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INTRODUCTION
Plant disease continues to threaten crop production in modern agriculture and plays a direct role in the destruction of natural resources in agriculture. In particular, soil borne plant pathogens especially fungi cause important losses and are most aggressive. Some of the important soil borne plant pathogens such as Pythium, Phytopthora, Botrytis, Rhizoctonia, Fusarium and Meloidogyne has spread very fast and have detrimental effects on crops of economic importance. With the advent of chemical compounds it was thought that a permanent and reliable solution of soil borne plant pathogens have been achieved but it was realized that pesticide application is not safe to the environment as the toxicants cause environmental pollution and has harmful effects on human beings. Unfortunately to control a target soil borne plant pathogen with a pesticide, over 100 species of non target organisms are adversely affected (Alabouvette and Couteadier, 1992). Despite realization of adverse effects of chemical pesticides on plants, animals and environment they are being applied indiscriminately to control soil borne plant pathogens. Moreover, some efficacious pesticides have been banned for use in agriculture (Table 1, 2). Hence, to reduce the use or dose of chemicals, one possibility is to utilize the disease suppressing activity of certain microorganisms which should be highly antagonistic against the targeted soil borne plant pathogens. Such microorganisms are commonly referred to as biological control (biocontrol) agents and their commercial formulations as biopesticides.
| Table 1: | List of pesticides banned in India |
| Source: G:\ NESAC-Integrated Plant Protection Banned Pesticides.htm | |
| Table 2: | Pesticides refused registration |
| Source: G:\NESAC-Integrated Plant Protection Banned Pesticides.htm | |
Recent researches on biological control have been conducted with greater systemic approach and practical utility. Various microorganisms viz., fungi, bacteria, mycorrhizae etc. have been tested for their ability to suppress plant pathogens. As most of the soil borne plant pathogens are fungi, biocontrol by fungi has been attempted extensively (Henis et al., 1979; Baker, 1987; Suarez et al., 2004).
Trichoderma species are among the most frequently isolated soil fungi and present in plant root ecosystems (Harman et al., 2004). The fungi are opportunistic, avirulent plant symbionts and function as parasites and antagonists of many phytopathogenic fungi, thus protecting plants from diseases. So far Trichoderma sp. are among the most studied fungal biocontrol agents and commercially marketed as a potent biopesticides, biofertilizer and also used in soil amendments (Harman, 2000; Harman et al., 2004). Depending upon the strain the use of Trichoderma in agriculture can provide numerous advantages: (1) Colonization of the rhizosphere (rhizosphere competence) allowing rapid establishment within the stable microbial communities in the rhizosphere, (2) control of pathogenic and competitive/deleterious microflora by using a variety of mechanism, (3)iImproving of the plant health and (4) stimulation of root growth (Harman et al., 2004).
Systematics of Trichoderma: Trichoderma is classified as under:
History of Trichoderma: The genus Trichoderma was described in 1791 in Germany and four species were originally described. In 1927 Gilman and Abbott recognized four species. These species were distinguished on the basis of color and shape of their conidia and on the colony appearance. Most species were identified as T. lignorum (= T. viride) because of its globose conidia or as T. koningii because of its oblong conidia. The potential for use of Trichoderma sp. as biocontrol agents was suggested more than 75 years ago by Weindling (1932) who was the first to demonstrate the parasitic activity of members of this genus to pathogens such as R. solani. Trichoderma is perhaps the best known mycoparasite suggested as a biocontrol agent against many soil borne plant pathogens (Table 3).
Factors influencing biocontrol agent: Rhizosphere competence: Rhizosphere competence is the ability to colonize and grow in association with plant roots. This is possibly the most important factor in considering the potential of any given isolate for biological control because it is a measure of the ability of an isolate to survive in the soil.
Temperature: It is important to understand the cycle of the pathogen in order to determine the best time for application of a biocontrol agent. Potential biocontrol strains need to cover the thermal spectrum of the target organism, e.g., Botrytis has a very wide spectrum. Crinipellis seems to grow at a higher optimum temperature than T. stromaticum thus limiting the ability of the Trichoderma to control the plant parasite.
Moisture: Moisture can limit the ability of a biocontrol agent to colonize the habitat. Lack of moisture can limit the ability of the biocontrol spores to germinate. Moisture controls availability of nutrients essential to growth of the biocontrol agent. Applications can be timed to periods when there will be enough moisture to stimulate spore germination.
Nutrients: Trichoderma conidia are very small. They must take up water and swell before germination. That process requires the presence of nutrients (carbon, nitrogen). Hyphae and chlamydospores are less sensitive to soil fungistasis.
Mechanism of disease suppression: The activities of biocontrol agents mainly depends on different physicochemical environmental conditions to which they are subjected. Understanding both the genetic diversity of strains within Trichoderma species and their mechanisms of biocontrol will lead to improved application of the different strains as biocontrol agents. These mechanisms are complex and what has been defined as biocontrol is the final result of different mechanisms acting synergistically to achieve disease control (Howell, 2003). Biocontrol results either from competition for nutrients and space or as a result of the ability of Trichoderma biocontrol agents to produce and/or resist metabolites that either impede spore germination (fungistatis), kill the cells (antibiosis) or modify the rhizosphere, e.g., by acidifying the soil, so that pathogens cannot grow. Biocontrol may also result from a direct interaction between the pathogen itself and the biocontrol agent, as in mycoparasitism, which involves physical contact and synthesis of hydrolytic enzymes, toxic compounds and/or antibiotics that act synergistically with the enzymes. Trichoderma sp. can even exert positive effects on plants with an increase in plant growth (mineralization) and the stimulation of plant defense mechanisms.
| Table 3: | Various soil borne plant pathogens controlled by Trichoderma species |
Mechanism of disease suppression may be due to competition, antibiosis or mycoparasitism.
Competition
Fungistatis: The nature of competition is fungistatic (inhibitor). Good antagonists are usually able to overcome the fungistatic effect of soil that results from the presence of metabolites produced by other species including plants and to survive under very extreme competitive conditions.
Trichoderma strains grow rapidly when inoculated in the soil because they are naturally resistant to many toxic compounds including herbicides, fungicides and pesticides such as DDT and phenolic compounds (Chet et al., 1997). Resistance to toxic compounds may be due to the presence of ABC transport systems in Trichoderma strains (Harman et al., 2004). Trichoderma strains are very efficient in controlling several phytopathogens such as R. solani, P. ultimum and S. rolfsii when alternated with methyl bromide, benomyl, captan or other chemicals due to the presence of ABC transport system (Vyas and Vyas, 1995).
Competition for nutrients: Starvation is the most common cause of death for microorganisms, so that competition for limiting nutrients results in biological control of fungal phytopathogens (Chet et al., 1997). For instance, in most filamentous fungi iron uptake is essential for viability (Eisendle et al., 2004) and under iron starvation most fungi excrete low molecular weight ferric iron specific chelators termed as siderophores to mobilize environmental iron (Eisendle et al., 2004). For this reason, soil composition influences the biocontrol effectiveness of Pythium by Trichoderma according to iron availability. Some Trichoderma biological agents produce highly efficient siderophores that chelate iron and stop the growth of other fungi (Chet and Inbar, 1994). One of the most sensitive stages for nutrient competition in the life cycle of Fusarium is chlamydospore germination (Baker, 1986). In soil the chlamydospores of F. oxysporum, need nutrition to maintain a germination rate of 20-30%. The germination may decrease due to sharing of nutrients by other microorganisms. Root exudates are major source of nutrients in soil which are excreted from the root tips. Thus, colonization in the rhizosphere of root tip by an antagonist might reduce infection by Fusarium-like pathotypes (Cook and Baker, 1983). In addition, T. harzianum T35 controls F. oxysporum by competing for both rhizosphere colonization and nutrients with biocontrol becoming more effective as the nutrient concentration decreases (Alabouvette and Couteadier, 1992). Competition for carbon has also been involved in the determination of the antagonism expressed by different strains of Trichoderma sp. against several plant pathogens, especially F. oxysporum (Sivan and Chet, 1989). T. viride controlled Chondrostereum purpureum, the silver leaf pathogen of plum trees due to competition exerted by the former (Corke and Hunter, 1979). Competition has proved to be particularly important for the biocontrol of phytopathogens such as Botrytis cinerea, the main pathogenic agent during the pre and post-harvest in many countries (Latorre et al., 2001). The advantage of using Trichoderma to control Botrytis cinerea is the coordination of several mechanisms, the most important is nutrient competition, since Botrytis cinerea is particularly sensitive to the lack of nutrients.
Trichoderma has a superior capacity to mobilize and take up soil nutrients compared to other organisms. The efficient use of available nutrients is based on the ability of Trichoderma to obtain ATP from the metabolism of different sugars, such as those derived from polymers wide spread in fungal environments: cellulose, glucan and chitin among others, all of them rendering glucose (Chet et al., 1997). While, the role of the glucose transport systems remains to be discovered, its efficiency may be crucial in competition (Delgado-Jarana et al., 2003) as supported by the isolation of a high affinity glucose transporter, Gtt 1, in T. harzianum CECT 2413. This strain is present in environments very poor in nutrients and it relies on extracellular hydrolases for survival. The Gtt 1 is only expressed at very low glucose concentrations, i.e., when sugar transport is expected to be limiting in nutrient competition (Delgado-Jarana et al., 2003).
Antibiosis: Antibiosis is required as one of the most important attribute in deciding the competitive saprophytic ability of Trichoderma sp. Our first knowledge of toxic metabolite production by species of Trichoderma was largely due to Weindling (1934, 1937) who showed the production of an antifungal metabolite by T. lignorum, later stated to be G. frimbiatum. The metabolite was named as gliotoxin. Antibiosis occurs during interactions involving low molecular weight diffusible compounds or antibiotics produced by Trichoderma strains that inhibit the growth of other microorganisms. Most Trichoderma strains produce volatile and non volatile metabolites (Table 4) that impede colonization by antagonized microorganisms; among these metabolites, the production of harzianic acid, alamethicins, tricholin, peptaibols, antibiotics, 6-penthyl-a-pyrone, massoilactone, viridin, gliovirin, glisoprenins, heptelidic acid and others have been described (Vey et al., 2001). In some cases, antibiotic production correlates with biocontrol ability and purified antibiotics mimic the effect of the whole agent. Volatile substances from Trichoderma sp. inhibited the mycelial growth of Macrophomina phaseolina by 22-51% (Angappan, 1992). The volatile antibiotics of T. harzianum and T. atroviride significantly decreased the growth of canker pathogen fungi of poplar, Cytospora chrysosperma and Dothiorella gregaria (Gao et al., 2001). Non-volatile metabolites in the culture filtrate of Trichoderma sp. inhibited the linear growth of pathogens (Deshmukh and Pant, 1992). Dwivedi (1992) reported that culture filtrate of T. harzianum inhibited the growth of F. solani and F. longipus by 60 and 64%, respectively.
Mycoparasitism: Mycoparasitism, the direct attack of one fungus on another, is a very complex process that involves sequential events including recognition, attack and subsequent penetration and killing of the host.
| Table 4: | Antibiotics or antibiotics-like effectors produced by Trichoderma species |
Trichoderma sp. may exert direct biocontrol by parasitizing a range of fungi detecting other fungi and growing towards them. The remote sensing is partially due to the sequential expression of cell wall degrading enzymes, mostly chitinases, glucanases and proteases (Harman et al., 2004). Trichoderma attaches to the pathogen with cell wall carbohydrates that bind to pathogen lectins. Once Trichoderma is attached, it coils around the pathogen and forms the appresoria. The following step consists of the production of cell wall degrading enzymes and peptaibols (Howell, 2003) which facilitate both the entry of Trichoderma hypha into the lumen of the parasitized fungus and the assimilation of the cell wall content. Trichoderma sp. react violently with hyphae of the Fusarium species. The hyphae of Trichoderma sp. when near to pathogen induce morphological deformalities in the host hyphae. Many a time bursting of hyphae and vacoulation has been observed (Komatsu, 1968; Gao et al., 2001). In addition, granulation, coagulation, disintegration and finally lysis of the pathogen occurs (Lim and The, 1990; Elad et al., 1983; Nigam et al., 1997; Gao et al., 2001). In vitro studies have revealed that purified endochitinase, chitobiosidase, n-acetyl-b-glucosidase and glucan 1,3-β-glucosidase and combinations thereof, greatly suppressed the spore germination and germ tube elongation in nine different fungal species (Lorito et al., 1993, 1994a, b; Di Pietro et al., 1993). T. harzianum TM transformants overexpressing chit36 chitinase inhibited F. oxysporum and S. rolfsii more strongly than the wild type. Moreover, culture filtrates inhibited the germination of B. cinerea almost completely (Viterbo et al., 2001).
Stimulation of host defence response: The ability of Trichoderma strains to protect plants against root pathogens has long been attributed to an antagonistic effect against the invasive pathogen (Chet et al., 1997). However, these root fungus associations also stimulate plant defensive mechanisms (Howell et al., 2000; Hanson and Howell, 2004). Strains of Trichoderma added to the rhizosphere protect plants against numerous classes of pathogens, e.g., those that produce aerial infections, including viral, bacterial and fungal pathogens, which point to the induction of resistance mechanisms similar to the Hypersensitive Response (HR), Systemic Acquired Resistance (SAR) and Induced Systemic Resistance (ISR) in plants (Harman et al., 2004). At a molecular level, resistance results in an increase in the concentration of metabolites and enzymes related to defensive mechanisms such as the enzymes Phenyl Alanine ammonia Lyase (PAL) and Chalcone Synthase (CHS), involved in the biosynthesis of phytoalexins (HR response), chitinases and glucanases. These comprise pathogenesis related proteins (SAR response) and enzymes involved in the response to oxidative stress. The addition to Trichoderma metabolites that may act as elicitors of plant resistance or the expression in transgenic plants of genes whose products act as elicitors, also results in the synthesis of phytoalexins, PR proteins and other compounds and in an increase in resistance against several plant pathogens, including fungi and bacteria (De Las Mercedes et al., 2001; Elad et al., 2000) as well as resistance to hostile abiotic conditions (Harman et al., 2004). Barley expressing Trichoderma atroviride endochitinase Ech 42 showed increased resistance towards Fusarium infection.
Cotton seedlings treated with efficient strain of T. virens had higher levels of defense related compounds such as terpenoids and peroxidase activity in the root (Howell et al., 2000). An ethylene-inducing xylanase produced by T. viride (Dean and Anderson, 1991) elicited the production of phytoalexin reversatrol in grapevine cells (Calderon et al., 1993). Hanson and Howell (2004) reported that culture filtrates from a strain of T. virens stimulated synthesis of terpenoid in cotton and the elicitors were presumably proteins or glycoproteins.
Plant growth promotion by Trichoderma species: Root colonization by Trichoderma strains frequently enhances root growth and development, crop productivity, resistance to abiotic stresses and the uptake and use of nutrients (Arora et al., 1992) (Table 5). Crop productivity in fields can increase upto 300% after the addition of T. hamatum or T. koningii. The experiments carried out in green houses with seed treatment with Trichoderma spores have shown significant greater yield (Chet et al.,1997). Equal degree of yield enhancement was observed when plant seeds were separated from Trichoderma by a cellophane membrane. This indicates that Trichoderma produces growth factors that enhanced the rate of seed germination, plant growth and yield (Benitez et al., 1998). Zimand et al. (1996) reported that T. harzianum T39 besides having inhibitory effect on the conidial germination and germ tube elongation of Botrytis cinerea, also reduced the production and activity of pathogen secreted pectolytic enzymes three days after inoculation. Reduced activities of pectolytic enzymes may increase the accumulation of pectic enzyme products, i.e., oligogalacturonides. These sugars can elicit the host plant (bean) defence mechanisms, thus checking the disease development. Activity of biocontrol agents could also reduce the concentration of substances in soil that are inhibitory to plant growth (Windham et al., 1986). Thus, the plant growth promotion may be due to production of plant hormones or increased uptake of nutrients by the plant (Chet et al., 1993); control of one or more sub potential pathogens (Baker, 1986) and/or strengthening plant’s own defense mechanism (Zimand et al., 1996).
| Table 5: | Percent yield increase of various crops by the application of Trichoderma sp. |
| Table 6: | Commercial formulations of Trichoderma species available in India |
Commercial formulations: For field application of a bioagent, an inert immobilizing substrate is essentially required which could carry maximum number of propagules of the biocontrol agent with minimum volume and necessarily maintain integrity of the organism. Various carriers viz., peat, seeds, meals, kernels, husks, brans, bagasse, FYM, cowdung, cake, compost, oilcakes, wood bark, vermiculite, sand, clay etc. have been tested to prepare commercial formulations of Trichoderma (Table 6).
ACKNOWLEDGMENTS
The author wish to dedicate this paper to the beloved Boba who breathed her last on 30 June, 2006. She was a great source of inspiration and strength for me. May her soul rest in the best place in heaven! Aameen.