Abstract

Mycoparasitic species of the fungal genus Trichoderma are among the most successful biofungicides in today’s agriculture although our understanding of the exact molecular mechanisms of their activity still is fragmentary. The biological control of fungal plant diseases by Trichoderma includes direct antagonism of phytopathogenic fungi by mycoparasitism. This mycoparasitic attack comprises pre-contact sensing of the prey followed by activation of “molecular weapons” such as cell wall-lytic enzymes, secondary metabolites, and infection structures finally resulting in attack and killing of the prey fungus.

We used the strong mycoparasite Trichoderma atroviride as a model to study the mycoparasitic fungus-fungus interaction. Investigation of the early interaction stages employing T. atroviride labeled with fluorescent CRIB (Cdc42/Rac1-interactive binding) reporters revealed a switch between positive and negative chemotropism in the mycoparasites’ hyphae during the pre-contact sensing phase, a behavior indicative of a stress response probably triggered by prey-derived substances. Accordingly, secondary metabolites released by both interaction partners could be visualized in the interaction zone by mass spectrometry imaging pointing to a chemical cross-talk between Trichoderma and the prey fungus. Our data support the current model of pre-contact prey sensing; consequently, the receptors and signaling pathways that are involved in sensing and in governing the mycoparasitic attack are of special interest. As indicated by our studies, T. atroviride relies on signaling via the Gpr1 G protein-coupled receptor as well as MAP kinase and TOR kinase pathways for triggering the mycoparasitic response in the presence of a fungal prey.

Abstract

Trichoderma is well known for the ability of some species to act as i) important biocontrol agents against phytopathogenic fungi; ii) biofertilizers; iii) increasing tolerance of plants to biotic and abiotic stresses; and iv) inducer of plant defense responses via the production and secretion of elicitor molecules. Proteins of the Cerato-platanin (CP) family are released during the early developmental stages of filamentous fungi. They can act as elicitors and induce defense responses in plants. In this study, we analyzed the effects of the Trichoderma harzianum Epl-1 protein in the interaction process with the phytopathogen Botrytis cinerea and with tomato and common bean plants in short and long periods after Trichoderma strains inoculation. The results showed that T. harzianum Epl-1 protein affected the eliciting 1) B. cinerea virulence genes; 2) tomato defense-related genes; 3) the activation of the primer effect in tomato plants; 4) the interaction at the first stage of tomato roots colonization; and 5) the growth promotion of bean plants.

Abstract

Soil fungi belonging to the ascomycete genus Trichoderma have the potential to provide environmental-friendly biocontrol of plant diseases. Biochemical and molecular genetic studies have clearly related the mycoparasitic behaviour of Trichoderma against phytopathogenic fungi and oomycetes with the secretion of chitinases, glucanases and proteases with the cell wall degrading activity. In addition, Trichoderma proteases can also hydrolyze nematode cuticles and eggs and inhibit enzymes produced by the pathogens to penetrate the plant. Comparative genome sequence analysis of biocontrol species of Trichoderma has revealed that the mycoparasitic activity in the rhizosphere facilitates the formation of endophytic associations and the evolution of positive interactions with plants, supporting the application of Trichoderma strains as plant biostimulants in agriculture and forestry. In this sense, it has been observed worldwide that, as a general rule, the Trichoderma positive impact is more apparent in plants subjected to some stress. Early transcriptomic responses of Trichoderma colonizing tomato roots have shown that genes related to the formation of infection structures in plant tissues resulted upregulated, and once the hyphal root attachment has already taken place, nutrient uptake and carbohydrate metabolism would be limited by plant defenses. This means that Trichoderma is capable of overcoming plant defense responses during the initial stages of the interaction, when the early systemic defense responses would not be reaching its full potential, allowing Trichoderma an intercellular apoplastic colonization. As a result, Trichoderma exerts beneficial effects on plants in terms of improvement or maintenance of soil productivity, increased percentages and rates of seed germination, nutrient uptake, growth promotion, alleviation of adverse effects caused by environmental damage and systemic defense stimulation against abiotic stress and pathogen attack, without the need of establishing any contact with the invader. Trichoderma-primed plants that have a priming memory are able to react more rapidly and more adequately when challenged by a stressor. We have recently observed that tomato progeny inherit resistance to pathogens linked to plant growth induced by Trichoderma, without compromising the level of defense.

Abstract

Biological control of plant diseases holds great promise for replacing chemical pesticides in future food production, as part of integrated pest management. The mycoparasitic fungus Clonostachys rosea is an efficient biological control agent under field conditions for a variety of plant diseases on agricultural crops. In order to improve our understanding of critical components of the mycoparasitic lifestyle of C. rosea, we sequenced the genome of C. rosea strain IK726 using Illumina/PacBio technology. Comparative genomics revealed a significant increase in the number of certain ABC-transporters, MFS-transporters, proteases, polyketide synthases, cytochrome P450 monooxygenases, pectin lyases and GMC oxidoreductases compared with other Hypocrealean fungi. Interestingly, the increase of membrane transporter gene number in C. rosea was primarily associated with efflux drug resistance transporters. Necrotrophic mycoparasites such as C. rosea are assumed to have broad host range with little specificity. However, transcriptomic analyses revealed that C. rosea responded with both common and specific gene expression during interactions with the plant pathogenic species Botrytis cinerea and Fusarium graminearum. In agreement with the data on increased gene copy numbers, the majority of the regulated genes were predicted to encode proteins involved in membrane transport, biosynthesis of secondary metabolites and carbohydrate-active enzymes. Whole-genome re-sequencing of 63 C. rosea strains followed by genome wide association studies of phenotypic variation related with biocontrol of Fusarium diseases on wheat further identified several membrane transporters, proteases and one polyketide synthase to be associated with biocontrol. Finally, gene deletion studies confirmed the involvement of several ABC-transporters, MFS-transporters and polyketide synthases in in vitro antagonism or biocontrol in C. rosea. In summary, our data emphasize the role of antibiosis in determining the outcome of biocontrol interactions. Efflux membrane transporters appear to play an important role in the biology of C. rosea, by providing tolerance towards secondary metabolites produced by the fungal prey or C. rosea itself.

Abstract

Powdery mildew fungi (Erysiphales) are obligate biotrophic plant pathogens, infecting around 10,000 dicot species and also some members of the Poaceae. Important crops, including wheat, barley, grapevine, apple and a number of cultivated and ornamental plants, are amongst the major targets of powdery mildew fungi. Pycnidial fungi belonging to the genus Ampelomyces are commonly found in powdery mildew colonies in the field, and some selected strains have been developed as biocontrol agents of different powdery mildew species. To improve visualization of the interaction between Ampelomyces spp. and their mycohosts, we produced GFP expressing Ampelomyces transformants with Agrobacterium-mediated transformation using Agrobacterium tumefaciens strain AGL1 carrying a plasmid with the hygromycin resistance and GFP genes. Transformants were selected on hygromycin-containing medium and were checked for fluorescence after being grown in culture. Growth characteristics and mycoparasitic activity of transformants were measured and compared to those of the wild type. Selected transformants were used in mycoparasitic tests using five different powdery mildew species. In these experiments, sporulating powdery mildew colonies were inoculated with spore suspensions of transformants. We have also conducted persistence tests, in which experimental plants were inoculated first with GFP expressing transformants, and one week later with powdery mildew conidia. The transformation method was effective as several transformants emerged on the selective medium and exhibited strong green fluorescent signal. Transformants were genetically stable as they emitted strong green fluorescence after several subculturing in the absence of selective pressure. Most transformants did not differ in growth characteristics and mycoparasitic activity from the wild type. In mycoparasitic tests we observed extensive intracellular colonization of powdery mildew hyphae, conidiophores and conidia; intracellular Ampelomyces hyphae, as well as pycnidia and conidia produced in powdery mildew structures exhibited strong green signals when examined with fluorescence microscopy. In persistence tests Ampelomyces germinated on plant leaves in the absence of their mycohosts, and parasitized powdery mildew colonies as soon as these were available on the inoculated leaves. This work showed that Ampelomyces is amenable to Agrobacterium tumefaciens-mediated transformation and the commonly used heterologous marker and reporter genes like hygromycin resistance and GFP can be efficiently used. Transformation with GFP is useful for improving direct observation of this interfungal parasitic relationship. Our persistence tests demonstrated that Ampelomyces strains can act as biocontrol agents even if the target pathogen infects plants one week after Ampelomyces application.

This work was supported by a grant of the Hungarian Research, Development and Innovation Office (NKFIH NN100415), a grant of the Austrian-Hungarian Action Foundation (90öu16) and Janos Bolyai Research Fellowship to AP.

Abstract

Pucciniomycotina is a subphylum with a high diversity in terms of habitat and life history strategies that include plant parasites, animal associates (including opportunistic human pathogens), saprobes and antagonists of other fungi. Antagonistic interactions can occur through: 1) direct physical contact between two fungi, i.e., mycoparasitism; or, 2) the production of killer toxins and other agents (known as the killer-toxin phenomenon). Killer toxins are the less studied of these two types of interactions, yet may play a significant role in the development of community structure in natural environments. The killer-toxin phenomenon was first described in Saccharomyces cerevisiae and has been more extensively studied in ascomycetous yeasts, while in Basidiomycota only 50 yeast species have been reported as producers of killer toxins, including a few species in the Cystobasidiomycetes. In this class, direct physical antagonistic interaction which is associated with sexual states has been reported in species of Cystobasidium, Naohidea, Cyphobasidium and Occultifur. On the other hand, the killer-toxin phenomenon which mainly occurs between the yeast stage of the fungi and other organisms has only been reported in Cystobasidium minutum, C. pallidum and Hasegawazyma lactosa. We hypothesize that the common ancestor to Cystobasidiomycetes is a mycoparasite that also produced killer toxins. To test this hypothesis, we evaluated the presence of killer toxins for 54 strains belonging to 24 species of Cystobasidiomycetes (including 14 species new to science). A sensitive strain was allowed to grow for 24h on media containing 0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1.0% glucose, and 2.0% agar, supplemented with 0.003% methylene blue and pH 4.2. Each strain in the Cystobasidiomycetes tested for killer activity was also incubated for 24h on Yeast Malt agar and inoculated by making a single streak on the plate containing the sensitive strain. Cultures were evaluated every 24h for 5 days for the presence of an inhibition zone with no growth. In addition, we constructed a resolved phylogeny for the class based on six loci (ITS-including 5.8 rDNA, LSU rDNA, SSU rDNA, and the protein coding genes RPB1, RPB2, TEFα) to determine the evolutionary origins of mycoparasitism through ancestral character reconstruction.