technologies include the establishment 1983). Trichoderma spp. are under intensive of product, formulation and delivery system for research because of their abundant natural

2 2 The Journal of Plant Protection Sciences, 2(2) : 1-8, 2010 microorganism that enable them for efficient suitable for small-scale production in cottage disease control. The mass production systems industries or at individual farmer level. The should be compatible with industrial and disadvantage of technique is laborious and commercial development methods and field results in a product which is bulky and prone for application. There are two major methods of contamination. Solid state fermentation inoculum production as (a)solid state technique for commercial/industrial scale fermentation and (b) Liquid state fermentation. production of biocontrol fungi has to worked out Solid-state fermentation in collaboration with industry. Solid fermentation is a very common method for Liquid fermentation mass production of Trichoderma. Various cheap In liquid fermentation system, Trichoderma is cereal grains like, sorghum, millets, ragi are grown in liquid media in stationary/shaker/ used as substrates (Jeyarajan 2006). The grains fermentor cultures and formulated and used for are moistened, sterilized and inoculated with field application. Different growth media used Trichoderma and incubated for days. for production of Trichoderma through liquid Trichoderma produces dark green spore coating fermentation are given in Table 2. on the grains. These grains can be powdered Maximum biomass of Trichoderma spp. can be finely and used as seed treatment or the grains realized in short-time by using appropriate can be used as it is for enriching FYM for soil medium in a fermentor with aeration, agitation, application. Other agriculture wastes and temperature, ph and antifoam controls than in byproducts used for mass production of shake-flask cultures and will be more suitable Trichoderma are given in Table 1. for industrial production of Trichoderma spp. Solid fermentation results in a product that is Studies at erstwhile Project Directorate of generally used as it is for soil application or for Biological Control (PDBC), Bangalore revealed enriching organic manures. This technique is Table 1. that maximum amount of biomass and viable Substrates successfully used to produce Trichoderma spp. by solid state fermentation Substrate Reference Sorghum grain Upadhyay and Mukhopadhyay 1986 Wheat bran-saw dust modified medium Mukhopadhyay et al Tapioca rind, Tapioca refuse, FYM, press mud Kousalya Gangadharan & Jeyarajan 1990 FYM, wheat bran, rice bran, peat soil, rice straw Sangeetha Panicker et al Groundnut shell medium Raguchander et al Spent tea leaf waste and coffee husk Bhai et al Wheat bran and biogas manure Jagadeesh & Geetha 1994 Pigeonpea husk, tapioca waste (after starch Jayaraj & Ramabadran 1996 extraction) and press mud. Coffee fruit skin, poultry manure and coffee Sawant & Sawant 1996 fruit skin composted with cow dung slurry Decomposed Coconut Coir pith Kumar & Marimuthu 1997 Spent malt Gopalakrishnan et al. 2003

3 The Journal of Plant Protection Sciences, 2(2) : 1-8, propagules of T. harzianum/t. viride can be obtained within 96h of fermentation in a fermentor with aeration, agitation, temperature controls (Prasad & Rangeswaran 1998). Table 2. Growth media used for production of Trichoderma in liquid state fermentation Growth media Reference Molasses and brewers yeast Sankar & Jeyarajan 1996 & Rangeshwaran 1998 Prasad Potato dextrose broth, V -8 juice and Molasses- yeast medium & Rangeshwaran 2000 Prasad Molasses soy medium Jaggery-soy medium Prasad et al (Nelson & Powelson 1988) and fluid drill gels (Conway 1986). The various types of Trichoderma formulations used in biological control of crop diseases are given below. 1. Talc based formulation: In India, talc based formulations of T. viride was developed at Tamil Nadu Agricultural University, Coimbatore for seed treatment of pulse crops and rice (Jeyarajan et al. 1994). Trichoderma is grown in the liquid medium is mixed with talc powder in the ratio of 1:2 and dried to 8% moisture under shade. The talc formulations of Trichoderma has shelf life of 3-4 months. It has become quite popular in India for management of several soil-borne diseases of various crops through seed treatment at 4-5g/kg seed. Several private industries produce large quantities of talc formulations in India for supply to the farmers. The annual requirement of Trichoderma has been estimated as 5,000 tones to cover 50 per cent area in India (Jeyarajan 2006). 2. Vermiculite-wheat bran formulation (Lewis et al. 1991) Ingredients: Formulations of Trichoderma spp. Vermiculite 100g In general, product formed from solid or semi Wheatbran 33g solid-state fermentation do not require Wet fermentor biomass 20g sophisticated formulation procedures prior to 0.05N HCL 175 ml use. For example, grain or other types of organic matter upon which Trichoderma is grown are Trichoderma is multiplied in molasses-yeast simply dried, ground and added to the area to be medium for 10 days. Vermiculite and wheat bran o treated. Biomass produced in liquid are sterilized in an oven at 70 C for 3 days. fermentation either can be separated from Then, 20 gms of fermentor biomass and 0.05N medium and concentrated or entire biomass with HCl are added, mixed well and dried in shade. medium can be incorporated into dusts, 3. Pesta granules (Connick et al. 1991) granules, pellets, wettable powders or Ingredients : emulsifiable liquids. The carrier material may be Wheat flour 100g inert or a food base or a combination of both. Fermentor biomass (FB) 52 ml Trichoderma spp. can be formulated as pelletts Sterile water sufficient enough (Papavizas & Lewis 1989), dusts and powders to form dough

4 4 The Journal of Plant Protection Sciences, 2(2) : 1-8, 2010 Fermentor biomass ( 52 ml) is added to wheat 7. Coffee husk: In Karnataka Sawant & flour (100g) and mixed by gloved hands to form Sawant (1996) developed a Trichoderma cohesive dough. The dough is kneaded, pressed formulation based on coffee husk which is a flat and folded by hand several times. Then one waste from coffee curing industry. This mm thick sheets (pesta) is prepared and air-dried product was very effective in managing till it breaks crisply. After drying, dough sheet Phytophthora foot rot of black pepper and is was ground and passed through a 18 mesh (1.0 widely used in Karnataka and Kerala. mm) sieve and granules were collected. 8. Oil-based formulations: They are prepared 4. Wheat flour- kaolin (Prasad & by mixing the conidia harvested from the solid Rangeswaran 1998 ) state/liquid state fermentation with a Ingredients : combination of vegetable/mineral oils in stable Wheat flour 80gm emulsion formulation. In such formulations, Kaolin 20 gm microbial agents are suspended in a water Fermentor biomass 52 ml immiscible solvent such as a petroleum fraction (diesel, mineral oils), and vegetable 52 ml of Fermentor biomass is added to wheat oils (groundnut etc.) with the aid of a surfaceflour (100g) and mixed by gloved hands to form a active agent. This can be dispersed in water to cohesive dough. The rest of the procedure is as form a stable emulsion. Emulsifiable described for pesta granules concentrates require a high concentration of an 5. Alginate prills (Fravel et al. 1985) oil soluble emulsifying agent, to give instantaneous formation of a homogenous Ingredients: emulsion on dilution in water. The oils used Sodium Alginate 25 gm should not have toxicity to the fungal spores, Wheat flour 50 gm plants, humans and animals. Such Fermentor biomass 200 ml formulations of Trichoderma, Pseudomonas, Sodium alginate is dissolved in one portion of and Beauveria are now being used as foliar distilled water (25g/750 ml) and food base is sprays. Oil-based formulations are supposed to suspended in another portion (50g/250ml). These be suitable for foliar sprays under dry weather preparations are autoclaved and when cool are conditions and to have prolonged shelf life. The blended together with biomass. The mixture is spores can survive for longer time in the plant added drop wise into CaCl 2 solution to form surface even during the dry weather as the spherical beads, which are air-dried and stored at spores are covered by oil that protects them o 5 C. from drying. Batta (2005) developed an 6. Press mud based formulation: Press mud is emulsion formulation of T. harzianum for the available as a by product of the sugar factory and control of post harvest decay of apple caused this can be used as a substrate for mass by Botrytis cinerea. Invert-emulsion multiplication of Trichoderma. Trichoderma formulation of T. harzianum with a shelf life of produced and formulated on press mud is sold to 8 months has been developed using indigenous farmers as value-added organic manure by a constituents at the erstwhile Project sugar factory in south India. (Jeyarajan 2006). Directorate of Biological Control (PDBC), Bangalore in India and this his formulation has

5 The Journal of Plant Protection Sciences, 2(2) : 1-8, been and found to be effective against soil- is coated with dry powder/dusts of Trichoderma borne diseases of groundnut. just before sowing. For commercial purpose, Shelf life of Trichoderma formulations dry powder of antagonist is used at 3 to 10 g per kg seed based on seed size (Mukhopadhyay et Shelf life of the formulated product of a bioal. 1992). Propagules of biocontrol agents control agent plays a significant role in germinate on the seed surface and colonize roots successful marketing. In general the antagonists of germinated seedlings and rhizosphere multiplied in an organic food base have longer (Tiwari 1996). T. harzianum, T. virens and T. shelf life than the inert or inorganic food bases. viride were found to be effective seed Shelf life of Trichoderma in coffee husk was protectants against Pythium spp. and more than 18 months. Talc, peat, lignite and R h i z o c t o n i a s o l a n i ( M u k h e r j e e & kaolin based formulation of Trichoderma, have Mukhopadhyay 1995). a shelf life of 3-4 months. The viable propagules of Trichoderma in talc formulation Seed biopriming: Seed biopriming is treating was reduced by 50% after 120 days of storage of seeds with Trichoderma and incubating under (Sankar & Jeyarajan 1996). Studies on the warm and moist conditions until just prior to storage of T. viride formulation in poly radical emergence). This technique has propylene bags of various colours revealed that potential advantages over simple coating of the population of T. viride was maximum in seeds as it results in rapid and uniform seedling milky white bags of 100 micron thickness. At emergence. Trichoderma conidia germinate on PDBC, Bangalore work on increasing shelf life the seed surface and form a layer around of talc formulations of Trichoderma using bioprimed seeds. Such seeds tolerate adverse various ingredients (chitin and glycerol) in soil conditions better. Biopriming could also production medium and heat shock at the end of reduce the amount of biocontrol agents that is log phase of fermentation was carried out which applied to the seed. Seed biopriming was can extend the shelf of talc formulation of successfully used in tomato, brinjal, soybean Trichoderma up to one year. (Sriram et al, 2010; and chickpea in Tarai region of Uttaranchal Sriram et al, 2011) (Mishra et al. 2001). Delivery of Trichoderma for disease Root treatment: Seedling roots can be treated management with spore or cell suspension of antagonists For successful diseases control, delivery and either by drenching the bioagent in nursery beds establishment of Trichoderma to the site of or by dipping roots in bioagent suspension action is very important. The most common before transplanting. This method is generally methods of application of Trichoderma are by used for the vegetable crops, rice where seed treatment, seedling dip, soil application transplanting is practiced (Singh & Zaidi 2002). and wound dressing. There are also reports on the reduction of sheath blight disease of rice by root dip of seedlings Seed Treatment: Seed coating with before transplantation (Vasudevan et al. 2002). Trichoderma is one of the easy and effective Root dipping of tomato seedlings reduces the methods of delivering the antagonist for the severity of root knot caused by Meloidogyne management of seed/soil-borne diseases. Seed incognita. Root dipping in antagonist's

6 6 The Journal of Plant Protection Sciences, 2(2) : 1-8, 2010 suspension not only reduces disease severity Insecticide Act. but also enhances seedling growth in rice, Standards for Trichoderma formulations: tomato, brinjal, chili and capsicum (Singh & 1.Colony Forming Units (CFUs) of Zaidi 2002). Trichoderma spp. should be a minimum of 2 x Soil treatment: There are several reports on the 6 10 CFU per ml or gm on selective medium application of biocontrol agents to the soil and 2.Pathogenic contaminants such as Salmonella, other growing media either before or at the time Shigella or Vibrio should not be present. Other of planting for control of a wide range of soil- 4 microbial contaminants not to exceed 1 x 10 borne fungal pathogens (Baby and count ml/gm Manibhushanrao 1996). Such applications are 3. Maximum moisture content should not be ideally suited for green house and nursery. more than 8% for dry formulation of fungi Trichoderma is capable of colonizing farm yard manure (FYM) and therefore application of Quality control parameters set by CIB are colonized FYM to the soil is more appropriate inadequate for knowing potentiality of a and beneficial. This is the most effective bioagent. It is time now for Ministry of method of application of Trichoderma Agriculture to identify a Central Agency for particularly for the management of soil-borne quality testing not only in terms of amount of diseases. live propagules in the formulation but also their bioefficacy against plant pathogens. Quality of Aerial spraying / Wound dressing: the products in these lines should be Trichoderma has been successful applied to the periodically checked by the identified agency aerial plant parts for the biocontrol of decay and that will ensure availability of better fungi in wounds on shrubs and trees (Papavizas products to farmers. 1985). Future Research: The future research should Registration and Quality Control focus on the following aspects for better To ensure that the products of microbial BCAs utilization of Trichoderma as a biocontrol agent do not affect the environment, human beings for crop disease management. and other living organisms adversely and to! Suitability of Trichoderma for control of prevent the sale of poor quality products to the foliar/aerial pathogens farmers, the Central Insecticide Board (CIB) of! Development of liquid/oil formulations the Government of India has made registration suitable for foliar applications of microbial pesticides mandatory before! Formulations with prolonged shelf life, field commercial production/import /sale. persistence and suitable for dry weather Guidelines and Data requirements for conditions registration of microbial pesticides (Data on Biological, Physical, Chemical properties and! Scaling up of Solid state production systems Bio-efficacy to the target pathogen, Effect on with Industry collaboration non-target organisms, Toxicological reports on! Large scale demonstration of biocontrol l a b o r a t o r y a n i m a l s, E c o - t o x i c i t y, technologies in farmers fields Manufacturing process, Packing and labeling)! Fast Track Registration have been provided in the Annexure of

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