Molecular analysis of the mating type (MAT1) locus in strains of the heterothallic ascomycete Botrytis cinerea

Botrytis cinerea shows a heterothallic bipolar mating‐type system; homothallism has been occasionally observed. MAT1 genes and flanking regions in the reference strains SAS56 (MAT1‐1) and SAS405 (MAT1‐2) and their monoascosporic progeny were analysed. The two mating types confirmed different sequences of 2513 bp (MAT1‐1) and 2776 bp (MAT1‐2), flanked by near identical regions. In all isolates, each idiomorph included two mating‐type specific genes: MAT1‐1‐1 (1161 bp), encoding an alpha‐domain containing protein, and MAT1‐1‐5 (1301 bp); or MAT1‐2‐1 (1236 bp), encoding a HMG‐domain protein, and MAT1‐2‐4 (712 bp); the latter genes encode putative proteins of unknown function. Truncated MAT1‐1‐1 (670 bp) and MAT1‐2‐1 (92 bp) sequences of the opposite mating‐type were found in the flanking regions. Idiomorph‐specific PCR primer pairs were used to explore the structure of the MAT1 locus in ascospore progeny and field isolates showing homothallic behaviour, and the locus organization in all of them did not differ from that of heterothallic strains. Constitutive expression of all the four mating‐type genes was ascertained by RT‐PCR at four different developmental stages (mycelium, sclerotia at two different stages and apothecia). Antisense transcription of the MAT1‐2‐1 gene with isoforms from alternative splicing was detected. Comparative analysis of MAT1 loci in B. cinerea and in the closely related homothallic Sclerotinia sclerotiorum led to the identification of short nearly identical sequences

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 Remedier™ (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 × 103and 4 × 104conidia g-1of 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

Tracking of Diversity and Evolution in the Brown Rot Fungi Monilinia fructicola, Monilinia fructigena, and Monilinia laxa

Monilinia species are among the most devastating fungi worldwide as they cause brown rot and blossom blight on fruit trees. To understand the molecular bases of their pathogenic lifestyles, we compared the newly assembled genomes of single strains of Monilinia fructicola, M. fructigena and M. laxa, with those of Botrytis cinerea and Sclerotinia sclerotiorum, as the closest species within Sclerotiniaceae. Phylogenomic analysis of orthologous proteins and syntenic investigation suggest that M. laxa is closer to M. fructigena than M. fructicola, and is closest to the other investigated Sclerotiniaceae species. This indicates that M. laxa was the earliest result of the speciation process. Distinct evolutionary profiles were observed for transposable elements (TEs). M. fructicola and M. laxa showed older bursts of TE insertions, which were affected (mainly in M. fructicola) by repeat-induced point (RIP) mutation gene silencing mechanisms. These suggested frequent occurrence of the sexual process in M. fructicola. More recent TE expansion linked with low RIP action was observed in M. fructigena, with very little in S. sclerotiorum and B. cinerea. The detection of active non-syntenic TEs is indicative of horizontal gene transfer and has resulted in alterations in specific gene functions. Analysis of candidate effectors, biosynthetic gene clusters for secondary metabolites and carbohydrate-active enzymes, indicated that Monilinia genus has multiple virulence mechanisms to infect host plants, including toxins, cell-death elicitor, putative virulence factors and cell-wall-degrading enzymes. Some species-specific pathogenic factors might explain differences in terms of host plant and organ preferences between M. fructigena and the other two Monilinia species

First Report of Pseudomonas Grapevine Bunch Rot Caused by Pseudomonas syringae pv. syringae .

Pseudomonas syringae pv. syringae, a Gammaproteobacterium belonging to genomospecies 2 within the P. syringae complex, is distributed worldwide, and it is responsible for bacterial canker on >100 different hosts, including the grapevine. P. syringae pv. syringae induces necrotic lesions in the leaf blades, veins, petioles, shoots, rachis, and tendrils on grapevine cultivars in different areas. P. syringae pv. syringae has been associated with severe economic losses in different grape cultivars in Australia, where it causes inflorescence rot. In midsummer to late summer 2017, symptoms of berry rots differing from those caused by the common berry rots agents were observed in different cultivar Red Globe vineyards of Apulia (southern Italy). As proven by fulfillment of Koch's postulates, these symptoms were caused by a bacterium that, according to the results of biochemical, physiological, nutritional, antimicrobial activity, and pathogenicity tests and sequencing of 16S ribosomal DNA, gyrB, rpoB, and rpoD genes, was identified as P. syringae pv. syringae. This is the first report of Pseudomonas grapevine bunch rot

Specific SCAR Primers for Fungi Associated with Wood Decay of Grapevine

RAPD (Random Amplified Polymorphic DNA) analysis, a technique based on the polymerase chain reaction, was applied to explore variation in 178 isolates of Fomitiporia punctata, 94 of Phaeomoniella chlamydospora and 34 of Phomopsis viticola, selected as being representative of fungal populations from different vineyards and locations. The analysis showed a broad genetic variability in F. punctata and a very high genetic uniformity in P. chlamydospora. With P. viticola, isolates belonging to different vegetative compatibility groups were investigated; the analysis evidenced high genetic similarity among isolates within groups and broad inter-group variation. For each pathogen, specific RAPD markers were selected, cloned and sequenced. The obtained sequences were used to design sequence-characterised amplified region (SCAR) primers specific for each pathogen. These are being used to develop molecular diagnostic tools

A new semi-selective medium for the ochratoxigenic fungus Aspergillus carbonarius

Aspergillus carbonarius (Bainier) Thom. and Aspergillus niger Van Tieghem are common fungal contaminants of several commodities, including grapes. A. carbonarius is the most important, if not the exclusive, responsible for wine contamination by ochratoxin A (OTA) in Mediterranean areas. Identification of A. carbonarius is made difficult by its high similarity with A. niger, the Aspergillus species most common on grapes, as well as with other species of the section Nigri of this genus. Hence, it requires deep knowledge of fungal taxonomy. A semi-selective medium based on Malt Extract Agar amended with appropriate antibiotics (chloramphenicol and chlorretracycline) and fungicides (dichloran and boscalid) was developed in order to speed up the quantitative detection of A. carbonarius in grapes and musts and improve risk assessment of OTA contamination in wine