Evaluation of agro-industrial wastes for conidia based inoculum production of bio-control agent: Trichoderma harzianum

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1 Journal of Scientific & Industrial Research Vol. 63, October 2004, pp Evaluation of agro-industrial wastes for conidia based inoculum production of bio-control agent: Trichoderma harzianum Lakshmi Tewari* and Chandra Bhanu Department of Microbiology, College of Basic Sciences & Humanities, G B Pant University of Agriculture & Technology, Pantnagar Received 29 March 2004; accepted 02 August 2004 For management of agro-industrial wastes and selection of a suitable cheaper substrate for bio-pesticide development, three categories of agro-industrial wastes, viz. (i) Cellulosic wastes, (ii) Organic manures, and (iii) Cereal brans are evaluated for growth responses and conidial yield of the bio-control agent Trichoderma harzianum. All the substrates support fairly good growth and sporulation of the bioagent, but conidial counts are maximum in rice bran in 15 d followed by paddy straw and wheat straw in 20 d. Supplementation of all the substrates with chickpea flour as organic nitrogen supplement enhances the conidial yield, maximum increase in spore count being recorded in the case of paper waste. The optimum level of chickpea flour supplementation for paper waste is recorded as 12 per cent ( w / w ), thereby showing maximum conidiospore production (17.07x10 8 cfu/g) in 20 d, a further increment in cfu count is recorded upon its supplementation with rice bran. Paper waste based mixed substrate combination containing paper waste (80 per cent), rice bran (8 per cent) and chickpea flour (12 per cent) shows considerably higher conidial yield (11.73x10 9 cfu/g) after 10 d incubation period, exploring the possibility of utilizing it for mass multiplication and large-scale inoculum production of ecofriendly bio-control agent for sustainable environment. Keywords: Trichoderma harzianum, Bio-control agent, Mass multiplication, Supplementation, Biological control IPC Code: Int. Cl. 7 : A 01 N 65/00 Introduction Biological control by an antagonistic organism is a potential, non-chemical and ecofriendly approach for managing plant diseases 1. Among several groups of plant diseases, major amount of work has been done on the biological/integrated control of soil borne fungal plant pathogens by using fungal antagonists like, Trichoderma sp and Gliocladium sp 2. Trichoderma harzianum, one of the common fungal biocontrol agents, is being used worldwide for sustainable management of various foliar and soil borne plant pathogens 3. The renewed interest in biocontrol is due to its environmental friendliness, long lasting effects and safety features. Some of the bacterial and fungal antagonists have however also been found to show distinct plant growth promoting effects. Fungicidal control of pathogens under field conditions is beset with practical problems on account of cost to user, environmental pollution, and persistence of chemicals in nature. Besides, increased use of potentially hazardous chemical pesticides has *Author for correspondence become a matter of growing concern among both environmentalists and public health authorities. Plant disease control has now become heavily dependent on chemical fungicides, which are highly expensive, carcinogenic non-ecofriendly, recalcitrant leaving some residual toxic effects and thereby polluting the environment. Therefore, considering the cost of chemical pesticides and hazards involved, biological control of plant diseases appears to be an effective and ecofriendly approach being practiced world over. Further, biological control strategy is highly compatible with sustainable agriculture and has a major role to play as a component of integrated pest management (IPM) programme. Large-scale production, along with shelf life and establishment of bioagent in targeted niche, determine the success of biological control 4. Therefore, costeffective large-scale production, shelf-life of formulation, establishment of bio-agent in to targeted niche and consistency in disease control are the primary concern with augmentative biological control 5. Adaptation of technology in the bio-control arsenal needs to be investigated. Development of acceptable,

2 808 J SCI IND RES VOL 63 OCTOBER 2004 easily prepared and cost-effective formulations for delivery should be the major goal. For mass multiplication of the bioagent through solid-state fermentation technology, an enormous quantity of spore biomass is needed. Various substrates, viz. cereal grains, meal, brans, straw, plant residues and organic manures are being used for mass multiplication of T. harzianum with various degrees of success 3, 6. Moreover, a huge amount of solid paper waste (sludge) from pulp and paper mills and other agro-industrial wastes, viz. cellulosic plant residues are released every year, increasing pollution and disposal problems. Therefore, looking towards need for large-scale cost-effective production of ecofriendly bio-pesticide, present investigation is carried out to evaluate locally available cheaper substrates for mass multiplication of Tharzianum and also to explore the possibility of utilizing pulp and paper mill waste (sludge) for mass multiplication of the bioagent, both for sustainable environment and sustainable agriculture. Materials and Methods Biological Control Agent and Fungal Pathogens Standard culture of the fungal biocontrol agent Tharzianum and three root rot/wilt causing fungal pathogens of chickpea, viz. Rhizoctonia solani, Sclerotium rolfsii, and Fusarium oxysporum were obtained from the Biocontrol Laboratory, Department of Plant Pathology, College of Agriculture of the university. T. harzianum culture was maintained on Potato Dextrose Agar (PDA) and Trichoderma Selective Medium (TSM)-slants, while all the pathogens were maintained on PDA slants in refrigerator. Evaluation of in vitro Antagonistic Potential of T. harzianum Dual culture plate technique 7 was used to evaluate in vitro antagonistic potential of the bioagent against three root rot/wilt causing fungal pathogens of chickpea, viz. Rhizoctonia solani, Sclerotium rolfsii, and Fusarium oxysporum. Dual culture plates were prepared by inoculating two mycelial discs (5mm diam), each of a pathogen and a bioagent culture, 60 mm apart on PDA plates. Monoculture control plates of the bioagent (T. harzianum) were also made by inoculating single disc on PDA plates. The plates were incubated at 28±1 0 C and periodically observed for radial growth of the bioagent and its ability to antagonize and parasitize the pathogens. Screening of Cellulosic Wastes for Conidial Yield and Mass Multiplication of Bioagent Three different categories of substrates, viz. agroindustrial cellulosic wastes (wheat straw, paddy straw, shelled maize cob, saw dust, paper waste and sugarcane baggase), organic manures (Farm Yard Manure, spent compost of Button mushroom and spent straw of Oyster mushroom) and cereal brans (wheat and rice bran) were evaluated for growth responses and conidial yield of T. harzianum through solid state fermentation technique. For determination of conidiospore production of the bioagent on different substrates, 200 g of each substrate adjusted with 70 per cent moisture content, was filled into (21x 6) poly propylene (PP) bags fitted with PP- rings at necks. The bags were then sterilized by autoclaving at 15 lb psi pressure for 1.5 h. One-millilitre spore suspension (10 6 conidia/ml) of Tharzianum in sterilized distilled water was used to inoculate each bag with the help of disposable syringe. Inoculated bags were incubated at 28 ± 1 0 C for 20 d. Samples withdrawn periodically at 5 d interval from each bag were air-dried in shade, powdered, and sieved through 80-mesh sieve. The spore powders were stored in refrigerator at 4 0 C before use. Spore suspensions were prepared by suspending spore powder in sterile distilled 0.1g/100mL and analyzed for conidial counts by serial dilution plate count method using 1.0 per cent glucose agar. Influence of Supplementation of Substrate on Conidial Yield Supplementation studies were made in order to enhance growth and conidial yield of the bioagent on various cellulosic wastes. All the cellulosic substrates prepared, as described above, were supplemented with chickpea flour as organic nitrogen 4 per cent ( w / w, on dry weight basis). The processed substrates (200 g each) were sterilized in PP-bags at 15 lb psi pressure for 1.5 h. The sterilized substrates were inoculated aseptically with 1.0 ml spore suspension (10 6 conidia/ml) of the bioagent and incubated at 28±1ºC for 20 d. The samples withdrawn at 5 d interval from each bag were air-dried, powdered and analyzed for conidial counts, as described above. Optimization of Rate of Chickpea Flour Supplementation For optimizing rate of chickpea flour supplementation of cellulosic solid wastes, a solid waste of pulp and paper industry (sludge) was selected as it is released in huge amounts by pulp and paper mills with big disposal problem. Paper waste was soaked in

3 TEWARI & BHANU: EVALUATION OF AGRO-INDUSTRIAL WASTES 809 fresh water for 18 h, then excess water was drained off, dried and moisture content was adjusted to 70 per cent. It was then supplemented with different levels of chickpea flour ranging from 2 to 20 per cent on dry wt basis. Unsupplemented paper waste was considered as control. For measurement of radial growth, 20.0 g of substrate from each supplementation group was sterilized in Petri plates by autoclaving at 15 lb psi for 1.0 h. Each plate was then inoculated in the center with a fungal disc (5mm) of actively growing mycelial culture. Radial growth of the bioagent was measured up to 64 h at an interval of 8 h. Conidial yields in different samples incubated in PP-bags were determined by serial dilution plate count method, as described above. Table 1 Radial Growth of T. harzianum in dual culture with chickpea wilt pathogens Incubation Period (h) Radial Growth of T. harzianum (mm) in dual culture plates R. solani F. oxysporum S. rolfsii Control Critical Difference ( CD) (P=0.05) for: Incubation period (A) = 1.25, Pathogens (B)= 1.02, A x B = 2.50 Results and Discussion When evaluated for in vitro antagonistic potential against three root rot/wilt causing fungal pathogens of chickpea, viz. Rhizoctonia solani, Sclerotium rolfsii, and Fusarium oxysporum by dual culture technique, Trichoderma harzianum inhibited the mycelial growth of all the fungal pathogens (Table 1). In dual culture plates, the bioagent completely colonized R. solani and F. oxysporum while S. rolfsii was not parasitized by the bio-agent although its mycelial growth was inhibited. The dual culture plates with R. solani and F. oxysporum were completely covered by the bioagent in 120 and 144 h, respectively, indicating its higher antagonistic potential against R. solani as compared to that for F. oxysporum. It is thus evident from results that T. harzianum can effectively colonize R. solani and F. oxysporum and can therefore be used as a biocontrol agent effectively against these two fungal pathogens. T. harzianum is a well-known biocontrol agent and is used for managing various plant diseases including foliar and soil borne plant pathogens 3, 8. For selection of a suitable, cheaper and easily available substrate for mass multiplication of the bioagent, three different categories of substrates, viz. agro-industrial cellulosic wastes, organic manures, and cereal brans were evaluated for conidial counts. Viable conidial counts in spore powders of T. harzianum raised on various cellulosic substrates, ranged from 0.51 to 4.95 x10 8 /g powder in 5 to 20 d old cultures (Table 2). Maximum and significantly higher conidial counts (40.80x10 8 /g) among all the substrates evaluated were recorded on rice bran at Table 2 Conidiospore production (cfu / g) of T hrazianum on different cellulosic substrates Substrates Cfu (X10 8 /g) at different time intervals (d) Wheat straw Paddy straw Shelled maize cob Paper waste Saw dust Sugarcane baggase FYM Spent compost Spent straw Wheat bran Rice bran CD (P=0.05) for: Substrate (A) = 0.26, Incubation (B) = 0.22, AXB= 0.52

4 810 J SCI IND RES VOL 63 OCTOBER d (Fig. 1). Among various cellulosic substrates evaluated, paddy straw and wheat straw produced maximum and at-par conidial counts (4.95x10 8 and 4.86x10 8 /g powder, respectively), in 20 d old cultures. Lowest cfu counts were recorded on spent compost of button mushroom (Agaricus bisporus) and Farm Yard Manure (FYM) (0.55 and 0.65x10 8 /g, respectively). For development of conidia based powdery formulations, a suitable cheaper substrate favouring enormous quantities of conidiospore production is needed 9. Higher mycelial growth but lower conidial yield on organic manures suggested that a mixed substrate combination of various categories of substrates might be used for powdery formulation development of the bioagent with higher conidial counts. Thus, all the cellulosic agro-wastes favoured growth of T. harzianum thereby indicating its high cellulolytic activity and exploring the possibility of exploiting these wastes as substrates for cost-effective mass multiplication of the bioagent for sustainable environment. Several workers have also reported Trichoderma as a potential cellulose degrader 2,10. Faster mycelial growth on wheat and paddy straw might be attributed to easily available nutrients and lower C: N ratio than saw dust, paper waste and shelled maize cob and enhanced cellulase enzymatic activity 11. Organic manures and cereal brans are known to enhance early growth and establishment of the bioagent in augmentative biological control with T harzianum 12. Cereal brans supported slower mycelial growth but higher sporulation, which might be due to suppressive effect of excess nutrients in pure form of rice and wheat bran. Supplementation of all the cellulosic substrates with chickpea flour (as organic nitrogen supplement) significantly enhanced the conidial yield of T. harzianum invariably (Fig. 2). Maximum cfu counts (11.96x10 8 /g) were observed with paddy straw that was at par with sugarcane baggase (11.79x10 8 /g), wheat straw (11.52x10 8 /g) and paper waste (11.07x10 8 /g) at 4 per cent supplementation level. Saw dust was found inferior in terms of spore yield as it showed lowest counts with or without chickpea flour supplementation. Although minimum conidial counts (1.16x10 8 /g) were recorded on paper waste but considerable increase in counts upon its 4 per cent chickpea flour supplementation was recorded showing 11.07x10 8 counts/g. Further enhancement of sporulation of T. harzianum upon chickpea flour supplementation might be due to the abundant Fig. 1 Sporulation of T. harzianum on organic manures and cereal brans Fig. 2 Influence of chickpea flour supplementation of cellulosic substrates on sporulation of T. harzianum nutrients and growth factors present in the chickpea flour 13. Thus, significant enhancement in conidial count in paper waste upon chickpea flour supplementation indicated the possibility of utilizing it as substrate for mass multiplication of the bioagent. Since a huge quantity of paper waste (sludge) is released from pulp and paper mills every year, creating environmental pollution and its disposal problem. Therefore, growth and sporulation of T. harzianum on this agro-industrial waste may solve both these problems along with cost-effective mass production of the bio-agent for augmentative biological control for sustainable agriculture and environment. As maximum enhancement in spore production upon chickpea flour supplementation was recorded in

5 TEWARI & BHANU: EVALUATION OF AGRO-INDUSTRIAL WASTES 811 Table 3 Radial growth of T. harzianum on paper waste (sludge) supplemented with different levels of chickpea flour Supplementation rate Radial growth (mm) of T. hrazianum at different time intervals (h) (Per cent) CD (P= 0.05) for: Incubation period (A)=1.30, Supplementation rate (B)=1.38, AXB= 3.91 Table 4 Influence of level of chickpea flour supplementation on conidiospore production of T. harzianum on paper waste (sludge) Supplementation rate the case of paper waste, therefore further efforts were made to optimize the rate of chickpea flour supplementation for maximum growth and conidial yield of the bio-agent. It is evident from mycelial growth of T. harzianum measured on paper waste supplemented with different levels (2 to 20 per cent) of chickpea flour, that the fungus grew faster on 12 per cent supplemented samples covering the full plate in 64 h (Table 3). It is evident from the data shown in Table 4 that conidial counts of the bio-agent on paper waste increased upon chickpea flour supplementation up to 12 per cent level, maximum counts (17.07x10 8 /g) being recorded at 12 per cent supplementation level at 20 d. However, thereafter a decrease in conidial counts was recorded upon further increase in chickpea flour level beyond 12 per cent and only 12.40x10 8 and 12.27x10 8 /g counts were recorded at 16 and 20 per cent supplementation levels, respectively, at 20 d. Trichoderma sp is a model organism among the cellulose decomposing c.f.u. ( x 10 8 )/g at different time intervals (d) (Per cent) CD (P=0.05) For: Incubation period (A) = 0.21, Supplementation rate (B) = 0.32, AXB = 0.64 communities 14. Enhancement of sporulation of the bioagent on paper waste upon chickpea flour supplementation might be due to decreased C:N ratio of supplemented substrates, other nutrients and growth factors present in chickpea flour. Deacon 4 has described that the presence of an appropriate C:N ratio in the substrate is necessary for the growth and development of fungi. Suitability of nitrogen rich leguminous substrates for mass multiplication of T. harzianum has also been described earlier 15,16. Low cost and easy availability of these cellulosic agro wastes may prove an asset for mass production of T. harzianum. Slow growth rate with abundant green sporulation of the bio-agent on rice and wheat brans, as observed during present investigation suggests, that these brans may be used as supplements for other substances of low nutritive value for higher conidial yield. Cheap and easily available substrates have also been evaluated by other workers for mass multiplication of

6 812 J SCI IND RES VOL 63 OCTOBER 2004 T. harzianum with great success 17. Organic manures though resulted in inferior sporulation, may serve as substrates in combination with other nutrient rich supplements for mass multiplication of T. harzianum. Considering these observations a mixed substrates combination was developed by mixing paper waste (80 per cent) with rice bran (8 per cent) and chickpea flour (12 per cent) and evaluated for conidial counts. This substrate-combination exhibited considerably higher conidial counts (11.73x10 9 /g) and that too in reduced time (10 d). Thus, efficient combinations of substrates have been found superior for spore yield of T. harzianum than on individual components as also seen earlier 18,19. Increased spore yield due to chickpea flour supplementation and combination of various substrates during present investigation would be a forward step towards augmentative biological control. These findings thus explore the possibility of developing a suitable substrate combination by mixing various categories of substrates with chickpea flour and rice bran for higher conidial yield and mass multiplication of the biological control agent. Acknowledgements The authors are grateful to U P Council of Agricultural Research for financial assistance and G B Pant University of Agriculture and Technology, Pantnagar for providing research facilities. References 1 Andrews J H, Biological control in the phyllosphere, Annu Rev Phytopathol, 30 (1992) Bellows T S & Fisher T W, Handbook of biological control (Academic press, London ) 1999, pp Butt T M, Jackson C W & Magan N, In Fungi as bio-control agent: Progress, problems and potentia (CABI, Press Oxon, UK) 2001, pp Deacon J W, In Modern mycology ( Blackwell Science Ltd.), 1997, pp Dennis C & Webster J, Antagonistic properties of species, groups of Trichoderma: production of non- volatile antibiotics, Trans Br Mycol Soc, 206 (1971) Elad Y, Chet I & Henis Y, Degradation of plant pathogenic fungi by Trichoderma harzianum, Can J Microbiol, 28 (1982) Gandhi N Kumar & Rangnathan K, Evaluation of different substrates on survival of Trichoderma viridae and Trichoderma harzianum, Proc Indian Phytopathol Soc Golden Jubilee, 1 ( 2001) Harman G E, Jin X, Stasz T E, Teruzzotti G, Leopold A C & Taylor A G, Production of conidial biomass of Trichoderma harzianum for biological control, Biol Control, 1 (1991) Jayaraj J & Rambadran R, Evaluation of certain organic substrates and adjuvant for mass multiplication of Trichoderma harzianum Refai, J Biol Control, 10 ( 1996) Laranjeira D, Olivera S M A, Menzes M & Neves R P, Effect of different substrates on sporulation of Trichoderma, Summa-Phytopathol, 22 (1996) Lewis J A & Papavizas G C, Production of chlamydospores and conidia by Trichoderma sp in liquid and solid growth media, Soil Biol Biochem, 15 ( 1983) Nevalainen K M N, Palva E T & Bailey M J, A high cellulase producing mutant strain of Trichoderma reesei, Enzs Microbial Technol, 2 ( 1980) Papavizas G C, Trichoderma and Gliocladium: Biology and potential for biological control, Annu Rev Phytopathol, 23 ( 1985) Papavizas G C & Lewis J A, Introduction and augmentation of microbial antagonists for the control of soil borne plant pathogens, In Biological control in crop production, edited by G C Papavizas (Allan held Osmun, Totowa, NJ), 1981, pp Patel S I & Ashok M, Evaluation of cheap and easily available substrates for mass multiplication of Trichoderma harzianum refai, Gujarat Agric Univ Res J, 20 ( 1994) Singh G, Singh U S & Mukhopadhyay A N, Mass production of spore powder of Trichoderma virens and its viability on coated lentil seed, Ann Pl Protect Sci, 9 (2001) Singhal S, Evolving cheap substrates for mass multiplication of Gliocladium virens and Trichoderma harzianum and standardizing biological seed treatment in pea (Pisum sativum L.), M Sc Thesis, G B Pant University of Agriculture & Technology, India, Tewari A K, & Mukhopadhyay A N, Testing of different formulations of Gliocladium virens against chickpea wilt complex, Indian Phytopath, 54 ( 2001) Tewari L & Bhanu C, Screening of various substrates for sporulation and mass multiplication of bio-control agent Trichoderma harzianum through solid state fermentation, Indian Phytopath, 56 (2003) 476.