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Diversity 2015, 7(4), 360-384; doi:10.3390/d7040360

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

1
Institute of Bioanalytical Sciences (IBAS), Anhalt University of Applied Sciences, Strenzfelder Allee 28, Bernburg 06406, Germany
2
Department of Applied Biosciences and Process Engineering, Anhalt University of Applied Sciences, Bernburger Straße 55, Köthen 06366, Germany
3
INSTAND e.V., Society for Promoting Quality Assurance in Medical Laboratories e.V., Ubierstr. 20, Düsseldorf 40223, Germany
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed.
Academic Editor: Michael Wink
Received: 2 September 2015 / Revised: 19 October 2015 / Accepted: 19 October 2015 / Published: 3 November 2015
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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. View Full-Text
Keywords: Hypocrea virens; simple sequence repeat; microsatellite; molecular marker; fragment analysis; root endophyte; biocontrol; strain identification; qPCR; monitoring Hypocrea virens; simple sequence repeat; microsatellite; molecular marker; fragment analysis; root endophyte; biocontrol; strain identification; qPCR; monitoring
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Geistlinger, J.; Zwanzig, J.; Heckendorff, S.; Schellenberg, I. SSR Markers for Trichoderma virens: Their Evaluation and Application to Identify and Quantify Root-Endophytic Strains. Diversity 2015, 7, 360-384.

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