Trichoderma for Biocontrol of Plant Pathogens:
From Basic Research to Commercialized Products
Gary E. Harman
Departments of Horticultural Science and of Plant Pathology
Cornell University NYSAES
Geneva, NY 14456
Trichoderma spp. are fungi that are present in substantial numbers in nearly all agricultural soils and in other environments such as decaying wood. Among their other activities, they grow tropically toward hyphae of other fungi, coil about them in a lectin-mediated reaction, and degrade cell walls of the target fungi. This process (mycoparastitism) limits growth and activity of plant pathogenic fungi. In addition to, or sometimes in conjunction with mycoparasitism, individual strains may produce antibiotics. However, numbers and the physiological attributes of wild strains are not sufficient for highly effective control of plant diseases. The antifungal abilities of these beneficial microbes have been known since the 1930s, and there have been extensive efforts to use them for plant disease control since then. However, they are only now beginning to be used commercially.
In my laboratory, we have been developing technology for the biocontrol of plant pathogens for about two decades. This paper will briefly discuss this effort, and the progress from basic research to products now available commercially.
The basic requirements of successful biocontrol.
Our early work demonstrated that Trichoderma spp. did indeed have the ability to control plant diseases. However, the level of efficacy and the reliability of simple approaches to biocontrol gave results that were substantially less than that of commercial fungicides. Over several years, we have concluded that biocontrol systems must be developed if effective results are to be obtained. There are three basic components to biocontrol systems. These are as follows:
A highly effective biocontrol strain or other material must be obtained or produced. Such strains must not only have appropriate mechanisms for biocontrol such as those just described, but it should also (a) be able to compete and persist in the environment in which it must operate, and (b) ideally, be able to colonize and proliferate on existing and newly formed plant parts at times well after application. In about 1982, we determined that our existing natural strains were insufficiently effective, so we decided to attempt to use protoplast fusion to combine useful derivatives from two strains into single more broadly effective strains. Protoplast fusion introduced great genetic diversity into progeny strains, and a very small percentage were of improved biocontrol ability over the wild strains. Strain 1295-22 (the 22nd progeny strain from a fusion of strains T12 and T95) exhibited substantial ability relative to either parental strain. In particular, it is extremely rhizosphere competent, i.e. it can colonize and protect the entire root system for the life of the crop. Moreover, it is effective against a wide range of plant pathogenic fungi including Pythium spp., Rhizoctonia solani , Fusarium spp., Botrytis cinerea, Sclerotium rolfsii, and Sclerotinia homoeocarpa.
However, development of biocontrol systems has only begun when an effective strain is identified. In our research, we have used only T. harzianum strain 1295-22 and T. virens strain 41 in our research for the past decade. The other components of biocontrol systems are of critical importance and require a great deal of detailed research.
Inexpensive production and formulation of the biocontrol agent or other material in question must be developed. The production process must result in biomass with excellent shelf life even under adverse storage conditions. In many respects, the requirements for production of products for agricultural use are more difficult to meet than those required for pharmaceutical products. If agricultural materials are to be successful, they must be very inexpensive, able to be produced in large quantities, and maintain good viability without specialized storage systems.
With Trichoderma spp. we have been studying the physiology of desiccation tolerance, the mechanisms of shelf life, and processes for large-scale production of these fungi. This research has been completed on the laboratory scale at Cornell and now has been transferred to TGT Inc, Geneva, NY for implementation and large-scale production.
In my opinion, the largest technological impediment to the use of biocontrol agents is the lack of both technology and facilities for biologically- and economically-effective production of biocontrol organisms. Nearly no public-sector research or funding is directed towards research on methods of production of biocontrol organisms.
Delivery and application methods that permit the full expression of the biocontrol agent. Delivery systems must ensure that biocontrol agents will grow well and achieve their purpose. In our experience, delivery and application processes must be developed on a crop by crop and application by application basis. No general solutions exist, and so biocontrol systems must be developed for each crop.
An analogy may be helpful to understand these requirements. We would achieve very poor results if we took even an excellent cultivar of wheat and did not understand the parameters of the agricultural system into which it is to be introduced. Imagine the results that would be obtained if we had wheat seed that had only ten percent germinable seeds, and then sowed the wheat into a deep woods. No wheat crop would result. Instead, we must have very good seed, produced and stored under the best conditions to provide high vigor, germinability, and freedom from contaminating weed seeds. Next, we plant the seed in a good seed bed to ensure that it has excellent conditions for germination. We provide adequate levels of nutrients in the form of fertilizers and control weeds through application of herbicides and tillage practices. In many cases, biocontrol researchers have attempted to use our biocontrol agents in a manner analogous to the sowing of wheat in the woods. We have not properly considered the biological and physiological requirements of our biological organisms, and the result has been, like wheat in the woods, at best, mixed, and often quite poor.
In our research at Cornell we have sought to produce effective and useful methods and concepts for the delivery of biocontrol agents. Some effective methods that have been developed for T. harzianum are summarized below:
- It is effective as a seed treatment with and without fungicides. It is very important to use the organism properly and to have appropriate expectations. It, or any biocontrol organism we know of, will be unable to protect seeds as well as chemical fungicides. However, it colonizes roots, increases root mass and health, and consequently frequently provides yield increases, which chemical fungicides applied at reasonable rates cannot do. An effective method of use is to use the biocontrol fungus in conjunction with chemical fungicides. The chemicals provide good short-term seed protection, and the biocontrol fungus provides long-term root protection. As a consequence, yields frequently are increased over use of the chemical alone.
It can also be used as an additive to greenhouse potting mixes. It can both reduce pesticide use in the greenhouse by limiting root-attacking diseases and protect transplants in the field by virtue of its ability to colonize roots.
When added to turf, the granular formulation results in establishment of the organism on roots and suppresses S. homoeocarpa (dollar spot), Pythium spp (Pythium blight) and R. solani (brown patch). However, the pathogens may survive in sufficient numbers to cause disease. Once the pathogens are established on the foliage, the soil-applied biocontrol agent no longer can protect the plant. This granular formulation can therefore result in disease reduction, but it must be used in conjunction with compatible chemical fungicides. A spray application of the biocontrol agent for control of foliar phases of the diseases is under development and has provided excellent results in field tests.
The organism is also highly effective when applied to blossoms or fruits for control of B. cinerea. Even low levels of the organism applied to strawberry blossoms by bee delivery or by sprays of liquid formulations are effective. For maximum control of the Botrytis bunch rot of grape, this initial application needs to be augmented by sprays as fruits mature, and addition of iprodione as a tank mix to this late application appears to have synergistic activity over either the biocontrol agent or the chemical fungicide alone.
Commercialization of biocontrol systems.
Once an effective biocontrol system has been identified, it can and should be commercialized. As a biocontrol researcher, it has become clear that solving the technological problems is perhaps the easiest part of developing biocontrol agents and systems. Legal and commercial considerations are also extremely important. Over the past 15 years, we filed a series of patents through the Cornell Research Foundation that covered the relevant technology. Protection of intellectual property was essential to further development. If the technology cannot be protected, few companies will be interested in developing it further.
By 1989, we had demonstrated that strain 1295-22 had several desirable features and also that a seed treatment using the process of solid matrix priming permitted this strain to be a highly effective seed protectant. At that time, a large national company was interested in adding an agricultural biotechnology line to its market mix and acquired the technology from Cornell University. They immediately began efforts to develop large-scale production systems using their liquid fermentation facilities and to register the product. For a variety of reasons, after two years, the company stopped development of this strain, along with a number of other larger ventures in biotechnology. In 1992, the company returned the entire technology package, including full EPA registrations and the trademark F-Stopª;, to Cornell. In the meantime, we had developed other technologies that were patented through CRF.
Consequntly, there was a body of knowledge and intellectual property available. Thomas Stasz, a PhD plant pathologist experienced in Trichoderma research and a J.D. degree recipient from Syracuse University, Rustin Howard, a Cornell MBA with good agricultural and startup biotechnology business experience, and I formed TGT Inc. This company acquired rights to the technology from my lab, and is designed to develop, produce, and market biocontrol products for agriculture.
TGT has developed large-scale production processes and facilities that now permit it to produce several hundred tons of biocontrol products each year. It currently is marketing several products primarily through distributors such as the Wilbur-Ellis Co, Seedway and Agway, AgChemServices, and Penn State Seed Co. The products are Bio-Trek 22G for application to golf course turf, T-22 planter box seed treatment for corn, soybean, Phaesolus bean, and cotton, and T-22 granular and T-22 drench biological fungicides which are two different formulations for treatment of greenhouse potting soils. Sales primarily are within the USA, but some products are being sold in Europe and Japan. Other products, including a spray material for control of Botrytis (gray mold) on fruits and vegetables and a spray formulation for turf, are in final development.
The company is succeeding because of its technical base, but critically, because of the business and legal abilities of Rustin Howard and Thomas Stasz. Biocontrol systems, once they are developed can be commercialized, but only if adequate business, legal, and financial expertise, and large-scale production techniques and facilities are available or can be constructed.
We are very interested in developing new methods of biocontrol. In particular, we have identified proteins, and the genes encoding them, from Trichoderma and other microbes. These proteins degrade chitin, which is a structural component of pathogenic fungi and herbivorous insects. We are investigating large-scale production of the enzymes themselves, and other uses of genes and gene products for the control of plant pests. This work is proceeding well in cooperation with researchers at Cornell and elsewhere and should represent a next generation of biocontrol products.
Jin, X., C. K. Hayes, and G. E. Harman. 1992. Principles in the development of biological control systems employing Trichoderma species against soil-borne plant pathogenic fungi. p. 174-195 In Leatham, G.F. (ed) Symposium on Industrial Mycology, Mycological Soc. Am., Brock/Springer Series in Contemporary Bioscience.
Harman, G. E. and T. E. Stasz. 1989. Combining effective strains of Trichoderma harzianum and solid matrix priming to provide improved biological seed treatment systems. Plant Disease 72:631-637.
Harman, G.E. and Hayes, C. K. 1993. The genetic nature and biocontrol ability of progeny from protoplast fusion in Trichoderma. In Chet, I. (ed). Biotechnology in Plant Protection. J. Wiley -Liss. p. 237-255.
Hayes, C. K., Peterbauer, C., Tronsmo, A., Klemsdal, S., and Harman, G. E. 1993. Antifungal chitinolytic enzymes from Trichoderma harzianum and Gliocladium virens purification, characterization, biological activity and molecular cloning. In Muzzarelli, R. A. A. (ed). Chitin Enzymology. Eur. Chitin Soc., Ancona, Italy. p.383-392.
Stasz, T. E., G. E. Harman, and N. F. Weeden. 1988. Protoplast preparation and fusion in two biocontrol strains of Trichoderma harzianum. Mycologia 80:141-150.
Smith, V. L., W. F. Wilcox, and G. E. Harman. 1990. Potential for biological control of Phytophthora root and crown rots of apple by Trichoderma and Gliocladium spp. Phytopathology 70:880-885.
Weindling, R. 1934. Studies on a lethal principle effective in the parasitic action of Trichoderma lignorum on Rhizoctonia solani and other soil fungi. Phytopathology 24: 1153-1179.
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