Sheridan L. Woo

Study of the three-way interaction between Trichoderma atroviride, plant and fungal pathogens by using a proteomic approach

The main molecular factors involved in the complex interactions occurring between plants (bean), two different fungal pathogens (Botrytis cinerea, Rhizoctonia solani) and an antagonistic strain of the genus Trichoderma were investigated. Two-dimensional (2-D) electrophoresis was used to analyze separately collected proteomes from each single, two- or three-partner interaction (i.e., plant, pathogenic and antagonistic fungus alone and in all possible combinations). Differential proteins were subjected to mass spectrometry and in silico analysis to search for homologies with known proteins. In the plant proteome, specific pathogenesis-related proteins and other disease-related factors (i.e., potential resistance genes) seem to be associated with the interaction with either one of the two pathogens and/or T. atroviride. This finding is in agreement with the demonstrated ability of Trichoderma spp. to induce systemic resistance against various microbial pathogens. On the other side, many differential proteins obtained from the T. atroviride interaction proteome showed interesting homologies with a fungal hydrophobin, ABC transporters, etc. Virulence factors, like cyclophilins, were up-regulated in the pathogen proteome during the interaction with the plant alone or with the antagonist too. We isolated and confidently identified a large number of protein factors associated to the multi-player interactions examined.

Identification of a New Biocontrol Gene in Trichoderma atroviride: The Role of an ABC Transporter Membrane Pump in the Interaction with Different Plant-Pathogenic Fungi

Successful biocontrol interactions often require that the beneficial microbes involved are resistant or tolerant to a variety of toxicants, including antibiotics produced by themselves or phytopathogens, plant antimicrobial compounds, and synthetic chemicals or contaminants. The ability of Trichoderma spp., the most widely applied biocontrol fungi, to withstand different chemical stresses, including those associated with mycoparasitism, is well known. In this work, we identified an ATP-binding cassette transporter cell membrane pump as an important component of the above indicated resistance mechanisms that appears to be supported by an extensive and powerful cell detoxification system. The encoding gene, named Taabc2, was cloned from a strain of Trichoderma atroviride and characterized. Its expression was found to be upregulated in the presence of pathogen-secreted metabolites, specific mycotoxins and some fungicides, and in conditions that stimulate the production in Trichoderma spp. of antagonism-related factors (toxins and enzymes). The key role of this gene in antagonism and biocontrol was demonstrated by the characterization of the obtained deletion mutants. They suffered an increased susceptibility to inhibitory compounds either secreted by pathogenic fungi or possibly produced by the biocontrol microbe itself and lost, partially or entirely, the ability to protect tomato plants from Pythium ultimum and Rhizoctonia solani attack.

Trichoderma-based Products and their Widespread Use in Agriculture

Governing bodies throughout the world, particularly in Europe, are now implementing legislative mandates with the objective of decreasing dependence on pesticides in agriculture to increase consumer and environmental safety. In order to reduce the risks associated with pesticide applications and reduce dependency on their use, Directives will promote low pesticide-input by implementing integrated pest management (IPM), and provide the means to establish the necessary conditions and measures to employ these practices, as well as to ensure security of commercial products. One approach includes the use of biological control agents and their products as alternatives to synthetic agro-chemicals. Trichoderma spp. are widely studied fungi and are among the most commonly used microbial biological control agents (MBCAs) in agriculture. They are presently marketed as bio-pesticides, biofertilizers, growth enhancers and stimulants of natural resistance. The efficacy of this fungus can be attributed to their ability to protect plants, enhance vegetative growth and contain pathogen populations under numerous agricultural conditions, as well as to act as soil amendments/inoculants for improvement of nutrient ability, decomposition and biodegradation. The living fungal spores (active substance) are incorporated in various formulations, both traditional and innovative, for applications as foliar sprays, pre-planting applications to seed or propagation material, post-pruning treatments, incorporation in the soil during seeding or transplant, watering by irrigation or applied as a root drench or dip. Trichoderma-based preparations are marketed worldwide and used for crop protection of various plant pathogens or increase the plant growth and productivity in diverse cultivated environments such as fields, greenhouses, nurseries; in the production of a variety of horticultural, fruits, trees and ornamental crops. A survey was conducted of Trichoderma-containing products found on the international market to obtain an overall perspective of the: 1) geographical distribution, 2) product composition and identity of Trichoderma species selected, 3) contents combined with Trichoderma in the products - other microbial species or substances in the mix, 4) number of products available globally and geographically, 5) number of products registered or having use specifications, 6) product formulations and applications, 7) manufacturer claims - target use, target pests, product type and effects of applications. The largest distribution of Trichoderma bioproducts is found in Asia, succeeded by Europe, South- Central America and North America. The majority of the labels indicated fungicidal properties, but only 38% of the marketed merchandise are registered. Ten Trichoderma species are specifically indicated, but many labels indicate a generic Trichoderma sp. or spp. mix in the list of ingredients. The most common formulation is a wettable powder, followed by granules. Generally, Trichoderma are applied to the seed or propagation material at the time of planting, then the secondary use is during plant development. On the whole, the target use is for the control of soilborne fungal pathogens such as Rhizoctonia, Pythium and Sclerotinia, and a few foliar pathogens such as Botrytis and Alternaria; whereas the minor use indication is for plant growth promotion. The use of Trichoderma-based biological products will have an important role in agricultural production of the future, in light of changing worldwide perspectives by consumers and governing bodies.

Calcium-mediated perception and defense responses activated in plant cells by metabolite mixtures secreted by the biocontrol fungus Trichoderma atroviride

BackgroundCalcium is commonly involved as intracellular messenger in the transduction by plants of a wide range of biotic stimuli, including signals from pathogenic and symbiotic fungi. Trichoderma spp. are largely used in the biological control of plant diseases caused by fungal phytopathogens and are able to colonize plant roots. Early molecular events underlying their association with plants are relatively unknown.ResultsHere, we investigated the effects on plant cells of metabolite complexes secreted by Trichoderma atroviride wild type P1 and a deletion mutant of this strain on the level of cytosolic free Ca2+ and activation of defense responses. Trichoderma culture filtrates were obtained by growing the fungus alone or in direct antagonism with its fungal host, the necrotrophic pathogen Botrytis cinerea, and then separated in two fractions (>3 and <3 kDa). When applied to aequorin-expressing soybean (Glycine max L.) cell suspension cultures, Trichoderma and Botrytis metabolite mixtures were distinctively perceived and activated transient intracellular Ca2+ elevations with different kinetics, specific patterns of intracellular accumulation of reactive oxygen species and induction of cell death. Both Ca2+ signature and cellular effects were modified by the culture medium from the knock-out mutant of Trichoderma, defective for the production of the secreted 42 kDa endochitinase.ConclusionNew insights are provided into the mechanism of interaction between Trichoderma and plants, indicating that secreted fungal molecules are sensed by plant cells through intracellular Ca2+ changes. Plant cells are able to discriminate signals originating in the single or two-fungal partner interaction and modulate defense responses.

Harzianic acid: a novel siderophore from Trichoderma harzianum

Agriculture-relevant microorganisms are considered to produce secondary metabolites during processes of competition with other micro- and macro-organisms, symbiosis, parasitism or pathogenesis. Many different strains of the genus Trichoderma, in addition to a direct activity against phytopathogens, are well-known producers of secondary metabolites and compounds that substantially affect the metabolism of the host plant. Harzianic acid is a Trichoderma secondary metabolite, showing antifungal and plant growth promotion activities. This report demonstrates the ability of this tetramic acid to bind with a good affinity essential metals such as Fe(3+) , which may represent a mechanism of iron solubilisation that significantly alters nutrient availability in the soil environment for other microorganisms and the host plant.

Trichoderma Secondary Metabolites Active on Plants and FungalPathogens

Beneficial microbes typically produce bioactive molecules that can affect the interactions of plants with their pathogens. Many secondary metabolites may also have antibiotic properties, which enable the producing microbe to inhibit and/or kill other microorganisms i.e. competing for a nutritional niche. Indeed, some of these compounds have been found to play an important role in the biocontrol of plant diseases by various beneficial microbes used world-wide for crop protection and bio-fertilization. In addition to direct toxic activity against plant pathogens, biocontrol-related metabolites may also increase disease resistance by triggering systemic plant defence activity, and/or enhance root and shoot growth. Fungi belonging to the Trichoderma genus are well known producers of secondary metabolites with a direct activity against phytopathogens and compounds that substantially affect the metabolism of the plant. The widescale application of selected metabolites to induce host resistance and/or to promote crop yield may become a reality in the near future and represents a powerful tool for the implementation of IPM strategies.

Multiple Roles and Effects of a Novel Trichoderma Hydrophobin

Fungi belonging to the genus Trichoderma are among the most active and ecologically successful microbes found in natural environments, because they are able to use a variety of substrates and affect the growth of other microbes and virtually any plant species. We isolated and characterized a novel type II hydrophobin secreted by the biocontrol strain MK1 of Trichoderma longibrachiatum. The corresponding gene (Hytlo1) has a multiple role in the Trichoderma-plant-pathogen three-way interaction, while the purified protein displayed a direct antifungal as well as a microbe-associated molecular pattern and a plant growth promotion (PGP) activity. Leaf infiltration with the hydrophobin systemically increased resistance to pathogens and activated defense-related responses involving reactive oxygen species, superoxide dismutase, oxylipin, phytoalexin, and pathogenesis-related protein formation or activity. The hydrophobin was found to enhance development of a variety of plants when applied at very low doses. It particularly stimulated root formation and growth, as demonstrated also by transient expression of the encoding gene in tobacco and tomato. Targeted knock-out of Hytlo1 significantly reduced both antagonistic and PGP effect of the wild-type strain. We conclude that this protein represents a clear example of a molecular factor developed by Trichoderma spp. to establish a mutually beneficial interaction with the colonized plant.

Potential of genes and gene products fromTrichoderma sp. andGliocladium sp. for the development of biological pesticides

Fungal cell wall degrading enzymes produced by the biocontrol fungiTrichoderma harzianum andGliocladium virens are strong inhibitors of spore germination and hyphal elongation of a number of phytopathogenic fungi. The purified enzymes include chitinolytic enzymes with different modes of action or different substrate specificity and glucanolytic enzymes with exo-activity. A variety of synergistic interactions were found when different enzymes were combined or associated with biotic or abiotic antifungal agents. The levels of inhibition obtained by using enzyme combinations were, in some cases, comparable with commercial fungicides. Moreover, the antifungal interaction between enzymes and common fungicides allowed the reduction of the chemical doses up to 200-fold. Chitinolytic and glucanolytic enzymes fromT. harzianum were able to improve substantially the antifungal ability of a biocontrol strain ofEnterobacter cloacae. DNA fragments containing genes encoding for different chitinolytic enzymes were isolated from a cDNA library ofT. harzianum and cloned for mechanistic studies and biocontrol purposes. Our results provide additional information on the role of lytic enzymes in processes of biocontrol and strongly suggest the use of lytic enzymes and their genes for biological control of plant diseases.

A novel fungal metabolite with beneficial properties for agricultural applications.

Trichoderma are ubiquitous soil fungi that include species widely used as biocontrol agents in agriculture. Many isolates are known to secrete several secondary metabolites with different biological activities towards plants and other microbes. Harzianic acid (HA) is a T. harzianum metabolite able to promote plant growth and strongly bind iron. In this work, we isolated from the culture filtrate of a T. harzianum strain a new metabolite, named isoharzianic acid (iso-HA), a stereoisomer of HA. The structure and absolute configuration of this compound has been determined by spectroscopic methods, including UV-Vis, MS, 1D and 2D NMR analyses. In vitro applications of iso-HA inhibited the mycelium radial growth of Sclerotinia sclerotiorum and Rhizoctonia solani. Moreover, iso HA improved the germination of tomato seeds and induced disease resistance. HPLC-DAD experiments showed that the production of HA and iso HA was affected by the presence of plant tissue in the liquid medium. In particular, tomato tissue elicited the production of HA but negatively modulated the biosynthesis of its analogue iso-HA, suggesting that different forms of the same Trichoderma secondary metabolite have specific roles in the molecular mechanism regulating the Trichoderma plant interaction.

Secondary metabolites from the endophytic fungus Talaromyces pinophilus

Abstract Endophytic fungi have a great influence on plant health and growth, and are an important source of bioactive natural compounds. Organic extracts obtained from the culture filtrate of an endophytic strain of Talaromyces pinophilus isolated from strawberry tree (Arbutus unedo) were studied. Metabolomic analysis revealed the presence of three bioactive metabolites, the siderophore ferrirubin, the platelet-aggregation inhibitor herquline B and the antibiotic 3-O-methylfunicone. The latter was the major metabolite produced by this strain and displayed toxic effects against the pea aphid Acyrthosiphon pisum (Homoptera Aphidiidae). This toxicity represents an additional indication that the widespread endophytic occurrence of T. pinophilus may be related to a possible role in defensive mutualism. Moreover, the toxic activity on aphids could promote further study on 3-O-methylfunicone, or its derivatives, as an alternative to synthetic chemicals in agriculture.