- Email: [email protected]
Advances in Formulation of Trichoderma for Biocontrol
C H A P T E R
39
Advances in Formulation of Trichoderma for Biocontrol Christian Joseph R. Cumagun College of Agriculture, University of the Philippines Los Baños, Los Baños, Laguna, Philippines, email: [email protected]
O U T L I N E Introduction527
Microencapsulation528
Compatibility with Other Biological Systems Modes of Delivery and Application Seed Treatment Solid Matrix Priming Liquid Coating Double Coating
529 529 529 529 530 530
Enhancement of Shelf Life and Application Efficiency528
Conclusion and Future Prospects
530
Types of Formulation Liquid Formulation Solid Formulation
528 528 528
INTRODUCTION Biological control of plant pathogens has become an integral component of pest management in light of the environmental and health issues attributed to the use of fungicides in agriculture. Renewed interest in biological control using Trichoderma, a soil-borne fungus and decomposer is in line with ensuring environmental sustainability and productive and sustainable agriculture by applying the principles of ecology to disease control (Cumagun, 2012). According to Harman (1991), the development of biological control systems depends on three crucial components: (1) a highly effective biocontrol agent; (2) production of a high level of effective and viable propagules; and (3) delivery systems conducive to the bioprotectant that provide a competitive advantage to the biocontrol agent relative to other microflora. The first step in the biocontrol study is the identification of promising biocontrol agent. Once the biocontrol agent is identified and is proven effective against plant pathogens over several reproducible results, the method of mass production, formulation and application should be taken
Biotechnology and Biology of Trichoderma http://dx.doi.org/10.1016/B978-0-444-59576-8.00039-4
into consideration to stabilize the product during storage and to facilitate its delivery to the plant. Both solid and liquid formulations are used to produce suitable quantities of active and viable inocula of Trichoderma. Three kinds of propagules can be used in formulations: hyphae, chlamydospores and conidia (Howell, 2003). The use of hyphae is not an option due to its lack of resistance to dehydration. Conidia and chlamydospores withstand adverse environmental conditions, which made them the natural choice as propagules in formulations (Jin et al., 1991; Papavizas, 1985). This review will focus with the recent advances made in the development of formulation and delivery systems of Trichoderma to enhance its biocontrol efficacy against plant pathogens. Other ideal properties of biomass of an antagonist as outlined by Harman et al. (1991) include the following: (1) economical, cost-effective production and preferably in submerged liquid fermentation; (2) preservation against microbial contamination in dry powder form and low water availability; (3) long shelf life biomass. A biocontrol preparation of an antagonist may not possess all these properties but it should have as many of
527
Copyright © 2014 Elsevier B.V. All rights reserved.
528
39. ADVANCES IN FORMULATION OF TRICHODERMA FOR BIOCONTROL
these desirable properties. For the end user point of view, it should be economically feasible to produce and readily adopted by farmers.
TYPES OF FORMULATION Liquid Formulation Deep tank fermentation system is employed in liquid formulation which make it a more preferred approach for biomass production in Europe and North America (Churchill, 1982). Inexpensive growth media such as molasses and brewer’s yeast are used for production in liquid formulation (Papavizas et al., 1984). The advantage of this formulation is the optimization of biomass production and quality, which allows control of nutrients, pH, temperature and other environmental factors thus reducing contamination (Whipps, 1997).
application efficiency (John et al., 2011). Microencapsulation is defined as a process in which tiny particles or droplets are surrounded by a coating, or embedded in a homogeneous or heterogeneous matrix, to give small capsules with many useful properties (Gharsallaoui et al., 2007). This formulation provides living cells with a physical barrier against the external environment (O’Riordan et al., 2001). Drying of conidia is essential in order to prevent spoilage by microbial contamination but it loses viability during the process of drying at elevated temperature (Jin and Custis, 2011). A dry product has the advantage of convenient storage and transport. Microencapsulation of conidia with sugars, such as sucrose, molasses or glycerol increased the survival of conidia of Trichoderma (Jin and Custis, 2011). Microencapsulation of Trichoderma harzianum in maltodextringum arabic biopolymer matrix obtained the highest conidia survival after spray-drying with 11-fold times higher than those of nonencapsulated conidia (MuñozCelaya et al., 2012).
Solid Formulation Solid formulation or fermentation is the alternative method for inoculum production. Agricultural waste materials such as wheat and rice straw, sugarcane bagasse, ground corn cobs, sawdust, rice bran are used as food base or substrate alone or in combination for the growth of Trichoderma. Cumagun and Lapis (1993) used rice bran as food base for Trichoderma spp. with tapioca flour as binding agent to produce pellets. Provision of food base in the formulation should in most cases favor the antagonist (Papavizas et al., 1984) or a food base that can only be utilized by antagonist in which the pathogen can be inhibited (Nelson et al., 1988). This method of formulation requires only minimal cost especially in small scale production but it is bulky as it requires large space for production, inoculation and storage including drying and milling. Both solid and liquid formulations require drying to obtain stable product with prolonged shelf life (Jin et al., 1992). Spray drying is preferred among the different drying techniques for large scale production of microorganisms containing dried powders due to its low cost (Morgan et al., 2006).
MICROENCAPSULATION The low viability of the biocontrol agent during storage and field application along with the lack of knowledge regarding the best carrier in conventional formulation are the major drawbacks for the two types of formulation. To address these, microencapsulation has been developed to prolong shelf-life and control microbial release from formulations and thus enhancing their
ENHANCEMENT OF SHELF LIFE AND APPLICATION EFFICIENCY Dried conidial pellets of T. harzianum were more effective antagonist formulation than liquid formulation in inhibiting sclerotial germination of Rhizoctonia solani (Cumagun and Ilag, 1998). Because liquid formulation of Trichoderma spp. is prone to desiccation compared to solid formulation, additives are added to prolong the antagonists’ survival. Sriram et al. (2011) studied the effect of glycerol, an osmoticant, on the shelf life of T. harzianum. The addition of glycerol in the production medium at 3% and 6% extended shelf-life prolonged the shelf-life of talc formulation to 7 and 12 months, respectively, compared to 4–5 months in formulations without glycerol. Sriram et al. (2010) added chitin in the production medium and talc formulation of T. harzianum which enhanced the shelf life by 2 months. The application of alginate formulation and paraffin oil in Trichoderma increased its shelf life (Al-Taweil et al., 2010). Trichoderma asperellum has shown biocontrol potential against Fusarium head blight by using liquid media of differing composition (Kolombet et al., 2008). The treatment with the greatest effect was the addition of starch and small amounts of copper with the latter as food base including lowering the pH of the biomass paste to reduce metabolic activity of T. asperellum. The biomass paste formulation remained viable for at least 6 months at room temperature (Kolombet et al., 2008). The invert emulsion (water-in-oil type) formulation of T. harzianum prolonged the postharvest shelf life of the fruit (Batta, 2007). For an alginate encapsulated Trichoderma spp. in Egypt, an addition of 10% cellulose increased the survival of the entrapped
G. BIOCONTROL AND PLANT GROWTH PROMOTION
Compatibility with Other Biological Systems
conidia better than without cellulose (Shaban and El-Komy, 2001). A chitin fortified bioformulation of Trichoderma/Hypocrea species allowed increased accumulation of total phenols, peroxidase, polyphenoloxidase and phenylalanine ammonia lyase in treated tomato plants when challenged with R. solani (Solanki et al., 2011). Kandula et al. (2010) supplemented commercial Trichoderma and powder formulations with NPK which improved growth response but with no reduction in root disease symptoms of specific apple replant disease (Kandula et al., 2010). The addition of organic fertilizer enhanced the performance of T. harzianum SQR-T037 as compared to conidial suspension alone in controlling Fusarium wilt of cucumbers (Yang et al., 2011). The reason for improved performance is the sustained colonization of T. harzianum SQR-T037 in the rhizosphere soil (Yang et al., 2011). To increase biomass and number of colony forming unit/mL of T. asperellum, Wijesinghe et al. (2011) incubated the biocontrol agent in yeast waste residue medium. The population peaked at 144 h and the formulation was used to treat fruits infected with black rot disease caused by Thielaviopsis paradoxa. Growing in bentonite–vermiculite formulation also increased the colony forming units of T. harzianum after 8 weeks and provided higher melon shoot weight and higher resistance to Fusarium wilt disease (Martinez-Medina et al., 2009). Bernal-Vicente et al. (2009) found the most effective treatment against Fusarium wilt of melon was the solid formulation bentonite and superficial vermiculite of T. harzianum isolate T-78. Thangavelu et al. (2003) found that dried banana leaves is the best carrier material to support the growth of Trichoderma spp. isolated from the rhizosphere of banana from Tamil, India. The addition of jaggery (10% w/v) promoted increase of T. harzianum and prolonged the survival for more than 6 months in storage. Immobilizing wet biomass of Trichoderma viride in gluten matrix reduced the amount of biomass needed and generated 106–107 colony forming units g-1 soil in the second week (Cho and Lee, 1999).
COMPATIBILITY WITH OTHER BIOLOGICAL SYSTEMS A single strain of Trichoderma may not be effective in dealing with all diseases at all conditions. As explained by Cook (1993), biological control is highly disease specific and may require mixture of strains or other biological control agent for optimum disease control. De Jaeger et al. (2011) demonstrated the compatibility of the entrapped T. harzianum and Glomus in alginate beads. The presence of T. harzianum has no inhibitory effect but rather stimulated the spore production and fitness of Glomus. The combined application of Pseudomonas
529
fluorescens and T. viride in talc formulation resulted in significant control of sheath blight of rice caused by R. solani and comparable to the treatment of carbendazim (Mathivanan et al., 2005). Similarly Khan et al. (2004) found that the combination of T. harzianum and P. fluorescens decreased soil population of Fusarium wilt of chickpea. Gilardi et al. (2007) observed competitive effect of the combined application of T. viride and P. fluorescens. Treatments with T. harzianum and Pochonia chlamydosporia grown in sawdust:soil:5% molasses (15:5:1) and 20 parts carrier (fly ash: soil:5% molasses mixture (5:3:1) effectively controlled Fusarium wilt and root-knot nematode of pulses (Khan et al., 2011).
Modes of Delivery and Application The success of biological control of plant pathogens using Trichoderma does not rely solely on effective antagonists but also on the method of delivery or application on the seed, root and soil. In addition, all antagonists rely on their placement on the infection court to effect successful protection and control (Mathre et al., 1999). Timing of delivery and application is also crucial and Trichoderma is usually only effective as a preventative measure but can be integrated with other disease management options especially when the disease has already established. The following are examples of effective modes of delivery and application of Trichoderma focusing on seed treatment:
Seed Treatment Seed treatment or coating is the most effective method of application of Trichoderma into an agricultural system (Mathre et al., 1999). Trichoderma is delivered in the infection court (surface of seed coat) as protectant at planting. This method of delivery should limit growth of competitive microflora and provide conducive growth for the biocontrol agent. Seed treatment using seed dressing formulation, Pusa 5SD has been proven more effective than soil application formulations PusaBiogranule 6 (PBG 6) and PusaBiopellet 16G (PBP 16G) in managing wet root rot of mungbean caused by R. solani (Dubey et al., 2011).
Solid Matrix Priming Seed priming is the process in which the seeds are hydrated to allow metabolic process of germination to take place but not sprouting. Two priming systems are available. Solid matrix priming (SMP) was developed by J. Eastin, Kamterter, Inc. (Lincoln, NE) to enhance biocontrol of Trichoderma by regulating water levels in the seed. It allows effective colonization of seed surface before planting given the right pH and matrix material. Osmopriming makes use of aerated aqueous solutions of
G. BIOCONTROL AND PLANT GROWTH PROMOTION
530
39. ADVANCES IN FORMULATION OF TRICHODERMA FOR BIOCONTROL
salts or polyethylene glycols to generate osmotic potential in the primary solution. SMP is simple and more cost-effective than osmopriming (Harman and Taylor, 1988). Seed priming of three rhizosphere isolates of T. harzianum enhanced growth and induced resistance in sunflower against downy mildew caused by Plasmopara halstedii (Nagaraju et al., 2012).
Liquid Coating Liquid coating is a seed coating system which involves application of Trichoderma to the seed with an aqueous adhesive or binder (pelgel or polyox-N-10) and a particulate material (Agro-lig or muck soil) to optimize pH level including a bulking agent (Taylor et al., 1991). The Agro-lig is reported to have a physical and chemical characteristic which favor the growth of the fungus.
Double Coating A modification of liquid coating in which Trichoderma is applied directly to the seed coat followed by a particulate to form the second layer. The advantage of this system is that the biocontrol agent has access to seed exudates especially with seeds that have a low level of exudation (Harman, 1991). It is recommended to allow Trichoderma to colonize the seed surface at 100% relative humidity to improve its biocontrol efficacy (Taylor et al., 1991).
CONCLUSION AND FUTURE PROSPECTS The advances in Trichoderma formulation have f urther promoted biological control of plant pathogens for sustainable agriculture. Future research on the biological control system of Trichoderma should look into formulation suitable to control foliar and aerial pathogens considering its endophytic nature (Evans et al., 2003). There is a need to strengthen research-industry partnership to scale up production systems including large scale promotion of Trichoderma formulation in farmers’ fields particularly in the developing countries.
References Al-Taweil, H.I., Osman, M.B., Abdulhamid, A., Mohammad, N., Yussof, W.M.W., 2010. Comparison of different delivery system of Trichoderma and Bacillus as biofertilizer. Adv. Environ. Biol. 4, 31–33. Batta, Y.A., 2007. Control of postharvest diseases of fruit with an invert emulsion formulation of Trichoderma harzianum Rifai. Postharvest Biol. Technol. 43, 143–150. Bernal-Vicente, A., Ros, M., Pascual, J.A., 2009. Increased effectiveness of the Trichoderma harzianum isolate T-78 against Fusarium wilt on melon plants under nursery conditions. J. Sci. Food Agric. 89, 827–833.
Cho, C., Lee, W., 1999. Formulation of a biocontrol agent by entrapping biomass of Trichoderma viride in gluten matrix. J. Biosci. Bioeng. 87, 822–824. Churchill, B.W., 1982. Mass production of microorganisms for biological control. In: Charudattan, R., Walker, H.L. (Eds.), Biological Control of Weeds with Plant Pathogens, John Wiley & Sons, New York, pp. 139–156. Cook, R.J., 1993. The role of biological control in the 21st century. In: Lumsden, R.D., Vaughn, J.L. (Eds.), Pest Management: Biologically Based Technologies, American Chemical Society, Washington D.C., pp. 10–20. Cumagun, C.J.R., 2012. Managing plant diseases and promoting sustainability and productivity with Trichoderma. J. Agric Sci. Technol. 14, 699–714. Cumagun, C.J.R., Ilag, L.L., 1998. Parasitism of sclerotial bodies of Rhizoctonia solani Kuehn by Trichodema harzianum Rifai and Penicillium oxalicum Currie and Thom. Philipp. Phytopathol. 33, 17–26. Cumagun, C.J.R., Lapis, D.B., 1993. Note: practical approach in mass production of Trichoderma spp. as a means of biological control against sheath blight of rice. Philipp. Agric. 76, 251–257. De Jaeger, N., de la Providencia, I.E., Rouhier, H., Declerck, S., 2011. Coentrapment of Trichoderma harzianum and Glomus sp. within alginate beads: impact on the arbuscular mycorrhizal fungi life cycle. J. Appl. Microbiol. 111, 125–135. Dubey, S.C., Bhavani, R., Singh, B., 2011. Integration of soil application and seed treatment formulations of Trichoderma species for management of wet root rot of mungbean caused by Rhizoctonia solani. Pest Manage. Sci. 67, 1163–1168. Evans, H.C., Holmes, K.A., Thomas, S.E., 2003. Endophytes and mycoparasites associated with an indigenous forest tree, Theobroma gileri in Ecuador and a preliminary assessment of their potential as biocontrol agents of cocoa diseases. Mycol. Prog. 2, 149–160. Gharsallaoui, A., Roudaut, G., Chambin, O., Voilley, A., Saurel, R., 2007. Applications of spray-drying in microencapsulation of food ingredients: an overview. Food Res. Int. 40, 1107–1121. Gilardi, G., Garibaldi, A., Gullino, M.L., 2007. Effect of antagonistic Fusarium spp. and of different commercial biofungicide formulations on Fusarium wilt of lettuce. Phytoparasitica 35, 457–465. Harman, G.E., 1991. Seed treatments for biological control of plant disease. Crop Prot. 10, 166–171. Harman, G.E., Jin, X., Stasz, T.E., Peruzzotti, G., Leopold, A.C., Taylor, A.G., 1991. Production of conidial biomass of Trichoderma harzianum for biological control. Biol. Control 1, 23–28. Harman, G.E., Taylor, A.G., 1988. Improved seedling performance by integration of biological control agents at favourable pH levels with solid matrix priming. Phytopathology 78, 520–525. Howell, C.R., 2003. Mechanisms employed by Trichoderma species in the biological control of plant diseases: the history and evolution of current concepts. Plant Dis. 87, 4–10. Jin, X., Custis, D., 2011. Microencapsulating aerial conidia of Trichoderma harzianum through spray drying at elevated temperatures. Biol. Control 56, 202–208. Jin, X., Harman, G.E., Taylor, A.G., 1991. Conidial biomass and desiccation tolerance of Trichoderma harzianum produced at different medium water potentials. Biol. Control 1, 237–243. Jin, X., Hayes, K.C., Harman, G.E., 1992. Principles in the development of biological control systems employing Trichoderma species against soil-born plant pathogenic fungi. In: Lantham, G.F. (Ed.), Frontiers in Industrial Mycology, Chapman & Hall Inc., New York, pp. 174–195. John, R.P., Tyagi, R.D., Brar, S.K., Surampalli, R.Y., Prévost, D., 2011. Bio-encapsulation of microbial cells for targeted agricultural delivery. Crit. Rev. Biotechnol. 31, 211–226. Kandula, D.R.W., Jones, E.E., Horner, I.J., Stewart, A., 2010. The effect of Trichoderma bio-inoculants on specific apple replant disease (SARD) symptoms in apple rootstocks in New Zealand. Australas. Plant Pathol. 39, 312–318.
G. BIOCONTROL AND PLANT GROWTH PROMOTION
REFERENCES
Khan, M.R., Khan, S.M., Mohiddin, F.A., 2004. Biological control of Fusarium wilt of chickpea through seed treatment with the commercial formulation of Trichoderma harzianum and/or Pseudomonas fluorescens. Phytopathol. Mediterr. 43, 20–25. Khan, M.R., Majid, S., Mohidin, F.A., Khan, N., 2011. A new bioprocess to produce low cost powder formulations of biocontrol bacteria and fungi to control Fusarial wilt and root-knot nematode of pulses. Biol. Control 59, 130–140. Kolombet, L.V., Zhigletsova, S.K., Kosareva, N.I., Bystrova, E.V., Derbyshev, V.V., Krasnova, S.P., Schisler, D., 2008. Development of an extended shelf-life, liquid formulation of the biofungicide Trichoderma asperellum. World J. Microbiol. Biotechnol. 24, 123–131. Martínez-Medina, A., Roldán, A., Pascual, J.A., 2009. Performance of a Trichoderma harzianum bentonite-vermiculite formulation against Fusarium wilt in seedling nursery melon plants. Hortic. Sci. 44, 2025–2027. Mathivanan, N., Prabavathy, V.R., Vijayanandraj, V.R., 2005. Application of talc formulations of Pseudomonas fluorescens Migula and Trichoderma viride pers. ex S.F. gray decrease the sheath blight disease and enhance the plant growth and yield in rice. J. Phytopathol. 153, 697–701. Mathre, D.E., Cook, R.J., Callan, N.W., 1999. From discovery to use: traversing the world of commercializing biocontrol agents for plant disease control. Plant Dis. 83, 972–983. Morgan, C.A., Herman, N., White, P.A., Vesey, G., 2006. Preservation of microorganisms by drying: a review. J. Microbiol. Methods 66, 183–193. Muñoz-Celaya, A.L., Ortiz-García, M., Vernon-Carter, E.J., JaureguiRincón, J., Galindo, E., Serrano-Carreón, L., 2012. Spray-drying microencapsulation of Trichoderma harzianum conidia in carbohydrate polymers matrices. Carbohydr. Polym. 88, 1141–1148. Nagaraju, A., Sudisha, J., Murthy, S.M., Ito, S.-, 2012. Seed priming with Trichoderma harzianum isolates enhances plant growth and induces resistance against Plasmopara halstedii, an incitant of sunflower downy mildew disease. Australas. Plant Pathol. 41, 609–620. Nelson, E.B., Harman, G.E., Nash, G.T., 1988. Enhancement of Trichoderma-induced biological control of Pythium seed rot and pre- emergence damping-off of peas. Soil Biol. Biochem. 20, 145–150. O’Riordan, K., Andrews, D., Buckle, K., Conway, P., 2001. Evaluation of microencapsulation of a Bifido bacterium strain with starch as an approach to prolonging viability during storage. J. Appl. Microbiol. 91, 1059–1066.
531
Papavizas, G.C., 1985. Trichoderma and Gliocladium: biology, ecology, and potential for biocontrol. Annu. Rev. Phytopathol. 82, 178–184. Papavizas, G.C., Dunn, M.T., Lewis, J.A., Beagle-Ristaino, J., 1984. Liquid fermentation technology for experimental production of biocontrol fungi. Phytopathology 74, 1171–1175. Shaban, G.M., El-Komy, H.M.A., 2001. Survival and proliferation of alginate encapsulated Trichoderma spp. in Egyptian soil in comparison with allyl alcohol soil fumigation. Mycopathology 151, 139–146. Solanki, M.K., Singh, N., Singh, R.K., Singh, P., Srivastava, A.K., Kumar, S., Kashyap, P.L., Arora, D.K., 2011. Plant defense activation and management of tomato root rot by a chitin-fortified Trichoderma/ Hypocrea formulation. Phytoparasitica 39, 471–481. Sriram, S., Palanna, K.B., Ramanujam, B., 2010. Effect of chitin on the shelf-life of Trichoderma harzianum in talc formulation. Indian J. Agric. Sci. 80, 930–932. Sriram, S., Roopa, K.P., Savitha, M.J., 2011. Extended shelf-life of liquid fermentation derived talc formulations of Trichoderma harzianum with the addition of glycerol in the production medium. Crop Prot. 30, 1334–1339. Taylor, A.G., Min, T.-, Harman, G.E., Jin, X., 1991. Liquid coating formulation for the application of biological seed treatments of Trichoderma harzianum. Biol. Control 1, 16–22. Thangavelu, R., Palaniswami, A., Velazhahan, R., 2003. Mass production of Trichoderma harzianum for managing Fusarium wilt of banana. Agric. Ecosys. Environ. 103, 259–263. Whipps, J.M., 1997. Developments in the biological control of soilborne plant pathogens. In: Callow, J.A. (Ed.), Advances in Botanical Research Incorporating Advances in Plant Pathology, vol. 26. pp. 1–123. Wijesinghe, C.J., Wijeratnam, R.S.W., Samarasekara, J.K.R.R., Wijesundera, R.L.C., 2011. Development of a formulation of Trichoderma asperellum to control black rot disease on pineapple caused by (Thielaviopsis paradoxa). Crop Prot. 30, 300–306. Yang, X., Chen, L., Yong, X., Shen, Q., 2011. Formulations can affect rhizosphere colonization and biocontrol efficiency of Trichoderma harzianum SQR-T037 against Fusarium wilt of cucumbers. Biol. Fertil. Soils 47, 239–248.
G. BIOCONTROL AND PLANT GROWTH PROMOTION
39
Advances in Formulation of Trichoderma for Biocontrol Christian Joseph R. Cumagun College of Agriculture, University of the Philippines Los Baños, Los Baños, Laguna, Philippines, email: [email protected]
O U T L I N E Introduction527
Microencapsulation528
Compatibility with Other Biological Systems Modes of Delivery and Application Seed Treatment Solid Matrix Priming Liquid Coating Double Coating
529 529 529 529 530 530
Enhancement of Shelf Life and Application Efficiency528
Conclusion and Future Prospects
530
Types of Formulation Liquid Formulation Solid Formulation
528 528 528
INTRODUCTION Biological control of plant pathogens has become an integral component of pest management in light of the environmental and health issues attributed to the use of fungicides in agriculture. Renewed interest in biological control using Trichoderma, a soil-borne fungus and decomposer is in line with ensuring environmental sustainability and productive and sustainable agriculture by applying the principles of ecology to disease control (Cumagun, 2012). According to Harman (1991), the development of biological control systems depends on three crucial components: (1) a highly effective biocontrol agent; (2) production of a high level of effective and viable propagules; and (3) delivery systems conducive to the bioprotectant that provide a competitive advantage to the biocontrol agent relative to other microflora. The first step in the biocontrol study is the identification of promising biocontrol agent. Once the biocontrol agent is identified and is proven effective against plant pathogens over several reproducible results, the method of mass production, formulation and application should be taken
Biotechnology and Biology of Trichoderma http://dx.doi.org/10.1016/B978-0-444-59576-8.00039-4
into consideration to stabilize the product during storage and to facilitate its delivery to the plant. Both solid and liquid formulations are used to produce suitable quantities of active and viable inocula of Trichoderma. Three kinds of propagules can be used in formulations: hyphae, chlamydospores and conidia (Howell, 2003). The use of hyphae is not an option due to its lack of resistance to dehydration. Conidia and chlamydospores withstand adverse environmental conditions, which made them the natural choice as propagules in formulations (Jin et al., 1991; Papavizas, 1985). This review will focus with the recent advances made in the development of formulation and delivery systems of Trichoderma to enhance its biocontrol efficacy against plant pathogens. Other ideal properties of biomass of an antagonist as outlined by Harman et al. (1991) include the following: (1) economical, cost-effective production and preferably in submerged liquid fermentation; (2) preservation against microbial contamination in dry powder form and low water availability; (3) long shelf life biomass. A biocontrol preparation of an antagonist may not possess all these properties but it should have as many of
527
Copyright © 2014 Elsevier B.V. All rights reserved.
528
39. ADVANCES IN FORMULATION OF TRICHODERMA FOR BIOCONTROL
these desirable properties. For the end user point of view, it should be economically feasible to produce and readily adopted by farmers.
TYPES OF FORMULATION Liquid Formulation Deep tank fermentation system is employed in liquid formulation which make it a more preferred approach for biomass production in Europe and North America (Churchill, 1982). Inexpensive growth media such as molasses and brewer’s yeast are used for production in liquid formulation (Papavizas et al., 1984). The advantage of this formulation is the optimization of biomass production and quality, which allows control of nutrients, pH, temperature and other environmental factors thus reducing contamination (Whipps, 1997).
application efficiency (John et al., 2011). Microencapsulation is defined as a process in which tiny particles or droplets are surrounded by a coating, or embedded in a homogeneous or heterogeneous matrix, to give small capsules with many useful properties (Gharsallaoui et al., 2007). This formulation provides living cells with a physical barrier against the external environment (O’Riordan et al., 2001). Drying of conidia is essential in order to prevent spoilage by microbial contamination but it loses viability during the process of drying at elevated temperature (Jin and Custis, 2011). A dry product has the advantage of convenient storage and transport. Microencapsulation of conidia with sugars, such as sucrose, molasses or glycerol increased the survival of conidia of Trichoderma (Jin and Custis, 2011). Microencapsulation of Trichoderma harzianum in maltodextringum arabic biopolymer matrix obtained the highest conidia survival after spray-drying with 11-fold times higher than those of nonencapsulated conidia (MuñozCelaya et al., 2012).
Solid Formulation Solid formulation or fermentation is the alternative method for inoculum production. Agricultural waste materials such as wheat and rice straw, sugarcane bagasse, ground corn cobs, sawdust, rice bran are used as food base or substrate alone or in combination for the growth of Trichoderma. Cumagun and Lapis (1993) used rice bran as food base for Trichoderma spp. with tapioca flour as binding agent to produce pellets. Provision of food base in the formulation should in most cases favor the antagonist (Papavizas et al., 1984) or a food base that can only be utilized by antagonist in which the pathogen can be inhibited (Nelson et al., 1988). This method of formulation requires only minimal cost especially in small scale production but it is bulky as it requires large space for production, inoculation and storage including drying and milling. Both solid and liquid formulations require drying to obtain stable product with prolonged shelf life (Jin et al., 1992). Spray drying is preferred among the different drying techniques for large scale production of microorganisms containing dried powders due to its low cost (Morgan et al., 2006).
MICROENCAPSULATION The low viability of the biocontrol agent during storage and field application along with the lack of knowledge regarding the best carrier in conventional formulation are the major drawbacks for the two types of formulation. To address these, microencapsulation has been developed to prolong shelf-life and control microbial release from formulations and thus enhancing their
ENHANCEMENT OF SHELF LIFE AND APPLICATION EFFICIENCY Dried conidial pellets of T. harzianum were more effective antagonist formulation than liquid formulation in inhibiting sclerotial germination of Rhizoctonia solani (Cumagun and Ilag, 1998). Because liquid formulation of Trichoderma spp. is prone to desiccation compared to solid formulation, additives are added to prolong the antagonists’ survival. Sriram et al. (2011) studied the effect of glycerol, an osmoticant, on the shelf life of T. harzianum. The addition of glycerol in the production medium at 3% and 6% extended shelf-life prolonged the shelf-life of talc formulation to 7 and 12 months, respectively, compared to 4–5 months in formulations without glycerol. Sriram et al. (2010) added chitin in the production medium and talc formulation of T. harzianum which enhanced the shelf life by 2 months. The application of alginate formulation and paraffin oil in Trichoderma increased its shelf life (Al-Taweil et al., 2010). Trichoderma asperellum has shown biocontrol potential against Fusarium head blight by using liquid media of differing composition (Kolombet et al., 2008). The treatment with the greatest effect was the addition of starch and small amounts of copper with the latter as food base including lowering the pH of the biomass paste to reduce metabolic activity of T. asperellum. The biomass paste formulation remained viable for at least 6 months at room temperature (Kolombet et al., 2008). The invert emulsion (water-in-oil type) formulation of T. harzianum prolonged the postharvest shelf life of the fruit (Batta, 2007). For an alginate encapsulated Trichoderma spp. in Egypt, an addition of 10% cellulose increased the survival of the entrapped
G. BIOCONTROL AND PLANT GROWTH PROMOTION
Compatibility with Other Biological Systems
conidia better than without cellulose (Shaban and El-Komy, 2001). A chitin fortified bioformulation of Trichoderma/Hypocrea species allowed increased accumulation of total phenols, peroxidase, polyphenoloxidase and phenylalanine ammonia lyase in treated tomato plants when challenged with R. solani (Solanki et al., 2011). Kandula et al. (2010) supplemented commercial Trichoderma and powder formulations with NPK which improved growth response but with no reduction in root disease symptoms of specific apple replant disease (Kandula et al., 2010). The addition of organic fertilizer enhanced the performance of T. harzianum SQR-T037 as compared to conidial suspension alone in controlling Fusarium wilt of cucumbers (Yang et al., 2011). The reason for improved performance is the sustained colonization of T. harzianum SQR-T037 in the rhizosphere soil (Yang et al., 2011). To increase biomass and number of colony forming unit/mL of T. asperellum, Wijesinghe et al. (2011) incubated the biocontrol agent in yeast waste residue medium. The population peaked at 144 h and the formulation was used to treat fruits infected with black rot disease caused by Thielaviopsis paradoxa. Growing in bentonite–vermiculite formulation also increased the colony forming units of T. harzianum after 8 weeks and provided higher melon shoot weight and higher resistance to Fusarium wilt disease (Martinez-Medina et al., 2009). Bernal-Vicente et al. (2009) found the most effective treatment against Fusarium wilt of melon was the solid formulation bentonite and superficial vermiculite of T. harzianum isolate T-78. Thangavelu et al. (2003) found that dried banana leaves is the best carrier material to support the growth of Trichoderma spp. isolated from the rhizosphere of banana from Tamil, India. The addition of jaggery (10% w/v) promoted increase of T. harzianum and prolonged the survival for more than 6 months in storage. Immobilizing wet biomass of Trichoderma viride in gluten matrix reduced the amount of biomass needed and generated 106–107 colony forming units g-1 soil in the second week (Cho and Lee, 1999).
COMPATIBILITY WITH OTHER BIOLOGICAL SYSTEMS A single strain of Trichoderma may not be effective in dealing with all diseases at all conditions. As explained by Cook (1993), biological control is highly disease specific and may require mixture of strains or other biological control agent for optimum disease control. De Jaeger et al. (2011) demonstrated the compatibility of the entrapped T. harzianum and Glomus in alginate beads. The presence of T. harzianum has no inhibitory effect but rather stimulated the spore production and fitness of Glomus. The combined application of Pseudomonas
529
fluorescens and T. viride in talc formulation resulted in significant control of sheath blight of rice caused by R. solani and comparable to the treatment of carbendazim (Mathivanan et al., 2005). Similarly Khan et al. (2004) found that the combination of T. harzianum and P. fluorescens decreased soil population of Fusarium wilt of chickpea. Gilardi et al. (2007) observed competitive effect of the combined application of T. viride and P. fluorescens. Treatments with T. harzianum and Pochonia chlamydosporia grown in sawdust:soil:5% molasses (15:5:1) and 20 parts carrier (fly ash: soil:5% molasses mixture (5:3:1) effectively controlled Fusarium wilt and root-knot nematode of pulses (Khan et al., 2011).
Modes of Delivery and Application The success of biological control of plant pathogens using Trichoderma does not rely solely on effective antagonists but also on the method of delivery or application on the seed, root and soil. In addition, all antagonists rely on their placement on the infection court to effect successful protection and control (Mathre et al., 1999). Timing of delivery and application is also crucial and Trichoderma is usually only effective as a preventative measure but can be integrated with other disease management options especially when the disease has already established. The following are examples of effective modes of delivery and application of Trichoderma focusing on seed treatment:
Seed Treatment Seed treatment or coating is the most effective method of application of Trichoderma into an agricultural system (Mathre et al., 1999). Trichoderma is delivered in the infection court (surface of seed coat) as protectant at planting. This method of delivery should limit growth of competitive microflora and provide conducive growth for the biocontrol agent. Seed treatment using seed dressing formulation, Pusa 5SD has been proven more effective than soil application formulations PusaBiogranule 6 (PBG 6) and PusaBiopellet 16G (PBP 16G) in managing wet root rot of mungbean caused by R. solani (Dubey et al., 2011).
Solid Matrix Priming Seed priming is the process in which the seeds are hydrated to allow metabolic process of germination to take place but not sprouting. Two priming systems are available. Solid matrix priming (SMP) was developed by J. Eastin, Kamterter, Inc. (Lincoln, NE) to enhance biocontrol of Trichoderma by regulating water levels in the seed. It allows effective colonization of seed surface before planting given the right pH and matrix material. Osmopriming makes use of aerated aqueous solutions of
G. BIOCONTROL AND PLANT GROWTH PROMOTION
530
39. ADVANCES IN FORMULATION OF TRICHODERMA FOR BIOCONTROL
salts or polyethylene glycols to generate osmotic potential in the primary solution. SMP is simple and more cost-effective than osmopriming (Harman and Taylor, 1988). Seed priming of three rhizosphere isolates of T. harzianum enhanced growth and induced resistance in sunflower against downy mildew caused by Plasmopara halstedii (Nagaraju et al., 2012).
Liquid Coating Liquid coating is a seed coating system which involves application of Trichoderma to the seed with an aqueous adhesive or binder (pelgel or polyox-N-10) and a particulate material (Agro-lig or muck soil) to optimize pH level including a bulking agent (Taylor et al., 1991). The Agro-lig is reported to have a physical and chemical characteristic which favor the growth of the fungus.
Double Coating A modification of liquid coating in which Trichoderma is applied directly to the seed coat followed by a particulate to form the second layer. The advantage of this system is that the biocontrol agent has access to seed exudates especially with seeds that have a low level of exudation (Harman, 1991). It is recommended to allow Trichoderma to colonize the seed surface at 100% relative humidity to improve its biocontrol efficacy (Taylor et al., 1991).
CONCLUSION AND FUTURE PROSPECTS The advances in Trichoderma formulation have f urther promoted biological control of plant pathogens for sustainable agriculture. Future research on the biological control system of Trichoderma should look into formulation suitable to control foliar and aerial pathogens considering its endophytic nature (Evans et al., 2003). There is a need to strengthen research-industry partnership to scale up production systems including large scale promotion of Trichoderma formulation in farmers’ fields particularly in the developing countries.
References Al-Taweil, H.I., Osman, M.B., Abdulhamid, A., Mohammad, N., Yussof, W.M.W., 2010. Comparison of different delivery system of Trichoderma and Bacillus as biofertilizer. Adv. Environ. Biol. 4, 31–33. Batta, Y.A., 2007. Control of postharvest diseases of fruit with an invert emulsion formulation of Trichoderma harzianum Rifai. Postharvest Biol. Technol. 43, 143–150. Bernal-Vicente, A., Ros, M., Pascual, J.A., 2009. Increased effectiveness of the Trichoderma harzianum isolate T-78 against Fusarium wilt on melon plants under nursery conditions. J. Sci. Food Agric. 89, 827–833.
Cho, C., Lee, W., 1999. Formulation of a biocontrol agent by entrapping biomass of Trichoderma viride in gluten matrix. J. Biosci. Bioeng. 87, 822–824. Churchill, B.W., 1982. Mass production of microorganisms for biological control. In: Charudattan, R., Walker, H.L. (Eds.), Biological Control of Weeds with Plant Pathogens, John Wiley & Sons, New York, pp. 139–156. Cook, R.J., 1993. The role of biological control in the 21st century. In: Lumsden, R.D., Vaughn, J.L. (Eds.), Pest Management: Biologically Based Technologies, American Chemical Society, Washington D.C., pp. 10–20. Cumagun, C.J.R., 2012. Managing plant diseases and promoting sustainability and productivity with Trichoderma. J. Agric Sci. Technol. 14, 699–714. Cumagun, C.J.R., Ilag, L.L., 1998. Parasitism of sclerotial bodies of Rhizoctonia solani Kuehn by Trichodema harzianum Rifai and Penicillium oxalicum Currie and Thom. Philipp. Phytopathol. 33, 17–26. Cumagun, C.J.R., Lapis, D.B., 1993. Note: practical approach in mass production of Trichoderma spp. as a means of biological control against sheath blight of rice. Philipp. Agric. 76, 251–257. De Jaeger, N., de la Providencia, I.E., Rouhier, H., Declerck, S., 2011. Coentrapment of Trichoderma harzianum and Glomus sp. within alginate beads: impact on the arbuscular mycorrhizal fungi life cycle. J. Appl. Microbiol. 111, 125–135. Dubey, S.C., Bhavani, R., Singh, B., 2011. Integration of soil application and seed treatment formulations of Trichoderma species for management of wet root rot of mungbean caused by Rhizoctonia solani. Pest Manage. Sci. 67, 1163–1168. Evans, H.C., Holmes, K.A., Thomas, S.E., 2003. Endophytes and mycoparasites associated with an indigenous forest tree, Theobroma gileri in Ecuador and a preliminary assessment of their potential as biocontrol agents of cocoa diseases. Mycol. Prog. 2, 149–160. Gharsallaoui, A., Roudaut, G., Chambin, O., Voilley, A., Saurel, R., 2007. Applications of spray-drying in microencapsulation of food ingredients: an overview. Food Res. Int. 40, 1107–1121. Gilardi, G., Garibaldi, A., Gullino, M.L., 2007. Effect of antagonistic Fusarium spp. and of different commercial biofungicide formulations on Fusarium wilt of lettuce. Phytoparasitica 35, 457–465. Harman, G.E., 1991. Seed treatments for biological control of plant disease. Crop Prot. 10, 166–171. Harman, G.E., Jin, X., Stasz, T.E., Peruzzotti, G., Leopold, A.C., Taylor, A.G., 1991. Production of conidial biomass of Trichoderma harzianum for biological control. Biol. Control 1, 23–28. Harman, G.E., Taylor, A.G., 1988. Improved seedling performance by integration of biological control agents at favourable pH levels with solid matrix priming. Phytopathology 78, 520–525. Howell, C.R., 2003. Mechanisms employed by Trichoderma species in the biological control of plant diseases: the history and evolution of current concepts. Plant Dis. 87, 4–10. Jin, X., Custis, D., 2011. Microencapsulating aerial conidia of Trichoderma harzianum through spray drying at elevated temperatures. Biol. Control 56, 202–208. Jin, X., Harman, G.E., Taylor, A.G., 1991. Conidial biomass and desiccation tolerance of Trichoderma harzianum produced at different medium water potentials. Biol. Control 1, 237–243. Jin, X., Hayes, K.C., Harman, G.E., 1992. Principles in the development of biological control systems employing Trichoderma species against soil-born plant pathogenic fungi. In: Lantham, G.F. (Ed.), Frontiers in Industrial Mycology, Chapman & Hall Inc., New York, pp. 174–195. John, R.P., Tyagi, R.D., Brar, S.K., Surampalli, R.Y., Prévost, D., 2011. Bio-encapsulation of microbial cells for targeted agricultural delivery. Crit. Rev. Biotechnol. 31, 211–226. Kandula, D.R.W., Jones, E.E., Horner, I.J., Stewart, A., 2010. The effect of Trichoderma bio-inoculants on specific apple replant disease (SARD) symptoms in apple rootstocks in New Zealand. Australas. Plant Pathol. 39, 312–318.
G. BIOCONTROL AND PLANT GROWTH PROMOTION
REFERENCES
Khan, M.R., Khan, S.M., Mohiddin, F.A., 2004. Biological control of Fusarium wilt of chickpea through seed treatment with the commercial formulation of Trichoderma harzianum and/or Pseudomonas fluorescens. Phytopathol. Mediterr. 43, 20–25. Khan, M.R., Majid, S., Mohidin, F.A., Khan, N., 2011. A new bioprocess to produce low cost powder formulations of biocontrol bacteria and fungi to control Fusarial wilt and root-knot nematode of pulses. Biol. Control 59, 130–140. Kolombet, L.V., Zhigletsova, S.K., Kosareva, N.I., Bystrova, E.V., Derbyshev, V.V., Krasnova, S.P., Schisler, D., 2008. Development of an extended shelf-life, liquid formulation of the biofungicide Trichoderma asperellum. World J. Microbiol. Biotechnol. 24, 123–131. Martínez-Medina, A., Roldán, A., Pascual, J.A., 2009. Performance of a Trichoderma harzianum bentonite-vermiculite formulation against Fusarium wilt in seedling nursery melon plants. Hortic. Sci. 44, 2025–2027. Mathivanan, N., Prabavathy, V.R., Vijayanandraj, V.R., 2005. Application of talc formulations of Pseudomonas fluorescens Migula and Trichoderma viride pers. ex S.F. gray decrease the sheath blight disease and enhance the plant growth and yield in rice. J. Phytopathol. 153, 697–701. Mathre, D.E., Cook, R.J., Callan, N.W., 1999. From discovery to use: traversing the world of commercializing biocontrol agents for plant disease control. Plant Dis. 83, 972–983. Morgan, C.A., Herman, N., White, P.A., Vesey, G., 2006. Preservation of microorganisms by drying: a review. J. Microbiol. Methods 66, 183–193. Muñoz-Celaya, A.L., Ortiz-García, M., Vernon-Carter, E.J., JaureguiRincón, J., Galindo, E., Serrano-Carreón, L., 2012. Spray-drying microencapsulation of Trichoderma harzianum conidia in carbohydrate polymers matrices. Carbohydr. Polym. 88, 1141–1148. Nagaraju, A., Sudisha, J., Murthy, S.M., Ito, S.-, 2012. Seed priming with Trichoderma harzianum isolates enhances plant growth and induces resistance against Plasmopara halstedii, an incitant of sunflower downy mildew disease. Australas. Plant Pathol. 41, 609–620. Nelson, E.B., Harman, G.E., Nash, G.T., 1988. Enhancement of Trichoderma-induced biological control of Pythium seed rot and pre- emergence damping-off of peas. Soil Biol. Biochem. 20, 145–150. O’Riordan, K., Andrews, D., Buckle, K., Conway, P., 2001. Evaluation of microencapsulation of a Bifido bacterium strain with starch as an approach to prolonging viability during storage. J. Appl. Microbiol. 91, 1059–1066.
531
Papavizas, G.C., 1985. Trichoderma and Gliocladium: biology, ecology, and potential for biocontrol. Annu. Rev. Phytopathol. 82, 178–184. Papavizas, G.C., Dunn, M.T., Lewis, J.A., Beagle-Ristaino, J., 1984. Liquid fermentation technology for experimental production of biocontrol fungi. Phytopathology 74, 1171–1175. Shaban, G.M., El-Komy, H.M.A., 2001. Survival and proliferation of alginate encapsulated Trichoderma spp. in Egyptian soil in comparison with allyl alcohol soil fumigation. Mycopathology 151, 139–146. Solanki, M.K., Singh, N., Singh, R.K., Singh, P., Srivastava, A.K., Kumar, S., Kashyap, P.L., Arora, D.K., 2011. Plant defense activation and management of tomato root rot by a chitin-fortified Trichoderma/ Hypocrea formulation. Phytoparasitica 39, 471–481. Sriram, S., Palanna, K.B., Ramanujam, B., 2010. Effect of chitin on the shelf-life of Trichoderma harzianum in talc formulation. Indian J. Agric. Sci. 80, 930–932. Sriram, S., Roopa, K.P., Savitha, M.J., 2011. Extended shelf-life of liquid fermentation derived talc formulations of Trichoderma harzianum with the addition of glycerol in the production medium. Crop Prot. 30, 1334–1339. Taylor, A.G., Min, T.-, Harman, G.E., Jin, X., 1991. Liquid coating formulation for the application of biological seed treatments of Trichoderma harzianum. Biol. Control 1, 16–22. Thangavelu, R., Palaniswami, A., Velazhahan, R., 2003. Mass production of Trichoderma harzianum for managing Fusarium wilt of banana. Agric. Ecosys. Environ. 103, 259–263. Whipps, J.M., 1997. Developments in the biological control of soilborne plant pathogens. In: Callow, J.A. (Ed.), Advances in Botanical Research Incorporating Advances in Plant Pathology, vol. 26. pp. 1–123. Wijesinghe, C.J., Wijeratnam, R.S.W., Samarasekara, J.K.R.R., Wijesundera, R.L.C., 2011. Development of a formulation of Trichoderma asperellum to control black rot disease on pineapple caused by (Thielaviopsis paradoxa). Crop Prot. 30, 300–306. Yang, X., Chen, L., Yong, X., Shen, Q., 2011. Formulations can affect rhizosphere colonization and biocontrol efficiency of Trichoderma harzianum SQR-T037 against Fusarium wilt of cucumbers. Biol. Fertil. Soils 47, 239–248.
G. BIOCONTROL AND PLANT GROWTH PROMOTION