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

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

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 Botrytis cinerea

Botrytis cinerea displays an extraordinary variability in phenotypic traits, making it a model for studying sources of variation in filamentous fungi and in particular in plant pathogens. The whole genome sequence was recently made available and is sustaining an impressive progress of knowledge. The present review aims at giving an updated picture on the genetic features of this fungal pathogen and, in particular, on mechanisms underlying its broad variation and adaptation capability, i.e. mating system and sexual behavior and other sources of variation (chromosome number, mycoviruses, transposons, vegetative compatibility, etc.), as well as on tools available for its genetic manipulation

Genetics of fungicide resistance

Acquired resistance to fungicides in fungal plant pathogens is a challenge in modern crop protection. Fungi are indeed very able to adapt to changing environmental conditions, such as the introduction of a new fungicide in the agricultural practice. Several genetic mechanisms may underlay fungicide resistance and influence the chance and time of its appearance and spreading in fungal populations. Resistance may be caused by mutations in major genes (monogenic or oligogenic resistance) or in minor genes (polygenic resistance) which may occur in nuclear genes as well as in cytoplasmic genes. They are immediately expressed in haploid fungi, while they may be dominant or recessive in diploid fungi. Allelic variants may cause different levels of resistance and/or different negative pleiotropic effects on the fitness of resistant mutants. The sexual process, where occurring, plays an important role in releasing new recombinant genotypes in fungal populations. Heterokaryosis provides multinucleate fungi with a further mechanism of adaptation. Resistant mutants can be obtained from samples representative of field population of a pathogen or under laboratory conditions through selection of spontaneous mutations or following chemical or physical mutagenesis. Nowadays, molecular tools, such as gene cloning, sequencing, site-directed mutagenesis and gene replacement, make genetic studies on fungicide resistance amenable even in asexual fungi for which classical genetic analysis of meiotic progeny is not feasible