BackgroundGrapevine downy mildew, caused by Plasmopara viticola, is a very serious disease affecting mainly Vitis vinifera cultivated varieties around the world. Breeding for resistance through the crossing with less susceptible species is one of the possible means to reduce the disease incidence and the application of fungicides. The hybrid Bianca and some of its siblings are considered very promising but their resistance level can vary depending on the pathogen strain. Moreover, virulent strains characterized by high fitness can represent a potential threat to the hybrid cultivation.ResultsThe host response and the pathogen virulence were quantitatively assessed by artificially inoculating cv Chardonnay, cv Bianca and their siblings with P. viticola isolates derived from single germinating oospores collected in various Italian viticultural areas. The host phenotypes were classified as susceptible, intermediate and resistant, according to the Area Under the Disease Progress Curve caused by the inoculated strain. Host responses in cv Bianca and its siblings significantly varied depending on the P. viticola isolates, which in turn differed in their virulence levels. The fitness of the most virulent strain did not significantly vary on the different hybrids including Bianca in comparison with the susceptible cv Chardonnay, suggesting that no costs are associated with virulence. Among the individual fitness components, only sporangia production was significantly reduced in cv Bianca and in some hybrids. Comparative histological analysis revealed differences between susceptible and resistant plants in the pathogen diffusion and cytology from 48 h after inoculation onwards. Defence mechanisms included callose depositions in the infected stomata, increase in peroxidase activity, synthesis of phenolic compounds and flavonoids and the necrosis of stomata and cells immediately surrounding the point of invasion and determined alterations in the size of the infected areas and in the number of sporangia differentiated.ConclusionsSome hybrids were able to maintain an intermediate-resistant behaviour even when inoculated with the most virulent strain. Such hybrids should be considered for further field trials.

Observations on cytological and ultrastructural changes in Plasmopara viticola oospores were carried out during the overwintering period. Three types of oospores were observed. Type I, characterized by a thin inner oospore wall (IOW), large lipid globules and two nuclei, was recovered only in samples collected in October. These oospores were considered to be immature. Maturation occurred during November and involved a noticeable increase in thickness of the IOW, fusion of nuclei, formation of an ooplast and break up of large lipid globules into smaller ones (type II oospores). A few oospores (type III) showed abnormal organization with very large lipid globules and less frequently discernible nuclei. IOW solubilization, dissolution of the ooplast and lipid globules and nuclear division were the first detectable events during oospore germination. Germinating oospores produce a germ tube which was terminated by a sporangium. In its young stage, the sporangium had a thick wall and an unusual multi-layered membrane. During this phase, nuclear divisions took place in the sporangium. While sporangium development progressed, the ribosome density in the cytoplasm decreased and mitochondria, initially roundish with evident cristae, became their usual tubular profile. The plasma membrane had a typical structure and storage organelles, such as finger print vacuoles and lipid globules, became more numerous in the cytoplasm. Larger vacuoles contained the flagella of differentiating zoospores.

Investigations carried out on secondary metabolites produced in culture by a hypovirulent strain of Cryphonectria parasitica allowed the isolation of several compounds which were characterized by NMR analysis and derivatization reactions. The most abundant metabolite was a new compound, called cryphonectric acid (1). Other metabolites were diaporthin, the only known phytotoxic compound isolated from both virulent and hypovirulent strains of C. parasitica, (+)-orthosporin, and L-p-hydroxyphenyllactic acid (HOPLA). Root growth activity of the purified compounds was evaluated both on tomato seedlings and maize subapical segments.

QoI fungicides, inhibitors of mitochondrial respiration at the Qo site of cytochrome b in the mitochondrial bc(1) enzyme complex, are commonly applied in vineyards against Plasmopara viticola (Berk. & MA Curtis) Berl. & De Toni. Numerous treatments per year with QoI fungicides can lead to the selection of resistant strains in the pathogen population owing to the very specific and efficient mode of action. In order to evaluate the resistance risk and its development, two different methods, biological and molecular, were applied to measure the sensitivity of oospores differentiated in vineyards, both treated and untreated with azoxystrobin, from 2000 to 2004. Assays using oospores have the advantage of analysing the sensitivity of bulked samples randomly collected in vineyards, describing accurately the status of resistance at the end of the grapevine growing season. Both methods correlated well in describing the resistance situation in vineyards. QoI resistance was not observed in one vineyard never treated with QoI fungicides. In the vineyard where azoxystrobin had been used in mixture with folpet, the selection of QoI-resistant strains was lower, compared with using solely QoI. In vineyards where QoI treatments have been stopped, a decrease in resistance was generally observed.

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