RNA interference of endochitinases in the sugarcane endophyte Trichoderma virens 223 reduces its fitness as a biocontrol agent of pineapple disease.
ABSTRACT: The sugarcane root endophyte Trichoderma virens 223 holds enormous potential as a sustainable alternative to chemical pesticides in the control of sugarcane diseases. Its efficacy as a biocontrol agent is thought to be associated with its production of chitinase enzymes, including N-acetyl-ß-D-glucosaminidases, chitobiosidases and endochitinases. We used targeted gene deletion and RNA-dependent gene silencing strategies to disrupt N-acetyl-ß-D-glucosaminidase and endochitinase activities of the fungus, and to determine their roles in the biocontrol of soil-borne plant pathogens. The loss of N-acetyl-ß-D-glucosaminidase activities was dispensable for biocontrol of the plurivorous damping-off pathogens Rhizoctonia solani and Sclerotinia sclerotiorum, and of the sugarcane pathogen Ceratocystis paradoxa, the causal agent of pineapple disease. Similarly, suppression of endochitinase activities had no effect on R. solani and S. sclerotiorum disease control, but had a pronounced effect on the ability of T. virens 223 to control pineapple disease. Our work demonstrates a critical requirement for T. virens 223 endochitinase activity in the biocontrol of C. paradoxa sugarcane disease, but not for general antagonism of other soil pathogens. This may reflect its lifestyle as a sugarcane root endophyte.
Project description:Plants respond to pathogens and insect attacks by inducing and accumulating a large set of defense-related proteins. Two homologues of a barley wound-inducible protein (BARWIN) have been characterized in sugarcane, SUGARWIN1 and SUGARWIN2 (sugarcane wound-inducible proteins). Induction of SUGARWINs occurs in response to Diatraea saccharalis damage but not to pathogen infection. In addition, the protein itself does not show any effect on insect development; instead, it has antimicrobial activities toward Fusarium verticillioides, an opportunistic fungus that usually occurs after D. saccharalis borer attacks on sugarcane. In this study, we sought to evaluate the specificity of SUGARWIN2 to better understand its mechanism of action against phytopathogens and the associations between fungi and insects that affect plants. We used Colletotrichum falcatum, a fungus that causes red rot disease in sugarcane fields infested by D. saccharalis, and Ceratocystis paradoxa, which causes pineapple disease in sugarcane. We also tested whether SUGARWIN2 is able to cause cell death in Aspergillus nidulans, a fungus that does not infect sugarcane, and in the model yeast Saccharomyces cerevisiae, which is used for bioethanol production. Recombinant SUGARWIN2 altered C. falcatum morphology by increasing vacuolization, points of fractures and a leak of intracellular material, leading to germling apoptosis. In C. paradoxa, SUGARWIN2 showed increased vacuolization in hyphae but did not kill the fungi. Neither the non-pathogenic fungus A. nidulans nor the yeast S. cerevisiae was affected by recombinant SUGARWIN2, suggesting that the protein is specific to sugarcane opportunistic fungal pathogens.
Project description:An endochitinase gene 'ech42' from the biocontrol fungus 'Trichoderma virens' was introduced to Brassica juncea (L). Czern and Coss via Agrobaterium tumefaciens mediated genetic transformation method. Integration and expression of the 'ech42' gene in transgenic lines were confirmed by PCR, RT-PCR and Southern hybridization. Transgenic lines (T1) showed expected 3:1 Mendelian segregation ratio when segregation analysis for inheritance of transgene 'hpt' was carried out. Fluorimetric analysis of transgenic lines (T0 and T1) showed 7 fold higher endochitinase activity than the non-transformed plant. Fluorimetric zymogram showed presence of endochitinase (42 kDa) in crude protein extract of transgenic lines. In detached leaf bioassay with fungi Alternaria brassicae and Alternaria brassicicola, transgenic lines (T0 and T1) showed delayed onset of lesions as well as 30-73 % reduction in infected leaf area compared to non-transformed plant.
Project description:Plant-associated fungi are considered a vast source for biotechnological processes whose potential has been poorly explored. The interactions and diversity of sugarcane, one of the most important crops in Brazil, have been rarely studied, mainly concerning fungal communities and their interactions with transgenic plants. Taking this into consideration, the purpose of this study was, based on culture dependent strategy, to determine the structure and diversity of the fungal community (root endophytes and rhizosphere) associated with two varieties of sugarcane, a non-genetically modified (SP80-1842) variety and its genetically modified counterpart (IMI-1, expressing imazapyr herbicide resistance). For this, the sugarcane varieties were evaluated in three sampling times (3, 10 and 17 months after planting) under two crop management (weeding and herbicide treatments). In addition, a strain of Trichoderma virens, an endophyte isolated from sugarcane with great potential as a biological control, growth promotion and enzyme production agent, was selected for the fungal-plant interaction assays. The results of the isolation, characterization and evaluation of fungal community changes showed that the sugarcane fungal community is composed of at least 35 different genera, mostly in the phylum Ascomycota. Many genera are observed at very low frequencies among a few most abundant genera, some of which were isolated from specific plant sites (e.g., the roots or the rhizosphere). An assessment of the possible effects upon the fungal community showed that the plant growth stage was the only factor that significantly affected the community's structure. Moreover, if transgenic effects are present, they may be minor compared to other natural sources of variation. The results of interaction studies using the Green fluorescent protein (GFP)-expressing T. virens strain T.v.223 revealed that this fungus did not promote any phenotypic changes in the host plant and was found mostly in the roots where it formed a dense mycelial cover and was able to penetrate the intercellular spaces of the root epidermis upper layers. The ability of T. virens to colonize plant roots suggests a potential for protecting plant health, inhibiting pathogens or inducing systemic resistance.
Project description:BACKGROUND:Nitrogen is an essential element for sugarcane growth and development and is generally applied in the form of urea often much more than at recommended rates, causing serious soil degradation, particularly soil acidification, as well as groundwater and air pollution. In spite of the importance of nitrogen for plant growth, fewer reports are available to understand the application and biological role of N2 fixing bacteria to improve N2 nutrition in the sugarcane plant. RESULTS:In this study, a total of 350 different bacterial strains were isolated from rhizospheric soil samples of the sugarcane plants. Out of these, 22 isolates were selected based on plant growth promotion traits, biocontrol, and nitrogenase activity. The presence and activity of the nifH gene and the ability of nitrogen-fixation proved that all 22 selected strains have the ability to fix nitrogen. These strains were used to perform 16S rRNA and rpoB genes for their identification. The resulted amplicons were sequenced and phylogenetic analysis was constructed. Among the screened strains for nitrogen fixation, CY5 (Bacillus megaterium) and CA1 (Bacillus mycoides) were the most prominent. These two strains were examined for functional diversity using Biolog phenotyping, which confirmed the consumption of diverse carbon and nitrogen sources and tolerance to low pH and osmotic stress. The inoculated bacterial strains colonized the sugarcane rhizosphere successfully and were mostly located in root and leaf. The expression of the nifH gene in both sugarcane varieties (GT11 and GXB9) inoculated with CY5 and CA1 was confirmed. The gene expression studies showed enhanced expression of genes of various enzymes such as catalase, phenylalanine-ammonia-lyase, superoxide dismutase, chitinase and glucanase in bacterial-inoculated sugarcane plants. CONCLUSION:The results showed that a substantial number of Bacillus isolates have N-fixation and biocontrol property against two sugarcane pathogens Sporisorium scitamineum and Ceratocystis paradoxa. The increased activity of genes controlling free radical metabolism may at least in part accounts for the increased tolerance to pathogens. Nitrogen-fixation was confirmed in sugarcane inoculated with B. megaterium and B. mycoides strains using N-balance and 15N2 isotope dilution in different plant parts of sugarcane. This is the first report of Bacillus mycoides as a nitrogen-fixing rhizobacterium in sugarcane.
Project description:BACKGROUND: Trichoderma is a genus of mycotrophic filamentous fungi (teleomorph Hypocrea) which possess a bright variety of biotrophic and saprotrophic lifestyles. The ability to parasitize and/or kill other fungi (mycoparasitism) is used in plant protection against soil-borne fungal diseases (biological control, or biocontrol). To investigate mechanisms of mycoparasitism, we compared the transcriptional responses of cosmopolitan opportunistic species and powerful biocontrol agents Trichoderma atroviride and T. virens with tropical ecologically restricted species T. reesei during confrontations with a plant pathogenic fungus Rhizoctonia solani. RESULTS: The three Trichoderma spp. exhibited a strikingly different transcriptomic response already before physical contact with alien hyphae. T. atroviride expressed an array of genes involved in production of secondary metabolites, GH16 ß-glucanases, various proteases and small secreted cysteine rich proteins. T. virens, on the other hand, expressed mainly the genes for biosynthesis of gliotoxin, respective precursors and also glutathione, which is necessary for gliotoxin biosynthesis. In contrast, T. reesei increased the expression of genes encoding cellulases and hemicellulases, and of the genes involved in solute transport. The majority of differentially regulated genes were orthologues present in all three species or both in T. atroviride and T. virens, indicating that the regulation of expression of these genes is different in the three Trichoderma spp. The genes expressed in all three fungi exhibited a nonrandom genomic distribution, indicating a possibility for their regulation via chromatin modification. CONCLUSION: This genome-wide expression study demonstrates that the initial Trichoderma mycotrophy has differentiated into several alternative ecological strategies ranging from parasitism to predation and saprotrophy. It provides first insights into the mechanisms of interactions between Trichoderma and other fungi that may be exploited for further development of biofungicides.
Project description:BACKGROUND: Sugarcane is one of the most important crops in Brazil, mainly because of its use in biofuel production. Recent studies have sought to determine the role of sugarcane endophytic microbial diversity in microorganism-plant interactions, and their biotechnological potential. Epicoccum nigrum is an important sugarcane endophytic fungus that has been associated with the biological control of phytopathogens, and the production of secondary metabolites. In spite of several studies carried out to define the better conditions to use E. nigrum in different crops, little is known about the establishment of an endophytic interaction, and its potential effects on plant physiology. METHODOLOGY/PRINCIPAL FINDINGS: We report an approach based on inoculation followed by re-isolation, molecular monitoring, microscopic analysis, plant growth responses to fungal colonization, and antimicrobial activity tests to study the basic aspects of the E. nigrum endophytic interaction with sugarcane, and the effects of colonization on plant physiology. The results indicate that E. nigrum was capable of increasing the root system biomass and producing compounds that inhibit the in vitro growth of sugarcane pathogens Fusarium verticillioides, Colletotrichum falcatum, Ceratocystis paradoxa, and Xanthomomas albilineans. In addition, E. nigrum preferentially colonizes the sugarcane surface and, occasionally, the endophytic environment. CONCLUSIONS/SIGNIFICANCE: Our work demonstrates that E. nigrum has great potential for sugarcane crop application because it is capable of increasing the root system biomass and controlling pathogens. The study of the basic aspects of the interaction of E. nigrum with sugarcane demonstrated the facultative endophytism of E. nigrum and its preference for the phylloplane environment, which should be considered in future studies of biocontrol using this species. In addition, this work contributes to the knowledge of the interaction of this ubiquitous endophyte with the host plant, and also to a better use of microbial endophytes in agriculture.
Project description:Mycoparasitism, a lifestyle where one fungus is parasitic on another fungus, has special relevance when the prey is a plant pathogen, providing a strategy for biological control of pests for plant protection. Probably, the most studied biocontrol agents are species of the genus Hypocrea/Trichoderma.Here we report an analysis of the genome sequences of the two biocontrol species Trichoderma atroviride (teleomorph Hypocrea atroviridis) and Trichoderma virens (formerly Gliocladium virens, teleomorph Hypocrea virens), and a comparison with Trichoderma reesei (teleomorph Hypocrea jecorina). These three Trichoderma species display a remarkable conservation of gene order (78 to 96%), and a lack of active mobile elements probably due to repeat-induced point mutation. Several gene families are expanded in the two mycoparasitic species relative to T. reesei or other ascomycetes, and are overrepresented in non-syntenic genome regions. A phylogenetic analysis shows that T. reesei and T. virens are derived relative to T. atroviride. The mycoparasitism-specific genes thus arose in a common Trichoderma ancestor but were subsequently lost in T. reesei.The data offer a better understanding of mycoparasitism, and thus enforce the development of improved biocontrol strains for efficient and environmentally friendly protection of plants.
Project description:Web blight/wet root rot caused by Rhizoctonia solani is one of the major constraints for mung bean (Vigna radiata) production. Growing of resistant varieties and use of biocontrol agents are the feasible options available to manage the disease. The present study was conducted to determine the variation in the expression of various defense-related genes in susceptible and resistant mung bean varieties in response to biocontrol agent Trichoderma virens and R. solani interactions. The primers were designed using sequences of defense-related genes, namely PR 10, epoxide hydrolase (EH), catalase and calmodulin available in NCBI database and evaluated against cDNA obtained from both susceptible and resistant mung bean plants at 1-4 days post-inoculation (dpi) with the test pathogen R. solani and biocontrol agent T. virens using conventional PCR and qPCR analyses. R. solani inoculation upregulated the mean expression of PR 10 and calmodulin in susceptible and resistant varieties, respectively, whereas downregulated in the rest of the treatments. Quantitative PCR analysis showed that except catalase in the susceptible variety, which is downregulated, the expression of PR 10, EH, catalase and calmodulin was upregulated in both resistant and susceptible varieties in response to T. virens alone and in the presence of R. solani. In general, the expression of PR 10 and calmodulin was highest at 1 dpi whereas EH and catalase expression were maximum at 4 dpi. The application of T. virens suppressed the development of disease in the presence of R. solani in both susceptible and resistant varieties with more pronounced effect in resistant variety. Thus, the application of biocontrol agent T. virens upregulated the expression of defense-related genes and reduced disease development.
Project description:Trichoderma virens (synonym, Gliocladium virens), a deuteromycete fungus, suppresses soilborne plant diseases caused by a number of fungi and is used as a biocontrol agent. Several traits that may contribute to the antagonistic interactions of T. virens with disease-causing fungi involve the production of peptide metabolites (e.g., the antibiotic gliotoxin and siderophores used for iron acquisition). We cloned a 5,056-bp partial cDNA encoding a putative peptide synthetase (Psy1) from T. virens using conserved motifs found within the adenylate domain of peptide synthetases. Sequence similarities with conserved motifs of the adenylation domain, acyl transfer, and two condensation domains support identification of the Psy1 gene as a gene that encodes a peptide synthetase. Disruption of the native Psy1 gene through gene replacement was used to identify the function of this gene. Psy1 disruptants produced normal amounts of gliotoxin but grew poorly under low-iron conditions, suggesting that Psy1 plays a role in siderophore production. Psy1 disruptants cannot produce the major T. virens siderophore dimerum acid, a dipetide of acylated N(delta)-hydroxyornithine. Biocontrol activity against damping-off diseases caused by Pythium ultimum and Rhizoctonia solani was not reduced by the Psy1 disruption, suggesting that iron competition through dimerum acid production does not contribute significantly to disease suppression activity under the conditions used.
Project description:Even though beta-1,6-glucanases have been purified from several filamentous fungi, the physiological function has not been conclusively established for any species. In the present study, the role of Tvbgn3, a beta-1,6-glucanase from Trichoderma virens, was examined by comparison of wild-type (WT) and transformant strains in which Tvbgn3 was disrupted (GKO) or constitutively overexpressed (GOE). Gene expression analysis revealed induction of Tvbgn3 in the presence of host fungal cell walls, indicating regulation during mycoparasitism. Indeed, while deletion or overexpression of Tvbgn3 had no evident effect on growth and development, GOE and GKO strains showed an enhanced or reduced ability, respectively, to inhibit the growth of the plant pathogen Pythium ultimum compared to results with the WT. The relevance of this activity in the biocontrol ability of T. virens was confirmed in plant bioassays. Deletion of the gene resulted in levels of disease protection that were significantly reduced from WT levels, while GOE strains showed a significantly increased biocontrol capability. These results demonstrate the involvement of beta-1,6-glucanase in mycoparasitism and its relevance in the biocontrol activity of T. virens, opening a new avenue for biotechnological applications.