Abstract
Trichoderma asperellum strain icc012 and Trichoderma gamsii strain icc080, the microbial active ingredients of RemedierTM (ISAGRO, Novara, Italy), are biocontrol agents (BCAs) employable for crop protection against a wide range of fungal pathogens, including soil-borne pathogens and fungi involved in grapevine trunk disease. In this study, single and duplex real-time quantitative PCR (qPCR) methods to detect and quantify T. asperellum and T. gamsii were developed. Primers/probe sets were designed on the T. asperellum and T. gamsii rpb2 genes and tested for specificity on a panel of microorganisms commonly associated with grape wood and soil. No differences were observed comparing single- and duplex-qPCR assays on different BCAs, 1 pg of target DNA was detected approximately at Cq = 34. R2-values and the efficiency were always equal to 0.99 and >80%, respectively. The detection limit of the duplex-qPCR assay on artificially inoculated samples was 2 × 103 and 4 × 104 conidia g-1 of grape wood tissue and soil, respectively. The methods will be useful to better schedule BCA application in the field and in grapevine nurseries, as well as for investigating the dynamic of BCA populations.
Highlights
Since combining two or more beneficial microbes in a biopesticide would be advantageous to biocontrol agents (BCAs) management (Raupach and Kloepper, 1998), the mixture of T. asperellum strain icc012 and T. gamsii strain icc080 is used in RemedierTM to increase the activity and widening the environmental adaptability (Liguori, 2016)
This microbial pesticide is registered against soil-borne pathogens affecting horticultural crops and turfs, and it is the only BCA-based plant protection products (PPPs) allowed in Italy to control pathogens associated with grapevine trunk diseases (GTDs)
single-nucleotide polymorphisms (SNPs) identified in intra- and external species alignments of the rpb2 gene sequences were used for species-specific primers/probe sets design
Summary
The genus Trichoderma, a cosmopolitan inhabitant of soil and plant root ecological niches includes the most explored BCA species, representing over 60% of all the currently registered BCAs used for the management of plant pathogens (Benítez et al, 2004; Harman et al, 2004; Mukherjee et al, 2013; Hyder et al, 2017; Sharma et al, 2017). Their biological activity is closely related to the ability of: (i) producing a wide range of lysing enzymes; (ii) degrading substrates; (iii) possessing high resistance to microbial inhibitors; (iv) competing for nutrients and space, (v) acting directly through mycoparasitism, (vi) producing antifungal metabolites; (vii) inducing systemic resistance in plants This microbial pesticide is registered against soil-borne pathogens affecting horticultural crops and turfs, and it is the only BCA-based PPP allowed in Italy to control pathogens associated with grapevine trunk diseases (GTDs)
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