No abstract

Phytoplasmas, uncultivable phloem-limited phytopathogenic wall-less bacteria, represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. Phytoplasma membrane proteins are in direct contact with hosts and are presumably involved in determining vector specificity. Such a role has been proposed for phytoplasma transmembrane proteins encoded by circular extrachromosomal elements, at least one of which is a plasmid. Little is known about the interactions between major phytoplasma antigenic membrane protein (Amp) and insect vector proteins. The aims of our work were to identify vector proteins interacting with Amp and to investigate their role in transmission specificity. In controlled transmission experiments, four Hemipteran species were identified as vectors of “Candidatus Phytoplasma asteris”, the chrysanthemum yellows phytoplasmas (CYP) strain, and three others as non-vectors. Interactions between a labelled (recombinant) CYP Amp and insect proteins were analysed by far Western blots and affinity chromatography. Amp interacted specifically with a few proteins from vector species only. Among Amp-binding vector proteins, actin and both the α and β subunits of ATP synthase were identified by mass spectrometry and Western blots. Immunofluorescence confocal microscopy and Western blots of plasma membrane and mitochondrial fractions confirmed the localisation of ATP synthase, generally known as a mitochondrial protein, in plasma membranes of midgut and salivary gland cells in the vector Euscelidius variegatus. The vector-specific interaction between phytoplasma Amp and insect ATP synthase is demonstrated for the first time, and this work also supports the hypothesis that host actin is involved in the internalization and intracellular motility of phytoplasmas within their vectors. Phytoplasma Amp is hypothesized to play a crucial role in insect transmission specificity.

Studies of phytoplasma-insect vector interactions and epidemiological surveys of plant yellows associated with the stolbur phytoplasma (StolP) require the identification of relevant candidate genes and typing markers. A recent StolP genome survey identified a partial coding sequence, SR01H10, having no homologue in the "Candidatus Phytoplasma asteris" genome but sharing low similarity with a variable surface protein of animal mycoplasmas. The complete coding sequence and its genetic environment have been fully characterized by chromosome walking. The vmp1 gene encodes a protein of 557 amino acids predicted to possess a putative signal peptide and a potential C-terminal transmembrane domain. The mature 57.8-kDa VMP1 protein is likely to be anchored in the phytoplasma membrane with a large N-terminal hydrophilic part exposed to the phytoplasma cell surface. Southern blotting experiments detected multiple sequences homologous to vmp1 in the genomes of nine StolP isolates. vmp1 is variable in size, and eight different vmp1 RsaI restriction fragment length polymorphism types could be distinguished among 12 StolP isolates. Comparison of vmp1 sequences revealed that insertions in largest forms of the gene encode an additional copy of a repeated domain of 81 amino acids, while variations in 11-bp repeats led to gene disruption in two StolP isolates. vmp1 appeared to be much more variable than three housekeeping genes involved in protein translation, maturation, and secretion and may therefore be involved in phytoplasma-host interactions.

A serious outbreak of flavescence dorée (FD) was reported in Piemonte, northwestern Italy, in 1998, and since then, the disease has compromised the economy of this traditional wine-growing area, even following the application of compulsory insecticide treatments to control Scaphoideus titanus, the vector of the causal phytoplasma. Affected vines show severe symptoms, varying according to the cultivar, and are rogued to reduce disease spread. Following winter and pruning, a previously affected vine may appear symptomless and free of phytoplasmas in its aerial as well as its root system, even by nested-polymerase chain reaction assays. Such plants are considered to be "recovered". Since 1998 homogenous data on the incidence of newly infected, healthy, or recovered plants productivity, presence of vectors, and treatment schedules have been collected in seven severely affected vineyards of southern Piemonte for 5 years (1999 to 2003). Infectivity and recovery rates were also calculated each year. From 1999 to 2003, the average number of healthy plants decreased and the numbers of recovered plants and those with symptoms increased. Productivity of recovered vines, although lower than that of healthy ones, was always higher than that of vines with symptoms and was not influenced by the time elapsed from date of recovery. The relationships between the ln-transformed number of vectors trapped in the vineyards the previous year and the infection and the recovery rates were fitted by an exponential (R(2) = 0.95) and an asymptotic (R(2) = 0.93) model, respectively.

Flavescence dorée phytoplasma (FDP) titre in two red grapevine cultivars, Barbera and Nebbiolo, was measured over the vegetative seasons of two consecutive years in two vineyards of the Piemonte Region (northwestern Italy), with a double absolute quantification of FDP cells and grapevine DNA in real‐time PCR. The relationships of pathogen concentration to cultivar susceptibility and symptom severity were investigated. FDP titre was always higher in cv. Barbera than in cv. Nebbiolo infected vines, and this difference was significant at early and late summer samplings of 2008 and at early summer sampling of 2009. A seasonal trend in FDP concentration (low in spring, high in early summer and intermediate in late summer) was conserved for cvs Barbera and Nebbiolo in both years and vineyards. Considering both cultivars and years from both vineyards, a significant positive correlation between FDP concentration and symptom severity was found in the spring samples. Regarding the FDP strains (‐C or ‐D), no differences in pathogen titres were detected for either cultivar. Similarly, the presence of another grapevine yellows phytoplasma, bois noir, a subgroup 16SrXII‐A phytoplasma, in mixed infection with FDP strains had no effect on FDP concentration. These results demonstrate for the first time that grapevine cultivars with different susceptibility to FDP support different pathogen titres.