Six stable hybridoma cell lines secreting monoclonal antibodies (MAbs) to Grapevine virus D (GVD) were obtained by fusing spleen cells of immunized BALB/c mice with mouse myeloma cell line Sp 2/0-Ag 14, In ELISA all MAbs detected the virus in Nicotiana leaf extracts or cortical shavings from mature grapevine canes, The use of a polyclonal antiserum for coating plates and of monoclonal antibodies and antimouse-conjugated antibodies for antigen detection, gave highly efficient and reproducible results for identification of GVD in field-grown grapevines. The reliability of the ELISA kit was confirmed by GVD-transmission tests to herbaceous hosts, using in vitro explants as inoculum, 223 vines affected by one or more of the 4 syndroms of the rugose wood complex (Kober stem grooving, Corky bark, LN stem grooving and Rupestris stem pitting) were tested in ELISA for the detection of Grapevine virus A (GVA), Grapevine virus B (GVB) and GVD and by Western blot for the detection of Grapevine rupestris stem pitting associated virus (GRSPaV). The possible cause-effect relationship between GVA and KSG, GVB and Co, and GRSPaV and RSP was confirmed, but no consistent association was found between GVD and any of the 4 above syndromes, Intriguingly, a reduction in the expression of stem pitting symptoms in V. rupestris (from 90 % to 75 %) and of stem grooving symptoms in Kober 5BB (from 95 % to 70 %) was observed when vitiviruses and GRSPaV were contemporarily present in the same indicator. Preliminary data of a survey involving 676 grapevine samples showed a high incidence (31 %) of GVD, regardless of the geographical origin of samples.

Research Note Grapevine trichovirus A (GVA) and grapevine trichovirus B (GVB) were successfully transferred with bulk transmission trials under controlled conditions, from infected grapevines to herbaceous hosts by Pseudococcus affinis MASK., a pseudococcid mealybug that may attack grapevines. P. affinis is the fourth mealybug species capable of vectoring GVA and GVB, confirming that transmission by mealybugs of grapevine trichoviruses may not be species-specific.

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AIM:Evaluation of the genetic variability of stolbur phytoplasma infecting grapevines, bindweeds and vegetables, collected in different central and southern Italian regions.MATERIALS AND RESULTS:Phytoplasma isolates belonging to stolbur subgroup 16SrXII-A were subjected to molecular characterization by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP), to investigate two different nonribosomal genes: tuf and vmp1. In grapevines, 32% of samples were infected by tuf-a type and 68% by tuf-b type, with different relative incidences in the regions surveyed. All herbaceous samples (bindweeds, tomato, tobacco, pepper, celery) were infected by tuf-b. The gene vmp1 showed higher polymorphism in grapevines (nine profiles) than herbaceous plants (six) by RFLP analysis, in agreement with nucleotide sequences' analysis and virtual digestions.CONCLUSIONS:The phylogenetic analysis of vmp1 gene sequences supports the RFLP data and demonstrates the accuracy of RFLP for preliminary assessments of genetic diversity of stolbur phytoplasmas and for screening different vmp types.SIGNIFICANCE AND IMPACT OF THE STUDY:Stolbur represents a serious phytosanitary problem in the areas under investigation, owing to heavy economic losses in infected grapevines and vegetables. Molecular information about the complex genotyping of the vmp1 gene provides useful data towards a better understanding of stolbur epidemiology. Moreover, this study clarifies some different vmp1 genotype classifications of stolbur, providing molecular data in comparison with previous investigations.

Studies were carried out in north Sardinia (Italy) in Chardonnay and Vermentino grapevines infected with "bois noir" to verify productive performance of the yields of plants healthy, infected and in recovery for one or two years. Statistical significant differences were observed in the yield among the symptomatic and the healthy plants, and among the healthy plants and those in recovery for one year, but not always between healthy plants and those in recovery for two years. Not statistically significant was the comparison between the two recovered groups. Productive increase was progressive in plants recovered for one year and in those in symptomatic remission for two years.

Myrtus communis is a bushy species of the Mediterranean area that produce very popular liquor: Red mirto and White mirto. Preliminary studies in North Sardinia, resulted in the observation of a severe symptomatology associated with phytoplasma presence. These studies continued, and recently a total of 33 symptomatic plants belonging to different cultivars in two plantations were mapped and repeatedly tested using molecular methods. Phytoplasmas belonging to the 16SrI, 16SrII-F, 16SrIII, 16SrV-A, 16SrX-A, and 16SrXII-A subgroups were identified. Molecular assays on potential insect vectors showed that 16SrX-A+16SrI-B, 16SrXII-A and 16SrIII subgroups were present.

A study was made on the recovery genotypes local grapevine with the aim to conserve them and to evaluate their health status and thus to improve the quality of the propagation material. Clone accessions of 61 vines were grafted and placed in a germoplasma field. The varieties that were considered enologically valuable were subjected to sanitary selection. Some vines showed indubitable healthy qualities, and should be immediately evaluated from an economical point view and then certificated. Possible sanitation treatments were also evaluated, with the aim of extending the number clone accessions to be included in experimental trials to access their yield .

Epidemiological surveys were performed in Northern Sardinia (Italy) in a 10-year-old vineyard affected by "Bois noir" disease. Samples collected between May and October 2003 from chlorotic and stunted weeds belonging to 14 different taxonomic groups were indexed molecularly for detection of phytoplasmas. Nested polymerase chain reaction (PCR) assays using primers specific for the phytoplasma 16SrDNA gene showed three of six Calendula arvensis, one of two Solanum nigrum, and one of seven Chenopodium spp. assayed positive. Restriction fragment length polymorphism analyses and sequencing of amplified 16SrDNA fragments identified a putative phytoplasma in the ribosomal subgroup 16SrII-E. Further characterization of the rps3 gene, coding a ribosomal protein, confirmed the identification. However. the weeds and leafhopper species collected in the vineyard tested negative by PCR assays for the Stolbur phytoplasma, the causal agent of "Bois noir". This is the first report of a phytoplasma of the 16SrII-E subgroup infecting C. arvensis, S. nigrum, and Chenopodium spp.

Epidemiological research was carried out in two vineyards affected by "Bois noir" (BN). Auchenorrhyncha potential vectors of BN, were monitored periodically between May to November 2004 in a vineyard and between May to June 2005 in the other. Auchenorrhyncha samples were tested to assess phytoplasmas presence using PCR and RFLP. Euscelis ttneolatus was positive to 16SrI-C ("Clover phyllody" reference strain) in 2004 while, in 2005, at the preimaginal age, at the 16SrXII -A ("Stolbur" reference strain) phytoplasmas. Exitianus taeniaticeps acquired 16SrI-B ("Maryland aster yellow" reference strain), 16SrV-A ("Elm yellow" reference strain) and 16SrX-C ("Pear decline" reference strain) phytoplasmas in 2004. Resulted news host of phytoplasmas: Psammotettix alienus was positive to 16SrI-B, 16SrV-A, 16SrX- A phytoplasmas ("Apple proliferation" reference strain) in 2004 and to 16SrXII -A in 2005. K lineolatus and P. alienus for 16SrXII -A E. taeniaticeps for 16SrV-A 16SrX-C and P. alienus for 16SrV-A and 16SrX-A Researches on the effective epidemiological role of K lineolatus and P. alienus in BN are in progress.