Trichoderma spp. are free-living fungi that are common in soil and root ecosystems. Recent discoveries show that they are opportunistic, avirulent plant symbionts, as well as being parasites of other fungi. At least some strains establish robust and long-lasting colonizations of root surfaces and penetrate into the epidermis and a few cells below this level. They produce or release a variety of compounds that induce localized or systemic resistance responses, and this explains their lack of pathogenicity to plants. These root-microorganism associations cause substantial changes to the plant proteome and metabolism. Plants are protected from numerous classes of plant pathogen by responses that are similar to systemic acquired resistance and rhizobacteria-induced systemic resistance. Root colonization by Trichoderma spp. also frequently enhances root growth and development, crop productivity, resistance to abiotic stresses and the uptake and use of nutrients.

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Trichoderma species--opportunistic, avirulent plant symbionts.

The loss of organic material from the roots provides the energy for the development of active microbial populations in the rhizosphere around the root. Generally, saproptrophs or biotrophs such as mycorrhizal fungi grow in the rhizosphere in response to this carbon loss, but plant pathogens may also develop and infect a susceptible host, resulting in disease. This review examines the microbial interactions that can take place in the rhizosphere and that are involved in biological disease control. The interactions of bacteria used as biocontrol agents of bacterial and fungal plant pathogens, and fungi used as biocontrol agents of protozoan, bacterial and fungal plant pathogens are considered. Whenever possible, modes of action involved in each type of interaction are assessed with particular emphasis on antibiosis, competition, parasitism, and induced resistance. The significance of plant growth promotion and rhizosphere competence in biocontrol is also considered. Multiple microbial interactions involving bacteria and fungi in the rhizosphere are shown to provide enhanced biocontrol in many cases in comparison with biocontrol agents used singly. The extreme complexity of interactions that can occur in the rhizosphere is highlighted and some potential areas for future research in this area are discussed briefly.

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Microbial interactions and biocontrol in the rhizosphere

Fungal species belonging to the genus Trichoderma are worldwide in occurrence and easily isolated from soil, decaying wood, and other forms of plant organic matter. They are, for the most part, classified as imperfect fungi, in that they have no known sexual stage. Rapid growth rate in culture and the production of numerous spores (conidia) that are varying shades of green characterize fungi in this genus. The reverse side of colonies is often uncolored, buff, yellow, amber, or yellow-green, and many species produce prodigious quantities of thick-walled spores (chlamydospores) in submerged mycelium (8). The potential of Trichoderma species as biocontrol agents of plant diseases was first recognized in the early 1930s (31), and in subsequent years, control of many diseases has been added to the list (1,3,5,7,9,11,19, 23,29,34,37,40). This has culminated in the commercial production of several Trichoderma species for the protection and growth enhancement of a number of crops in the United States (24), and in the production of Trichoderma species and mixtures of species in India, Israel, New Zealand, and Sweden (D. R. Fravel, personal communication). One of the most interesting aspects of the science of biological control is the study of the mechanisms employed by biocontrol agents to effect disease control. Past research indicates that the mechanisms are many and varied, even within the genus Trichoderma. In order to make the most effective use of biocontrol agents for the control of plant diseases, we must understand how the agents work and what their limitations are. We can then develop effective means of culturing, storing, applying, and utilizing biocontrol agents so that we harness their best effort for disease control. The selected research papers cited in this article were chosen because they illustrate what has been learned about mechanisms involved in biocontrol with Trichoderma species.

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Mechanisms Employed by Trichoderma Species in the Biological Control of Plant Diseases: The History and Evolution of Current Concepts.

Cellulases and related enzymes in biotechnology

Fungi in the genus Trichoderma have been known since at least the 1920s for their ability to act as biocontrol agents against plant pathogens. Until recently, the principal mechanisms for control have been assumed to be those primarily acting upon the pathogens and included mycoparasitism, antibiosis, and competition for resources and space. Recent advances demonstrate that the effects of Trichoderma on plants, including induced systemic or localized resistance, are also very important. These fungi colonize the root epidermis and outer cortical layers and release bioactive molecules that cause walling off of the Trichoderma thallus. At the same time, the transcriptome and the proteome of plants are substantially altered. As a consequence, in addition to induction of pathways for resistance in plants, increased plant growth and nutrient uptake occur. However, at least in maize, the increased growth response is genotype specific, and some maize inbreds respond negatively to some strains. Trichoderma spp. are beginning to be used in reasonably large quantities in plant agriculture, both for disease control and yield increases. The studies of mycoparasitism also have demonstrated that these fungi produce a rich mixture of antifungal enzymes, including chitinases and beta-1,3 glucanases. These enzymes are synergistic with each other, with other antifungal enzymes, and with other materials. The genes encoding the enzymes appear useful for producing transgenic plants resistant to diseases and the enzymes themselves are beneficial for biological control and other processes.

https://apsjournals.apsnet.org/doi/pdf/10.1094/PHYTO-96-0190

Overview of Mechanisms and Uses of Trichoderma spp.

Two chitinolytic enzymes from Trichoderma harzianum strain P1 were tested for their antifungal activity in bioassays against nine different fungal species. Spore germination (or cell replication) and germ tube elongation were inhibited for all chitin-containing fungi except T. harzianum strain P1. The degree of inhibition was proportional to the level of chitin in the cell wall of the target fungi. For most of the fungi tested, the ED50 values for the endochitinase and the chitobiosidase were 35-135 micrograms ml-1 and 62-180 micrograms ml-1, respectively. Complete inhibition occurred at 200-300 micrograms ml-1. Combining the two enzymes resulted in a synergistic increase of antifungal activity. The ED50 values for a 1:1 mixture of endochitinase and chitobiosidase were as low as 10 micrograms ml-1 for Botrytis cinerea, 34 micrograms ml-1 for Ustilago avenae, 13 micrograms ml-1 for Uncinula necator, and 30 micrograms ml-1 for Fusarium solani. T. harzianum strain P1 was resistant to its own chitinolytic enzymes up to 800 micrograms ml-1, with an ED50 value 1,000 micrograms ml-1. The chitinolytic enzymes from T. harzianum appeared to be biologically more active than enzymes from other sources and more effective against a wider range of fungi. The involvement of these chitinolytic enzymes in biocontrol is also discussed

Chitinolytic enzymes produced by trichoderma harzianum; antifungal activity of purified endochitinase and chitobiosidase

A glucan 1,3-β-glucosidase (EC 3.2.1.58) and an N-acetyl-β-glucosaminidase (EC 3.2.1.30) were purified to homogeneity from the culture filtrate of Tricloderma harzianum strain PI. The molecular masses and the pIs were 78 kDa and 6.2, respectively, for the glucan 1,3-β-glucosidase and 72 kDa and 4.6, respectively, for the N-acetyl-β-glucosaminidase. The glucan 1,3-β-glucosidase and the N-acetyl-β-glucosaminidase were tested against Botrytis cinerea, and their antifungal activity was compared with that obtained for an endochitinase and a chitin 1,4-β-chitobiosidase also purified from T. harzianun strain P1. The four cell wall-degrading enzymes were also tested as mixtures containing two, three, or all four proteins in all possible combinations [...]

Purification, characterization, and synergistic activity of a glucan 1,3-beta-glucosidase and an N-acetyl-beta-glucosaminidase from Trichoderma harzianum.

The goal of this research was to improve scab resistance of apple by transformation with genes encoding chitinolytic enzymes from the bio-control organism Trichoderma harzianum. The endochitinase gene, as cDNA and genomic clones, was transferred into apple cv. Marshall McIntosh by Agrobacterium-transformation. A total of 15 lines were identified as transgenic by NPTII enzyme-linked immunosorbent assay and polymerase chain reaction and confirmed by Southern analysis. Substantial differences in endochitinase activity were detected among different lines by enzymatic assay and western analysis. Eight lines propagated as grafted and own-rooted plants were inoculated with Venturia inaequalis. Six of these transgenic lines expressing endochitinase were more resistant than nontransformed cv. Marshall McIntosh. Disease severity compared with cv. Marshall McIntosh was reduced by 0 to 99.7% (number of lesions), 0 to 90% (percentage of leaf area infected), and 1 to 56% (conidia recovered) in the transgenic lines tested. Endochitinase also had negative effects on the growth of both inoculated and uninoculated plants. There was a significant negative correlation between the level of endochitinase production and both the amount of disease and plant growth.

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Expression of Endochitinase from Trichoderma harzianum in Transgenic Apple Increases Resistance to Apple Scab and Reduces Vigor.

Different classes of cell wall degrading enzymes produced by the biocontrol fungi Trichoderma harzianum and Gliocladium virens inhibited spore germination of Botrytis cinerea in a bioassay in vitro. The addition of any chitinolytic or glucanolytic enzyme to the reaction mixture synergistically enhanced the antifungal properties of five different fungitoxic compounds against B. cinerea. The chemicals tested were gliotoxin, flusilazole, miconazole, captan and benomyl. Dose response curves were determined for each combination of toxin and enzyme, and in all cases the ED50 values of the mixtures were substantially lower than ED50 values of the two compounds used alone. For instance, the addition of endochitinase from T. harzianum at a concentration of 10λg ml-1 reduced the ED50 values of toxins up to 86-fold. The level of synergism appeared to be higher when enzymes were combined with toxins having primary sites of action associated with membrane structure, compared with pesticides having multiple or cytoplasmic sites of action. Among enzymes tested, the highest levels of synergism with synthetic fungicides were detected for the endochitinase from T. harzianum strain P1, which, when used alone, was the most effective chitinolytic enzyme against phytopathogenic fungi of those tested. The use of hydrolytic enzymes to synergistically enhance the antifungal ability of fungitoxic compounds may reduce the impact of some chemical pesticides on plants and animals.

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Synergistic interaction between fungal cell wall degrading enzymes and different antifungal compounds enhances inhibition of spore germination

A number of cell wall degrading enzymes (CWDEs) and cell membrane affecting compounds (MACs) that alter cell membrane structure or permeability have been assayed in vitro against phytopathogenic fungi and bacteria. Osmotin, gramicidin, valinomycin, phospholipase B, trichorzianine A1, trichorzianine B1, gliotoxin, flusilazole, and miconazole were tested in combination with three endochitinases, four exochitinases, and one glucan 1,3-beta-glucosidase from fungi, bacteria, or plants. Every combination of MAC + CWDE showed a high level of inhibition against Botrytis cinerea and Fusarium oxysporum and the interaction between the two kinds of compounds was of a synergistic nature. Different levels of synergism were obtained among the compound combinations depending upon the antifungal activity of the enzyme. When the enzyme treatment was applied subsequent to the MAC, the level of synergism was lower, indicating that degradation of the cell wall is needed to establish the synergistic interaction. The synergism with MACs was also present when the fungal cell wall was altered in a non-enzymatic manner by including L-sorbose in the growth media. The sensitivity of bacterial strains to the two trichorzianines depended upon the nature of their cell wall and could be synergistically enhanced by partial digestion of the wall. Some of the combinations showed a high level of synergism, suggesting that the interaction between MACs and CWDEs could be involved in biocontrol processes and plant self-defense mechanisms.

Christopher K. Hayes

Papers

Chitinolytic enzymes produced by trichoderma harzianum; antifungal activity of purified endochitinase and chitobiosidase

Purification, characterization, and synergistic activity of a glucan 1,3-beta-glucosidase and an N-acetyl-beta-glucosaminidase from Trichoderma harzianum.

Expression of Endochitinase from Trichoderma harzianum in Transgenic Apple Increases Resistance to Apple Scab and Reduces Vigor.

Synergistic interaction between fungal cell wall degrading enzymes and different antifungal compounds enhances inhibition of spore germination

Synergistic interaction between cell wall degrading enzymes and membrane affecting compounds