Artemio Mendoza-Mendoza

Enhanced biocontrol activity of Trichoderma through inactivation of a mitogen-activated protein kinase

The production of lytic enzymes in Trichoderma is considered determinant in its parasitic response against fungal species. A mitogen-activated protein kinase encoding gene, tvk1, from Trichoderma virens was cloned, and its role during the mycoparasitism, conidiation, and biocontrol was examined in tvk1 null mutants. These mutants showed a clear increase in the level of the expression of mycoparasitism-related genes under simulated mycoparasitism and during direct confrontation with the plant pathogen Rhizoctonia solani. The null mutants displayed an increased protein secretion phenotype as measured by the production of lytic enzymes in culture supernatant compared to the wild type. Consistently, biocontrol assays demonstrated that the null mutants were considerably more effective in disease control than the wild-type strain or a chemical fungicide. In addition, tvk1 gene disruptant strains sporulated abundantly in submerged cultures, a condition that is not conducive to sporulation in the wild type. These data suggest that Tvk1 acts as a negative modulator during host sensing and sporulation in T. virens.

An injury-response mechanism conserved across kingdoms determines entry of the fungus Trichoderma atroviride into development

A conserved injury-defense mechanism is present in plants and animals, in which the production of reactive oxygen species (ROS) and lipid metabolism are essential to the response. Here, we describe that in the filamentous fungus Trichoderma atroviride, injury results in the formation of asexual reproduction structures restricted to regenerating cells. High-throughput RNA-seq analyses of the response to injury in T. atroviride suggested an oxidative response and activation of calcium-signaling pathways, as well as the participation of lipid metabolism, in this phenomenon. Gene-replacement experiments demonstrated that injury triggers NADPH oxidase (Nox)–dependent ROS production and that Nox1 and NoxR are essential for asexual development in response to damage. We further provide evidence of H2O2 and oxylipin production that, as in plants and animals, may act as signal molecules in response to injury in fungi, suggesting that the three kingdoms share a conserved defense-response mechanism.

Reproduction without sex: conidiation in the filamentous fungus Trichoderma.

Trichoderma spp. have served as models for asexual reproduction in filamentous fungi for over 50 years. Physical stimuli, such as light exposure and mechanical injury to the mycelium, trigger conidiation; however, conidiogenesis itself is a holistic response determined by the cells metabolic state, as influenced by the environment and endogenous biological rhythms. Key environmental parameters are the carbon and nitrogen status and the C : N ratio, the ambient pH and the level of calcium ions. Recent advances in our understanding of the molecular biology of this fungus have revealed a conserved mechanism of environmental perception through the White Collar orthologues BLR-1 and BLR-2. Also implicated in the molecular regulation are the PacC pathways and the conidial regulator VELVET. Signal transduction cascades which link environmental signals to physiological outputs have also been revealed.

Trichoderma atroviride LU132 promotes plant growth but not induced systemic resistance to Plutella xylostella in oilseed rape

Several species of the fungus Trichoderma can promote plant health and are widely used as commercial biopesticides. Beneficial effects of this fungus are attributed to various mechanisms such as mycoparasitism, plant-growth promotion, increased stress tolerance and elicitation of induced systemic resistance against pathogens via jasmonic acid/ethylene-dependent pathways. Despite such well-established effects on pathogens, surprisingly little is known about the influence of Trichoderma on plant defences against herbivorous insects. This study investigated whether soil-supplementation of the established biocontrol agent Trichoderma atroviride LU132 affected the performance of oilseed rape (Brassica napus) and the development of Plutella xylostella caterpillars. Furthermore, induction and priming of defence-related phytohormones, genes and secondary metabolites by fungus and herbivore were assessed. Plants colonized by T. atroviride LU132 had significantly larger root and shoot biomass than controls. No effects of fungal inoculation were found on herbivore development. Leaf feeding of the herbivore induced higher jasmonic acid levels, but this was not influenced by fungal treatment. Similarly, the defence-related genes MYC2 and TPI were induced by herbivory but not primed or induced by T. atroviride. Expression of the gene PDF1.2 was repressed by herbivore feeding while no effects on the gene ACO and glucosinolates were observed. We conclude that T. atroviride LU132 has positive effects on the growth of oilseed but it does not enhance above-ground insect defences.

Environmental Growth Conditions of Trichoderma spp. Affects Indole Acetic Acid Derivatives, Volatile Organic Compounds, and Plant Growth Promotion

Trichoderma species are soil-borne filamentous fungi widely utilized for their many plant health benefits, such as conferring improved growth, disease resistance and abiotic stress tolerance to their hosts. Many Trichoderma species are able to produce the auxin phytohormone indole-3-acetic acid (IAA), and its production has been suggested to promote root growth. Here we show that the production of IAA is strain dependent and diverse external stimuli are associated with its production. In in vitro assays, Arabidopsis primary root length was negatively affected by the interaction with some Trichoderma strains. In soil experiments, a continuum effect on plant growth was shown and this was also strain dependent. In plate assays, some strains of Trichoderma spp. inhibited the expression of the auxin reporter gene DR5 in Arabidopsis primary roots but not secondary roots. When Trichoderma spp. and A. thaliana were physically separated, enhancement of both shoot and root biomass, increased root production and chlorophyll content were observed, which strongly suggested that volatile production by the fungus influenced the parameters analyzed. Trichoderma strains T. virens Gv29.8, T. atroviride IMI206040, T. sp. “atroviride B” LU132, and T. asperellum LU1370 were demonstrated to promote plant growth through volatile production. However, contrasting differences were observed with LU1370 which had a negative effect on plant growth in soil but a positive effect in plate assays. Altogether our results suggest that the mechanisms and molecules involved in plant growth promotion by Trichoderma spp. are multivariable and are affected by the environmental conditions.

Trichoderma down under: species diversity and occurrence of Trichoderma in New Zealand

This is the first comprehensive survey of the species diversity of Trichoderma for a region within the temperate Southern Hemisphere. New Zealand makes an ideal target for such a survey because of the extensive historical collections of this genus from both native and human-modified ecosystems. From the 320 Trichoderma strains sequenced for the translation elongation factor-1α (tef1α) gene, in addition to the names associated with voucher specimens at the New Zealand Fungarium (PDD, Landcare Research), we recognise 71 Trichoderma species as present in New Zealand. Thirty-two species are reported for the first time from New Zealand and 14 of these appear to represent undescribed taxa. The New Zealand species are positioned across most Trichoderma clades, with terminal lineages related to T. viride, T. koningii and T. harzianum well represented. Of the 14 undescribed species, Trichoderma sp. “atroviride B”, a sister species to T. atroviride s.s., was the most commonly recovered species. Records of several species known only from fungarium specimens could not be confirmed by DNA analysis. Populations of Trichoderma in New Zealand are likely to represent a mixture of ancient indigenous lineages, more recent natural introductions, and species introduced as a result of human-mediated dispersal. Twenty-three Trichoderma species have been reported only from New Zealand or other Southern Hemisphere locations. The diversity of Trichoderma species in New Zealand, their phylogenetic relationships, distribution, ecology, and possible origins are discussed in this paper.

Arabidopsis thaliana polyamine content is modified by the interaction with different Trichoderma species

Plants are associated with a wide range of microorganisms throughout their life cycle, and some interactions result on plant benefits. Trichoderma species are plant beneficial fungi that enhance plant growth and development, contribute to plant nutrition and induce defense responses. Nevertheless, the molecules involved in these beneficial effects still need to be identify. Polyamines are ubiquitous molecules implicated in plant growth and development, and in the establishment of plant microbe interactions. In this study, we assessed the polyamine profile in Arabidopsis plants during the interaction with Trichoderma virens and Trichoderma atroviride, using a system that allows direct plant-fungal contact or avoids their physical interaction (split system). The plantlets that grew in the split system exhibited higher biomass than the ones in direct contact with Trichoderma species. After 3 days of interaction, a significant decrease in Arabidopsis polyamine levels was observed in both systems (direct contact and split). After 5 days of interaction polyamine levels were increased. The highest levels were observed with T. atroviride (split system), and with T. virens (direct contact). The expression levels of Arabidopsis ADC1 and ADC2 genes during the interaction with the fungi were also assessed. We observed a time dependent regulation of ADC1 and ADC2 genes, which correlates with polyamine levels. Our data show an evident change in polyamine profile during Arabidopsis - Trichoderma interaction, accompanied by evident alterations in plant root architecture. Polyamines could be involved in the changes undergone by plant during the interaction with this beneficial fungus.

The Ustilago maydis repetitive effector Rsp3 blocks the antifungal activity of mannose-binding maize proteins

To cause disease in maize, the biotrophic fungus Ustilago maydis secretes a large arsenal of effector proteins. Here, we functionally characterize the repetitive effector Rsp3 (repetitive secreted protein 3), which shows length polymorphisms in field isolates and is highly expressed during biotrophic stages. Rsp3 is required for virulence and anthocyanin accumulation. During biotrophic growth, Rsp3 decorates the hyphal surface and interacts with at least two secreted maize DUF26-domain family proteins (designated AFP1 and AFP2). AFP1 binds mannose and displays antifungal activity against the rsp3 mutant but not against a strain constitutively expressing rsp3. Maize plants silenced for AFP1 and AFP2 partially rescue the virulence defect of rsp3 mutants, suggesting that blocking the antifungal activity of AFP1 and AFP2 by the Rsp3 effector is an important virulence function. Rsp3 orthologs are present in all sequenced smut fungi, and the ortholog from Sporisorium reilianum can complement the rsp3 mutant of U. maydis, suggesting a novel widespread fungal protection mechanism.The fungus Ustilago maydis secretes many effector proteins to cause disease in maize. Here, Ma et al. show that the repetitive effector Rsp3 is required for virulence by inhibiting the antifungal activity of two mannose-binding proteins that are secreted by the plant cells.

Methods for the Evaluation of the Bioactivity and Biocontrol Potential of Species of Trichoderma.

Members of the genus Trichoderma comprise the majority of commercial fungal biocontrol agents of plant diseases. As such, there is a wealth of information available on the analysis of their biocontrol potential and the mechanisms behind their superior abilities. This chapter aims to summarize the most common methods utilized within a Trichoderma biocontrol program for assessing the biological properties of individual strains.

Detection of the entomopathogenic fungus Beauveria bassiana in the rhizosphere of wound-stressed Zea mays plants

Entomopathogenic fungi from the genus Beauveria (Vuillemin) play an important role in controlling insect populations and have been increasingly utilized for the biological control of insect pests. Various studies have reported that Beauveria bassiana (Bals.), Vuill. also has the ability to colonize a broad range of plant hosts as endophytes without causing disease but while still maintaining the capacity to infect insects. Beauveria is often applied as an inundative spore application, but little research has considered how plant colonization may alter the ability to persist in the environment. The aim of this study was to investigate potential interactions between B. bassiana and Zea mays L. (maize) in the rhizosphere following inoculation, in order to understand the factors that may affect environmental persistence of the fungi. The hypothesis was that different isolates of B. bassiana have the ability to colonize maize roots and/or rhizosphere soil, resulting in effects to the plant microbiome. To test this hypothesis, a two-step nested PCR protocol was developed to find and amplify Beauveria in planta or in soil; based on the translation elongation factor 1-alpha (ef1α) gene. The nested protocol was also designed to enable Beauveria species differentiation by sequence analysis. The impact of three selected B. bassiana isolates applied topically to roots on the rhizosphere soil community structure and function were consequently assessed using denaturing gradient gel electrophoresis (DGGE) and MicroRespTM techniques. The microbial community structure and function were not significantly affected by the presence of the isolates, however, retention of the inocula in the rhizosphere at 30 days after inoculation was enhanced when plants were subjected to intensive wounding of foliage to crudely simulate herbivory. The plant defense response likely changed under wound stress resulting in the apparent recruitment of Beauveria in the rhizosphere, which may be an indirect defensive strategy against herbivory and/or the result of induced systemic susceptibility in maize enabling plant colonization.