The need of accurate and reliable methods for DNA isolation and plant species identification in foodstuffs is of great importance, especially in the protection of high added value products. Fresh foods, which are not subjected to any modifications, are suitable for many kind of analysis; for processed products, such as musts, wines, olive oils, and pasta, the situation may be more complicated due to DNA fragmentation and, in the worst case, by its degradation. This work aimed to establish an exhaustive and reproducible analytical procedure for table grape DNA tracing in industrial musts. Three different DNA extraction methods were initially compared and DNA was tested in PCR for its suitability for the amplification reaction of microsatellite markers or simple sequence repeats (SSRs). An optimized DNA extraction method for microsatellite amplification was developed and adapted for industrial musts. Two SSR-based molecular methods, High Resolution Melting and capillary electrophoresis, were tested and the markers VrZAG62 and VrZAG79 were found to be the most informative. High Resolution Melting analysis, here applied for the first time on musts, proved to be the method of choice for a preliminary screening using four cultivars chosen as references and different DNA mixtures prepared in laboratory. Capillary electrophoresis, providing allele size, allowed a fine genotyping of musts in comparison with reference cultivars. The LOD6 of a single grape cultivar in mixture with other varieties was also determined at 2.5 ng. Merging the information of the two molecular analyses applied to real samples, we demonstrated that is possible to discover case of musts adulterated with table grapes, and we propose our procedure in controlling musts quality and origin certification.

Aspergillus carbonarius, belonging to the groupNigri, is the main species responsible for contamination by ochratoxin A (OTA) in grapes and derivative products. OTA can accumulate in the mycelium and in black conidia of the fungus and released into the matrix. Here, we have deleted inA. carbonariusthealb1orthologue gene ofA. fumigatus, involved in melanin biosynthesis. ThreeA. carbonariusΔalb1mutants were characterized for morphologic traits and OTA production on different media and temperatures. Δalb1mutants showed a fawn color of conidia associated with a significant reduction of the conidiogenesis and a statistically significant increase (p≤ 0.01) of total OTA production as compared to the wild type (WT) strain. Thealb1gene somehow affected OTA partitioning since in Δalb1mutants OTA amount was lower in conidia and was more abundantly secreted into the medium as compared to the WT. On grape berries the Δalb1mutants and the WT caused lesions with similar sizes but OTA amount in berry tissues was higher for the mutants. These results demonstrate thatA. carbonariusconidia pigmentation is largely dependent on polyketide biosynthesis. The gene is not directly involved in virulence and its deletion affects morphological features and OTA production in the fungus.

BACKGROUND: QoI fungicides, inhibitors of mitochondrial respiration, are considered to be at high risk of resistance development. In several phytopathogenic fungi, resistance is causedby mutations (most frequentlyG143A)inthemitochondrial cytochrome b (cytb) gene. The genetic and molecular basis of QoI resistance were investigated in laboratory and field mutants of Botryotinia fuckeliana (de Bary)Whetz. exhibiting in vitro reduced sensitivity to trifloxystrobin. RESULTS: B. fuckeliana mutants highly resistant to trifloxystrobin were obtained in the laboratory by spontaneous mutations in wild-type strains, or from naturally infected plants on a medium amended with 1–3 mg L−1 trifloxystrobin and 2mM salicylhydroxamic acid, an inhibitor of alternative oxidase. No point mutations were detected, either in the complete nucleotide sequences of the cytb gene or in those of the aox and Rieske protein genes of laboratory mutants, whereas all field mutants carried the G143A mutation in the mitochondrial cytb gene. QoI resistance was always maternally inherited in ascospore progeny of sexual crosses of field mutants with sensitive reference strains. CONCLUSIONS: The G143A mutation in cytb gene is confirmed to be responsible for field resistance to QoIs in B. fuckeliana. Maternal inheritance of resistance to QoIs in progeny of sexual crosses confirmed that it is caused by extranuclear genetic determinants. Inlaboratory mutants theheteroplasmic state of mutated mitochondria could likelyhampertheG143Adetection, otherwise other gene(s) underlying different mechanisms of resistance could be involved.

Monilinia fructigena (phylum Ascomycota, family Sclerotiniaceae) is a plant pathogen that causes brown rot and blossom blight in pome fruit and stone fruit of the Rosaceae family, which can cause significant losses in the field and mainly postharvest. The aim of this study was to create a high-quality draft of the M. fructigena genome assembly and annotation that provides better understanding of the epidemiology of the pathogen and its interactions with the host(s) and will thus improve brown rot management.

BACKGROUND: Succinate dehydrogenase inhibitors (SDHIs), interfering with fungal respiration, are considered to be fungicides at medium to high risk of resistance. Boscalid was the first molecule belonging to the SDHIs that was introduced for the control of Botryotinia fuckeliana. A range of different target-site mutations leading to boscalid resistance have been found in field populations of the fungus. The different types of mutation confer different cross-resistance profiles towards novel SDHIs, such as the recently introduced fungicide fluopyram. This study combines the determination of cross-resistance profiles and the setting-up of methods for fast molecular detection of the mutations. RESULTS: By means of in vitro tests, a range of SdhB mutations were characterised for resistance levels towards boscalid and fluopyram. SdhB mutations conferring P225L and P225F substitutions conferred high resistance to boscalid and high or moderate resistance to fluopyram respectively. Mutants carrying the N230I replacement were moderately resistant to both SDHIs. Substitutions at position H272 responsible for a high level of resistance to boscalid conferred sensitivity (H272R), hypersensitivity (H272Y) or moderate resistance (H272V) to fluopyram. Allele-specific (AS) PCR was developed and used for genotyping 135 B. fuckeliana isolates. The assay confirmed the strict association between resistance profiles and allelic variants of the SdhB gene. Real-time AS-PCR proved to be sensitive and specific for quantitative detection of different SDHI-resistant genotypes. CONCLUSION: Fluopyram-resistant mutants are currently rarely detected in the field sprayedwith boscalid, but thismay change with intensive exposureof the fungalpopulation tofluopyram.PCRassays/methodsdeveloped inthe study provide tools for fast monitoring of field populations and observing possible changes in population composition following fluopyram introduction, useful for the setting-up of appropriate preventivemeasures.