Epidemiological observations on ‘Bois Noir’ in Lambrusco grapevine

Lorenzo Milanesi(1), Roberto Bondavalli(2), Nicola Mori(3), Pasquale Mazio(2), Gianni Cavallini(1), Anselmo Montermini(2) and Assunta Bertaccini(4)

(1)Consorzio Fitosanitario Provinciale di Modena, Via Andreoli, 13 - 41100 Modena, Italy
(2)Consorzio Fitosanitario Provinciale di Reggio Emilia, Via Gualerzi, 32 - 42100
Reggio Emilia Italy
(3)Agrea Centro Studi, Via Garibaldi 5 - 37057 San Giovanni Lupatoto (VR) Italy
(4)Dipartimento di Scienze e Tecnologie Agroambientali - Patologia Vegetale, Alma Mater Studiorum, Università di Bologna, Viale Fanin 42 - 40127 Bologna Italy


Introduction
Bois Noir (BN) is a grapevine yellows (GY) associated with the presence of 16SrXII-A phytoplasma (‘Candidatus Phytoplasma solani') of increasing economic importance in the Northern Italy provinces where Lambrusco is cultivated (Bertaccini et al., 2003). BN phytoplasma is transmitted by Hyalesthes obsoletus Signoret (Homoptera Cixiidae) (Alma et al., 2002), and nettle (Urtica dioica) is considered its main host plant. Molecular identification of BN and Flavescence dorée (FD, ribosomal subgroup 16SrV-D, ‘Candidatus Phytoplasma vitis') phytoplasmas was achieved in routine testing in Modena and Reggio Emilia provinces since 2000 (Bertaccini et al., 2001). Further monitoring carried out by Provincial Inspection Services from 2000 to 2004 demostrated the widespread and increasing of grapevine plants showing yellows symptoms.


Materials and Methods
Insects and grapevine samples showing typical yellows symptoms were collected during routine inspections in the vineyards of Modena and Reggio Emilia provinces (Northern Italy). From a total of 1020 grapevine samples, nucleic acids were extracted using 1 g of leaf midribs following the protocol described by Prince et al. (1993). H. obsoletus samples were also extracted from batches of 1 to 4 following the protocol of Zhang et al. (1995). Nucleic acids were diluted to a final concentration of 20 ng/ml in sterile deionized water. One ml of this dilution was used in the amplification assays described below. Direct PCR was performed using universal primer pair P1/P7, both for plants and for insects, while nested PCR reactions on direct PCR products diluted 1:30 in sterile distilled water, were performed using primer pairs M1/B6 or R16F2/R2 (Martini et al., 1999; Lee et al., 1995). Each 25 ml PCR reaction mix contained 2,5 ml 10X PCR buffer, 0,8 U of Taq polymerase (Polymed, Florence, Italy), 0,2 mM dNTPs, 1,5 mM MgCl2 and 0,4 mM each primer. Thirty-five PCR cycles were performed in an automated thermocycler (Biometra, Uno Thermoblock, Gottingen, Germany, EU) as described (Bertaccini et al., 2003). Six ml of PCR products were analysed in a 1% agarose gel stained with ethidium bromide, and then visualized with an UV transilluminator. To differentiate among phytoplasmas, PCR products were digested with TaqI and TruI restriction enzymes; the RFLP fragments were separated in a 5% polyacrylamide gel stained with ethidium bromide, and then visualized with an UV transilluminator.
During 2003 and 2004, in some of the most symptomatic vineyards monitoring of H. obsoletus populations was performed using entomological nets and then aspirators. This method resulted to be more efficient than the use of yellow chromotropic traps (Cavallini et al., 2003). Insect collection by using entomological nets was also carried out every week between the border grapevine rows and inside the drains nearby to the vineyards, among weed plants belonging to genera Urtica, Convolvolus and Medicago. Insects then were classified and tested by nested-PCR as described above to verify phytoplasma presence.
Survival trials of H. obsoletus are carried out in green house using some weed species resulted positive to the BN phytoplasma (Credi et al., 2004) and cultivated in pots.

Results and Discussion
Molecular analysis (PCR/RFLP) carried out on symptomatic plants showed the striking prevalence of BN phytoplasma on FD. Out of 557 samples collected in the Reggio Emilia province, 55,7% were positive to BN whereas 14,4% of the tested plants resulted infected by FD. Regarding the 463 analyses on samples collected in the Modena province, 67,4% samples resulted positive to BN, while those positive to FD were 10,8%; the 2,3% of the Reggio Emilia collected samples appeared positive to both phytoplasmas. A number of symptomatic grapevines was negative: 27,3% in Reggio Emilia and 21% in Modena provinces.
High percentages of symptomatic plants were observed in the first years of BN occurence at vineyards borders with a decreasing gradient to the center of the field as already reported (Credi et al., 2004); however this gradient was progressively disappearing over the years.
Data of H. obsoletus adults' flying period in BN affected areas indicate their presence from the middle of June to the end of August, with a peak in the middle of July. Surveys on adult's capture have statistically shown that they are more present along the perimeter of vineyards rather than in the centre. The insects distribution is correlated to the presence of nettle and other weed species in the areas near to vineyards.
Molecular analysis carried out by PCR/RFLP revealed that in 2003 the 15,1% of field collected H. obsoletus adults were infected with BN phytoplasma, while in 2004 the 26,6% of specimens were positive to the same phytoplasma.
Green house studies were performed to verify the possibility that the vector is able to survive and complete its biological cycle on some perennial dicotyledon, chosen among the most widespread in our viticulture areas, and resulted positive to the BN phytoplasma. The tested species were: Cirsium arvense, Convolvolus arvensis, Medicago sativa, Plantago major, Taraxacum officinale and U. dioica. In our experimental conditions the insect has revealed good survival on P. major, capacity of laying eggs on all weeds, and to achieve nymphs development in C. arvense, C. arvensis, M. sativa, P. major and U. dioica. The cixiid survives only for a few days on grapevine.


References
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Per saperne di più:

- Flavescenza dorata e legno nero, a cura del Consorzio Fitosanitario Provinciale di Reggio Emilia