Study of bois noir disease epidemiology in experimental vineyards through phytoplasma molecular identification and data spatial analyses

Introduction Bois noir (BN) is a grapevine yellows disease associated with Stolbur group phytoplasmas (Quaglino et al., 2010) transmitted plant-to-plant by the vector Hyalesthes obsoletus Signoret (Hemiptera Cixiide) (Maixner, 1994), a polyphagous insect living preferentially on spontaneous weeds inside and/or around vineyards (Langer and Maixner, 2004; Berger et al., 2009). Recently, several researches were focused on BN epidemiology and development of disease control strategies (Navratil et al., 2009). Materials and Methods Investigation on BN epidemiology was carried out in two vineyards located in Ronco all’Adige and San Pietro di Lavagno, Verona province, North-Eastern Italy, in the years 2010 and 2011. The study was based on (i) monitoring and mapping diseased grapevines, spontaneous weeds and H. obsoletus specimens, (ii) BN phytoplasma (BNp) identification through real-time PCR analyses (Galetto et al., 2005) performed on leaf samples collected from grapevines and weeds and insect specimens captured by cromotropic traps and nets, (iii) statistic analyses of data spatial distribution by means of the software SADIE (Spatial Analysis by Distance Indices) (Perry et al., 1999) Results and Discussion In the years 2010 and 2011, diseased grapevines increased (8.2% to 9.8%) in Ronco all’Adige and decreased (5.7% to 3.3%) in San Pietro di Lavagno. Molecular analyses identified BNp in 7 and 10 weed species at Ronco all’Adige and San Pietro di Lavagno, respectively. In detail, Convolvolus arvensis, Urtica dioica, Polygonum persicaria, Taraxacum officinale, Plantago lanceolata, Chenopodium album, Amaranthus retroflexus, Malva sylvestris, Artemisia vulgaris and Sonchus oleracea, previously reported as BNp-host plants, (Langer and Maixner, 2004; Berger et al., 2009; Kessler et al., 2011), have been found frequently infected by BNp. Furthermore, BNp was identified for the first time in Conyza canadensis, Rumex acetosa and Portulaca oleracea. On the other hand, the species Potentilla reptans, Solanum nigrum, Trifolium pratense, Equisetum arvense, Lactuca spp., Veronica persica, Sorghum spp., Medicago sativa and Calystegia sepium did not host BNp in the examined vineyards. Moreover, 318 H. obsoletus specimens (126 in Ronco all’Adige and 192 in San Pietro di Lavagno) were captured; BNp was identified in 15% of analyzed insects (3% in Ronco all’Adige and 23% in San Pietro di Lavagno). Spatial Analysis by Distance Indices evidenced that spatial distribution of diseased grapevines and of 12 weed species were aggregated in Ronco all’Adige in 2010 and 2011. Diseased grapevine and Urtica dioica distributions were associated. These findings, along with the high BNp-infection rate of nettles, indicated that BNp spreading could be closely associated with U. dioica, the main host of H. obsoletus (Lessio et al., 2007). Diseased grapevines and U. dioica plants are aggregated mainly on vineyard borders, suggesting the possible role in BNp transmission of H. obsoletus feeding on weeds around the vineyard. In San Pietro di Lavagno, spatial distribution of diseased grapevines in 2011, ten weeds and insects were aggregated. A strong statistic association was observed between BNp-infected insects, captured in 2010, and grapevines newly infected in 2011, suggesting the involvement of BNp-infected H. obsoletus in BN diffusion. Moreover, association between diseased grapevines and weeds Amaranthus retroflexus, Portulaca oleracea, Plantago lanceolata, and Rumex acetosa was observed in 2011, suggesting the possible role of such weeds in BN spreading. BNp was identified in such weeds. No statistical relationships were found between weeds, associated with diseased grapevines, and BNp-infected insects, suggesting that H. obsoletus should live on other weeds randomly distributed, such as nettle. These evidences could suggest the possible involvement of other insect vectors that could live preferentially in weeds statistically associated with diseased grapevines. In conclusion, findings from the present work contributed to formulize the hypothesis that BN epidemics could be determined by diverse actors: (i) “H. obsoletus / additional vector(s) – U. dioica – grapevine” in Ronco all’Adige; (ii) “H. obsoletus – U. dioica / C. arvensis – grapevine” and “Additional vector(s) – A. retroflexus / P. oleracea / P. lanceolata / R. acetosa – grapevine” in San Pietro di Lavagno.