Epidemiology of "bois noir" disease in Veneto region through phytoplasma molecular identification and spatial analyses data

Introduction Bois noir (BN) is a grapevine yellows disease associated with 'Candidatus Phytoplasma solani' strains (Quaglino et al., 2013) 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 symptomatic grapevines, spontaneous weeds and H. obsoletus specimens, (ii) BN phytoplasmas (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). Moreover, the monitoring and the spatial distribution of symptomatic grapevines was carried out on 2012. Results and Discussion In the years 2010-2012, symptomatic grapevines increased (9% to 11%) in Ronco all’Adige and decreased (6.2% to 4.9%) 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 were 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. The H. obsoletus population density in the two vineyards was high. BNp were identified in 18-19% of analyzed insects in San Pietro di Lavagno and 3-21% in Ronco all’Adige. Spatial Analysis by Distance Indices evidenced that spatial distribution of symptomatic grapevines and of 12 weed species were aggregated in Ronco all’Adige in 2010. During 2011, aggregation was detected in the distribution of symptomatic grapevines and of 16 weeds. Symptomatic grapevine distribution was associated with those of U. dioica and P. reptans in both years. BNp-infected insects showed an aggregated distribution in 2011, but not in 2010. However, in 2011 their distribution were not associated with weeds or symptomatic grapevines. Considering new symptomatic grapevines, their distribution was found aggregated in 2012, and it was associated with the distribution of P. oleracea, M. sylvestris and A. retroflexus in 2011. 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). Symptomatic grapevines and U. dioica plants are aggregated mainly on vineyard borders, suggesting the role in BNp transmission of H. obsoletus feeding on weeds around the vineyard. (Mori et al., 2012) In San Pietro di Lavagno, spatial distribution of symptomatic grapevines in 2011 and 2012, 13 weeds in 2011, and insects in 2011 was aggregated. Considering newly symptomatic grapevines, their distribution was found aggregated in 2011 and 2012. 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 symptomatic grapevines and weeds A. retroflexus, P. oleracea, P. lanceolata, and R. acetosa was observed in 2011, suggesting the possible role of such weeds in BN spreading. BNp were identified in such weeds. No statistical relationships were found between weeds, associated with symptomatic 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 symptomatic grapevines. In particular, further studies will focus on the role of P. oleracea and A. retroflexus, associated with symptomatic grapevines and infected by BNp in both vineyards, in the BNp diffusion.