Gary E. Harman

Trichoderma species - opportunistic, avirulent plant symbionts.

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.

Overview of mechanisms and uses of Trichoderma spp

ABSTRACT 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.

INDUCED SYSTEMIC RESISTANCE AND PLANT RESPONSES TO FUNGAL BIOCONTROL AGENTS

Biocontrol fungi (BCF) are agents that control plant diseases. These include the well-known Trichoderma spp. and the recently described Sebacinales spp. They have the ability to control numerous foliar, root, and fruit pathogens and even invertebrates such as nematodes. However, this is only a subset of their abilities. We now know that they also have the ability to ameliorate a wide range of abiotic stresses, and some of them can also alleviate physiological stresses such as seed aging. They can also enhance nutrient uptake in plants and can substantially increase nitrogen use efficiency in crops. These abilities may be more important to agriculture than disease control. Some strains also have abilities to improve photosynthetic efficiency and probably respiratory activities of plants. All of these capabilities are a consequence of their abilities to reprogram plant gene expression, probably through activation of a limited number of general plant pathways.

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

ABSTRACT 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.

Interactions Between Trichoderma harzianum Strain T22 and Maize Inbred Line Mo17 and Effects of These Interactions on Diseases Caused by Pythium ultimum and Colletotrichum graminicola.

ABSTRACT Seed treatment with Trichoderma harzianum strain T22, which results in colonization of plant roots but little or no colonization of shoots or leaves, had substantial effects on growth of and disease expression in maize inbred line Mo17. Shoots and roots of 10-day-old seedlings grown in a sandy loam field soil were larger (roots were nearly twice as long) in the presence of T22 than in its absence. Both main and secondary roots were increased in size and area and the root hair area was greater with T22. However, root hair area per unit of root length was greater in control plants. Increased growth probably was due to direct stimulation of plant growth in addition to effects from biological control of deleterious microflora. Seedlings of Mo17 grown in autoclaved or mefenoxamtreated sandy loam field soil were larger than those produced in untreated soil. However, seedlings grown in the presence of T22, either in treated or untreated soil, were larger than those produced in its absence. Infestation of soil with Pythium ultimum had little effect upon growth of Mo17. The presence of T22 increased protein levels and activities of beta-1,3 glucanase, exochitinase, and endochitinase in both roots and shoots, even though T22 colonized roots well but colonized shoots hardly at all. With some enzymes, the combination of T22 plus P. ultimum gave the greatest activity. Plants grown from T22-treated seed had reduced symptoms of anthracnose following inoculation of leaves with Colletotrichum graminicola, which indicates that root colonization by T22 induces systemic resistance in maize.

Seed Treatment with Trichoderma harzianum Alleviates Biotic, Abiotic, and Physiological Stresses in Germinating Seeds and Seedlings

Trichoderma spp. are endophytic plant symbionts that are widely used as seed treatments to control diseases and to enhance plant growth and yield. Although some recent work has been published on their abilities to alleviate abiotic stresses, specific knowledge of mechanisms, abilities to control multiple plant stress factors, their effects on seed and seedlings is lacking. We examined the effects of seed treatment with T. harzianum strain T22 on germination of seed exposed to biotic stress (seed and seedling disease caused by Pythium ultimum) and abiotic stresses (osmotic, salinity, chilling, or heat stress). We also evaluated the ability of the beneficial fungus to overcome physiological stress (poor seed quality induced by seed aging). If seed were not under any of the stresses noted above, T22 generally had little effect upon seedling performance. However, under stress, treated seed germinated consistently faster and more uniformly than untreated seeds whether the stress was osmotic, salt, or suboptimal temperatures. The consistent response to varying stresses suggests a common mechanism through which the plant-fungus association enhances tolerance to a wide range of abiotic stresses as well as biotic stress. A common factor that negatively affects plants under these stress conditions is accumulation of toxic reactive oxygen species (ROS), and we tested the hypothesis that T22 reduced damages resulting from accumulation of ROS in stressed plants. Treatment of seeds reduced accumulation of lipid peroxides in seedlings under osmotic stress or in aged seeds. In addition, we showed that the effect of exogenous application of an antioxidant, glutathione, or application of T22, resulted in a similar positive effect on seed germination under osmotic stress or in aged seed. This evidence supports the model that T. harzianum strain T22 increases seedling vigor and ameliorates stress by inducing physiological protection in plants against oxidative damage.

The Molecular Basis of Shoot Responses of Maize Seedlings to Trichoderma harzianum T22 Inoculation of the Root: A Proteomic Approach

Trichoderma spp. are effective biocontrol agents for several soil-borne plant pathogens, and some are also known for their abilities to enhance systemic resistance to plant diseases and overall plant growth. Root colonization with Trichoderma harzianum Rifai strain 22 (T22) induces large changes in the proteome of shoots of maize (Zea mays) seedlings, even though T22 is present only on roots. We chose a proteomic approach to analyze those changes and identify pathways and genes that are involved in these processes. We used two-dimensional gel electrophoresis to identify proteins that are differentially expressed in response to colonization of maize plants with T22. Up- or down-regulated spots were subjected to tryptic digestion followed by identification using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry and nanospray ion-trap tandem mass spectrometry. We identified 91 out of 114 up-regulated and 30 out of 50 down-regulated proteins in the shoots. Classification of these revealed that a large portion of the up-regulated proteins are involved in carbohydrate metabolism and some were photosynthesis or stress related. Increased photosynthesis should have resulted in increased starch accumulation in seedlings and did indeed occur. In addition, numerous proteins induced in response to Trichoderma were those involved in stress and defense responses. Other processes that were up-regulated were amino acid metabolism, cell wall metabolism, and genetic information processing. Conversely, while the proteins involved in the pathways noted above were generally up-regulated, proteins involved in other processes such as secondary metabolism and protein biosynthesis were generally not affected. Up-regulation of carbohydrate metabolism and resistance responses may correspond to the enhanced growth response and induced resistance, respectively, conferred by the Trichoderma inoculation.

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

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 10 micrograms 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.

Synergistic interaction between cell wall degrading enzymes and membrane affecting compounds

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.

Combining effective strains of Trichoderma harzianum and solid matrix priming to improve biological seed treatments

In all crop-pathogen combinations, Trichoderma strains increased stands relative to the untreated control and were as effective as the chemical fungicides, even in the absence of solid matrix priming. Priming of seeds treated with Trichoderma strains increased plant stands in solid infested with F. graminearum and P. ultimum but not in soils infested with R. solani or S. rolfsii. In soils infested with P. ultimum, strain T95 generally gave the poorest results, T12 was intermediate, and the progeny strains (22 and 106) gave improved stands