Application of Trichoderma harzianum (T22) and Trichoderma atroviride (P1) as plant growth promoters, and their compatibility with copper oxychloride

Trichoderma strains are used in agriculture because they provide to the plants the following benefits: i) are rhizosphere competence and establish stable rhizosphere microbial communities; ii) control plant disease caused by pathogenic and competitive microflora, by using a variety of mechanisms; iii) improve vegetative growth, root development and yield; iv) make nutrients more available to the plant. In this work we have investigated the ability of T. harzianum T22 and T. atroviride P1 to improve plant growth of locally important horticultural crops: lettuce, tomatoes and peppers and to prevent disease in the greenhouse and field. The effect of the Trichoderma treatment was evaluated by determining the weight of fresh and dry roots and above ground plant biomass, measuring plants height, counting the number of emerged leaves (lettuce, tomatoes and peppers) and quantifying production (tomatoes and peppers). No disease symptoms were found during production, although Fusarium sp. strains pathogenic to tomato were detected in the soil. Compounds containing copper oxychloride are frequently used for fungal disease control in agriculture. In order to investigate the compatibility of T. harzianum T22 and T. atroviride P1 with copper oxychloride applications, the effect on mycelia growth was monitored in both liquid and solid medium. In general, the tests indicated a high level of tolerance of the Trichoderma strains to concentrations of copper oxychloride varying from 0.1 to 5 mmol/L.

Transcriptome reprogramming,epigenetic modifications and alternative splicing orchestrate the tomato root response to the beneficial fungus Trichoderma harzianum

Beneficial interactions of rhizosphere microorganisms are widely exploited for plant biofertilization and mitigation of biotic and abiotic constraints.To provide new insights into the onset of the roots–beneficial microorganisms interplay,we characterised the transcriptomes expressed in tomato roots at 24,48 and 72 h post inoculation with the beneficial fungus Trichoderma harzianum T22 and analysed the epigenetic and post-trascriptional regulation mechanisms.We detected 1243 tomato transcripts that were differentially expressed between Trichoderma-interacting and control roots and 83 T.harzianum transcripts that were differentially expressed between the three experimental time points.Interaction with Trichoderma triggered a transcriptional response mainly ascribable to signal recognition and transduction,stress response,transcriptional regulation and transport.In tomato roots,salicylic acid,and not jasmonate,appears to have a prominent role in orchestrating the interplay with this beneficial strain.Differential regulation of many nutrient transporter genes indicated a strong effect on plant nutrition processes,which,together with the possible modifications in root architecture triggered by ethylene/indole-3-acetic acid signalling at 72 h post inoculation may concur to the well-described growth-promotion ability of this strain.Alongside,T.harzianum-induced defence priming and stress tolerance may be mediated by the induction of reactive oxygen species,detoxification and defence genes.A deeper insight into gene expression and regulation control provided first evidences for the involvement of cytosine methylation and alternative splicing mechanisms in the plant–Trichoderma interaction.A model is proposed that integrates the plant transcriptomic responses in the roots,where interaction between the plant and beneficial rhizosphere microorganisms occurs.

Use of Trichoderma spp. in remediation of polluted soils and waters

Trichoderma spp. probably have a role in remediation of polluted soils and waters. Highly rhizosphere competent strains persist on roots for an extended period of time (at least months) and continuously interact with the plants. They can increase general plant and root growth and increase uptake of a variety of materials. This makes the Trichoderma-plant interaction highly attractive for use in phytoextraction technologies. Moreover, Trichoderma spp. are resistant to a wide range of toxic compounds and can degrade some of these. One highly attractive target for remediation are soils that are polluted with cyanide and metallocyanides. Shrub willows (genus Salix) have been shown to take up and degrade these compounds by unknown mechanisms. Thus, they remove these compounds from soil but there are no cyanide residues in the plants. Similarly, Trichoderma spp. degrade free cyanide via production of extracellular enzymes and take up and then degrade metallocyanides such as Prussian blue. The willow-Trichoderma system therefore provides a plant-microbe system for degradation of these toxic compounds. The fungi also can be used directly in remediation strategies; for example, they degrade polyphenols such as those found in large quantities in waste water from production of olive oil. Thus, the abilities of the fungi to interact and enhance plant growth, their ability to grow in the presence of toxicants and their enzymatic abilities to degrade polluting substances provide a number of opportunities for either plant-microbe or pure fungal systems to remove pollutants from lands and waters.

Heterologous expression of the glucose oxidase gene in Trichoderma atroviride leads enhanced ability to attack phytopathogenic fungi and induction of plant systemic disease resistance

A transgenic strain of Trichoderma atroviride that expresses the Aspergillus niger glucose oxidase gene goxA under a homologous pathogen-inducible promoter (nag1) has been constructed, with the aim of increasing the ability of this biocontrol agent (BCA) to attack phytopathogenic fungi and enhance plant systemic disease resistance. The sporulation and growth rate of the transgenic progenies were similar to the wild-type strain P1. goxA expression occurred immediately after contact with the plant pathogen, and the glucose oxidase formed was secreted extracellularly. The transformed strain SJ3 4, containing 12-14 copies of the transgene, produced significantly less N-acetyl-glucosaminidase and endochitinase then wild type. However, the ability of its culture filtrate to inhibit the germination of Botrytis cinerea spores was increased by about 3-fold. In comparison to P1, the transgenic strain more quickly overgrew and lysed in vitro the pathogens Rhizoctonia solani and Pythium ultimum. In assays in vivo SJ3 4 showed a highly improved biocontrol ability in soil heavily infested with those pathogens, where the wild type was unable to protect the plant and allow seeds to germinate. The Trichoderma-gox was able to induce a much higher level of systemic resistance against the foliar pathogen B. cinerea, as compared to the parent strain. This work demonstrate that i) heterologous genes driven by pathogen-inducible promoters can improve the biocontrol and Induced Systemic Resistance properties of fungal BCAs such as Trichoderma spp., and ii) these microbes can be used as vectors to provide the plant with useful molecules able, for instance, to increase pathogen

Novel understanding of Trichoderma interaction mechanisms

Trichoderma- based biofungicides are a reality in commercial agriculture, with more than 50 formulations registered worldwide as biopesticides or biofertilizers. Several research strategies have been applied to identify the main genes and compounds involved in the complex, three-way interactions between fungal antagonists, plants and microbial pathogens. Proteome and genome analyses have greatly enhanced our ability to conduct targeted and genome-based functional studies. We have obtained reproducible 2-D maps of the entire fungal proteome in various conditions of interaction, which permitted the isolation of many proteins related to specific functions. Many differential proteins from several biocontrol strains of Trichoderma spp. during the in vivo interaction with different plants and/or several phytopathogenic fungi have been isolated and analyzed by MALDI-TOF. Relevant genes have been cloned and specifically inactivated, to demonstrate their function in biocontrol and induction of disease resistance. GFP-based reporter systems with interaction-inducible promoters allowed the characterization of regulatory sequences activated by the presence of the pathogen or the plant. From extensive cDNA. and EST libraries of genes expressed during Trichoderma-pathogen-plant interactions, we are identified and determined the role of a variety of novel genes and gene-products, including ABC transporters specifically induced during antagonism with other microbes; enzymes and other proteins that produce or act as novel elicitors of Induced Resistance in plant and promote root growth and crop yield; proteins possibly responsible of a gene-for-gene avirulent interaction between Trichoderma and plants; mycoparasitism-related inducers released from fungal pathogens and that activate biocontrol in Trichoderma; fungal promoters specifically induced during mycoparasitism and plant colonization; plant proteins and a novel phytoalexin induced by the presence of the fungal antagonist; etc. We have also transgenically demonstrated the ability of Trichoderma to transfer heterologous proteins into plants during root colonization. Finally we have used GFP and other markers to monitor the interaction in vivo and in situ between Trichoderma and its host (s) (the fungal pathogen and the plant).

G protein signalling involved in host recognition and mycoparasitism-related chitinase expression in Trichoderma atroviride

Mycoparasitic species of Trichoderma are commercially applied as biological control agents against various fungal pathogens. The mycoparasitic interaction is host specific and includes recognition, attack and subsequent penetration and killing of the host. Investigations on the underlying events revealed that Trichoderma responds to multiple signals from the host (e.g. lectins or other ligands such as low molecular weight components released from the host’s cell wall) and host attack is accompanied by morphological changes and the secretion of hydrolytic enzymes and antibiotics. Degradation of the cell wall of the host fungus is-besides glucanases and proteases-mainly achieved by chitinases. In vivo studies showed that the ech42 gene (encoding endochitinase 42) is expressed before physical contact of Trichoderma with its host, probably representing one of the earliest events in mycoparasitism, whereas Nag1 (N-acetylglucosaminidase) plays a key role in the general induction of the chitinolytic enzyme system of T. atroviride . Investigations on the responsible signal transduction pathways of T. atroviride led to the isolation of several genes encoding key components of the cAMP and MAP kinase signaling pathways, as alpha and β subunits of heterotrimeric G proteins, the regulatory subunit of cAMP-dependent protein kinase, adenylate cyclase, and three MAP kinases. Analysis of knockout mutants, generated by Agrobacterium-mediated transformation, revealed that at least two alpha-subunits of heterotrimeric G proteins are participating in mycoparasitism-related signal transduction. The Tga1 G alpha subunit was shown to be involved in mycoparasitism-related processes such as chitinase expression and overproduction of toxic secondary metabolites, whereas Tga3 was found to be completely avirulent showing defects in chitinase formation and host recognition.

Trichoderma harzianum enhances tomato indirect defense against aphids

Many fungal root symbionts of the genus Trichoderma are well-known for their beneficial effects on agronomic performance and protection against plant pathogens; moreover, they may enhance protection from insect pests, by triggering plant resistance mechanisms. Defense barriers against insects are induced by the activation of metabolic pathways involved in the production of defense-related plant compounds, either directly active against herbivore insects, or exerting an indirect effect, by increasing the attrac- tion of herbivore natural enemies. In a model system composed of the tomato plant, the aphid Macrosiphum euphorbiae and the parasitoid Aphidius ervi, plant metabolic changes induced by Trichoderma harzianum and their effects on higher trophic levels have been assessed. T. harzianum T22 treatments induce a primed state that upon aphid attacks leads to an increased attraction of aphid parasitoids, mediated by the enhanced produc- tion of volatile organic compounds （VOCs） that are known to induce Aphidius ervi flight. Transcriptome sequencing of T22-treated plants infested by aphids showed a remarkable upregulation of genes involved in terpenoids biosynthesis and salicylic acid pathway, which are consistent with the observed flight response ofA. ervi and the VOC bouquet profile underlying this behavioral response.