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

Observations on the Fungi Associated with Esca and on Spatial Distribution of Esca-Symptomatic Plants in Apulian (Italy) Vineyards

The paper reports the results of observations on the fungi associated with deteriorated wood of esca affected vines and the spatial distribution of diseased plants in 21 vineyards located in Apulia (Southern Italy). Examination of over 43,000 plants revealed that the incidence of plants showing symptoms of esca was 12% (5-18%) in vineyards younger than 10 years and 22% (4-54%) in older ones. The most common deteriorations of the wood were a white rot of soft consistency and a dark brown discoloration of a hard consistency including longitudinal black streaks. Mycological analysis was carried out on 554 diseased vines from 5 vineyards. Phellinus sp. was frequently isolated from white rot, whereas Phaeoacremonium spp., Botryosphaeria spp. and Eutypa lata were more often isolated from dark brown tissues. Preliminary observations of maps of plants with symptoms of esca seem to indicate a tendency to aggregation, especially in young vineyards

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

Genetics of sexual compatibility and resistance to benzimidazole and dicarboximide fungicides in isolates of Botryotinia fuckeliana (Botrytis cinerea) from nine countries

Field isolates and laboratory mutants of Botryotinia fuckeliana (Botrytis cinerea) from nine different countries were crossed with reference strains of known mating type, to determine the genetic bases of differences in sexual fertility and in resistance to benzimidazole and dicarboximide fungicides. Most isolates were heterothallic, and carried the MA T1-1 or MA T1-2 mating-type allele. A few isolates were fertile with strains of opposite mating types, due to the presence of both mating-type alleles in heterokaryotic mycelia. The MA T1-1 and MA T1-2 alleles were fairly evenly distributed among the field isolates from most countries. Seventy-six isolates displayed resistance to benzimidazole and/or dicarboximide fungicides due to mutant alleles of the Mbc1 and/or Daf1 genes. Unlike wild-type and low-resistant isolates, those carrying the high-resistance Mbc1HR alleles were sensitive to N-phenylcarbamates. With one exception, isolates carrying the high-resistance Daf1HR alleles were hypersensitive to media of high osmolarity. Heterokaryons containing Daf1HR alleles together with sensitive Daf1S or low-resistance Daf1LR alleles in separate nuclei combined high resistance to dicarboximides with relative tolerance of media with high osmolarity