SSR Markers for Trichoderma virens: Their Evaluation and Application to Identify and Quantify Root-Endophytic Strains

Abstract

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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.

Keywords

• Hypocrea virens
• simple sequence repeat
• microsatellite
• molecular marker
• fragment analysis
• root endophyte
• biocontrol
• strain identification
• qPCR
• monitoring