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

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 OPEN ACCESS Diversity 2015, 7 361 confirmed, strains in roots were quantified and simultaneous colonization of roots by different strains was observed

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

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

Fungal genera containing relevant phytopathogens combated with effective fungicides.

<p><i>Fusarium</i>/<i>Gibberella</i> spp. were not effectively suppressed by fungicide application and accumulated in all intensive treatments, particularly in pre-crop maize samples (WW1). In contrast, <i>Mycospharella</i>/<i>Septoria</i> spp. were only enriched in pre-crop rapeseed samples (WW2) and more abundant in the MP extensive treatment that never received fungicides in the long-term field trial. <i>Fusarium</i>/<i>Gibberella</i> were detectable with the ITS1 (designated with a red dot) and ITS2 (designated with a blue triangle) primers, and <i>Mycosphaerella</i>/<i>Septoria</i> only with the ITS1 primers. For abbreviations refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195345#pone.0195345.g003" target="_blank">Fig 3</a>.</p

Fungal community diversity indices according to Simpson and Shannon.

<p>Fungal community diversity indices according to Simpson and Shannon.</p

Venn diagrams depicting the numbers and percentages of fungal genera in differently managed soils.

<p>For the two winter wheat fields WW1 pre-crop maize (left) and WW2 pre-crop rapeseed (right), absolute numbers and percentages are given. Soil samples were investigated from mould-board plough intensive (MP_int), mould-board plough extensive (MP_ext), conservation tillage intensive (CT_int) and CT extensive variants (CT_ext) in four replicates. Two threshold values were applied (minimum relative abundance 0.01% and reproducibility with minimum presence in two out of four replicates). Genera are listed in descending order of relative abundance (*genera including plant beneficial or ^<b>#</b>plant pathogenic species). Unique genera in distinct soil treatments are shown below the respective figure panels. Genera in bold appeared in both pre-crops.</p

Selected putative phytopathogenic or plant beneficial fungal genera.

<p>Selected putative phytopathogenic or plant beneficial fungal genera.</p

PermANOVA analysis of ITS1 and ITS2 data based on Bray-Curtis dissimilarities (10,000 permutations).

<p>PermANOVA analysis of ITS1 and ITS2 data based on Bray-Curtis dissimilarities (10,000 permutations).</p

Putative phytopathogenic and plant beneficial fungal genera present in different soil treatments.

<p>Putative phytopathogenic and plant beneficial fungal genera present in different soil treatments.</p

Wheat grain yield depending on pre-crop and soil management.

<p>MP, mould-board plough <i>vs</i>. CT, conservation tillage cultivator and int, intensive <i>vs</i>. ext, extensive N fertilization.</p

Influence of ITS1 and ITS2 data on significance levels of Simpson and Shannon diversity indices.

<p>Influence of ITS1 and ITS2 data on significance levels of Simpson and Shannon diversity indices.</p