Abstract
Using biological fertilizers and pesticides based on beneficial soil microbes in order to reduce mineral fertilizers and chemical pesticides in conventional agriculture is still a matter of debate. In this regard, a European research project seeks to elucidate the role of root-endophytic fungi and to develop molecular tools to trace and quantify these fungi in the rhizosphere and root tissue. To do this, the draft genome sequence of the biocontrol fungus Trichoderma virens (T. virens) was screened for simple sequence repeats (SSRs) and primers were developed for 12 distinct loci. Primers were evaluated using a global collection of ten isolates where an average of 7.42 alleles per locus was detected. Nei’s standard genetic distance ranged from 0.18 to 0.27 among the isolates, and the grand mean of haploid diversity in AMOVA analysis was 0.693 ± 0.019. Roots of tomato plants were inoculated with different strains and harvested six weeks later. Subsequent PCR amplification identified root-endophytic strains and co-colonization of roots by different strains. Markers were applied to qPCR to quantify T. virens strains in root tissue and to determine their identity using allele-specific melting curve analysis. Thus, the root-endophytic lifestyle of T. virens was confirmed, strains in roots were quantified and simultaneous colonization of roots by different strains was observed.
Highlights
DNA sequencing of the ITS1, 5.8S and ITS2 region identified the ascomycete Hypocrea virens (Chaverri, Samuels and Steward) as the teleomorph of Trichoderma virens (Miller, Giddens and Foster)Arx [1,2,3]
The 39 Mb draft genome sequence of T. virens harbors considerable amounts of repetitive DNA, especially simple sequence repeats suited for microsatellite marker development
Gene duplication events are rarely detected by microsatellite analysis of haploids, but examples do exist e.g., for Beauveria bassiana Vuill
Summary
DNA sequencing of the ITS1, 5.8S and ITS2 region identified the ascomycete Hypocrea virens (Chaverri, Samuels and Steward) as the teleomorph of Trichoderma virens (Miller, Giddens and Foster). The estimation of root colonization efficiencies of different Trichoderma strains, their ability to induce systemic resistance in host plants and their capability of inhibiting plant pathogenic fungi in the rhizosphere under field conditions requires precise re-identification to track and trace strains experimentally released to the root system In this context, diagnostic molecular markers are a prerequisite for studying plant-microbe interaction, the persistence of released strains in the rhizosphere and their impact on rhizosphere microbial communities. T. virens and T. atroviride) are available on the ISTH webpage (www.isth.info/links.php, International Subcommission on Trichoderma and Hypocrea Taxonomy) genomic data have not yet been intensively exploited for the development of locus-specific molecular markers Reports about this marker type are sparse in Trichoderma spp. and comprise solely the development of sequence characterized amplified regions (SCAR markers) obtained from sequence data of cloned random amplified polymorphic DNA (RAPD) fragments [22,23]. Transferability of the markers to other Trichoderma species, in particular to Trichoderma harzianum, was evaluated
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