A Ready-to-Use Single- and Duplex-TaqMan-qPCR Assay to Detect and Quantify the Biocontrol Agents Trichoderma asperellum and Trichoderma gamsii

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

Schede fitopatologiche: Funghi

In: Interreg II Italia-Albania Asse 6. Cooperazione transfrontaliera; Misura 6.2. Cooperazione in agricoltura; Sottomisura 6.2.C Introduzione di innovazioni tecnologiche nei processi produttivi; 10 -Difesa della vite (Boscia D., B. Di Terlizzi, G. Nuzzaci, V. Savino e G. Tauro, coord

Mating System in the Brown Rot Pathogens Monilinia fructicola, M. laxa, and M. fructigena

Monilinia fructicola, M. laxa, and M. fructigena are the most important pathogens responsible for brown rot disease of stone and pome fruits. Information on their mating system and sexual behavior is scant. A mating-type-specific PCR-based assay was developed and applied to 155 Monilinia isolates from 10 countries and 10 different host plants. We showed that single isolates carry only one of two opposite idiomorphs at the MAT1 locus consistent with a heterothallic mating system for all three species. MAT1-1 and MAT1-2 mating types were detected in similar proportions in samples of isolates of each species and hence there do not appear to be genetic obstacles to the occurrence of sexual reproduction in their populations. Inter simple sequence repeat markers suggested that asexual reproduction is prevalent, but that sexual recombination occurs in M. fructicola populations in Italy. The genetic architectures of the MAT1 loci of the three pathogens were analyzed. MAT1-1 and MAT1-2 idiomorphs are flanked upstream and downstream by the APN2 and SLA2 genes and resemble those of Botrytis cinerea and other heterothallic fungi in the family Sclerotiniaceae. Each idiomorph contains a specific couple of genes, MAT1-1-1 (with alpha-box domain) and MAT1-1-5 in MAT1-1, and MAT1-2-1 (with HMG-box domain) and MAT1-2-10 in MAT1-2. Small gene fragments (dMAT1-1-1 and dMAT1-2-1) from the opposite idiomorph were detected close to their flanking regions. Constitutive expression of the four MAT1 genes during vegetative growth was ascertained by transcriptomic analysis (RNA-Seq). Antisense transcription of the MAT1-1-1 and MAT1-2-1 genes and intergenic transcribed regions of the MAT1 locus were detected. These results represent new insights into the mating systems of these three economically-important pathogens which could contribute to improve the knowledge on their population biology

Phomopsis viticola is easily transformed with hph and Bmlr genes

Phomopsis viticola (Sacc.) Sacc. is the phytopathogenic fungus causing a severe disease of grapevine known as Phomopsis cane and leaf spot. Protoplasts from mycelium of R viticola were successfully transformed either with plasmids carrying the bacterial hph gene, conferring resistance to hygromycin B (pAN7-1, pOHT, pOHT-AMA1), or the Bml(r) gene from Neurospora crassa, causing resistance to benzimidazole fungicides (pBT6). Up to more than 300 transformants per mug of plasmid DNA were obtained with the hph marker gene. The highest effectiveness was obtained with pOHT, whereas pBT6 yielded around 25 transformants per mug of plasmid DNA. Southern blot analysis showed the occurrence of multiple integration events in the fungal genome of all tested plasmids. Experiments of co-transformation with pOHT and pBT6 were successful and about 70% of transformants were resistant to both hygromycin B and benomyl. The "Instant Gene Bank" technique and mutagenesis through Restriction Enzyme Mediated Integration (REMI) were attempted. As reported for other phytopathogenic fungi, the REMI technique proved to be a powerful method for obtaining mutant strains with variation in phenotypic traits