Role in “bois noir” epidemiology of inter-row vineyard groundcover vegetation used for green manure

‘Candidatus Phytoplasma solani’ (CaPsol), associated with grapevine ‘bois noir’ (BN), has a broad range of host plants and can be transmitted to grapevine by several insect vectors. Hyalesthes obsoletus Signoret, the main CaPsol vector in European and Mediterranean countries, feeds preferentially and completes its biological cycle on Convolvulus arvensis L. and Urtica dioica L., occasionally transmitting the phytoplasma to grapevine, a dead-end host for the pathogen. Recent studies evidenced that other weeds, naturally present within and around the vineyards, can play a role in BN spreading. This study investigated the role of eight groundcover plant species (Eruca sativa Miller, Sinapis arvensis L., Phacelia tanacetifolia Benth., Vicia sativa L., Vicia faba var. minor Beck, Trifolium incarnatum L., Trifolium alexandrinum L., Polygonum fagopyrum L.), commonly utilized for inter-row vineyard green manure in Franciacorta (North Italy), in BN epidemiology. The activities, conducted in the years 2019 and 2020 in two BN-affected vineyards in Gussago and Provaglio d’Iseo (Franciacorta), included: (i) monitoring and sampling groundcover plant species and C. arvensis (known host plant) in July, and symptomatic grapevines in September; (ii) total nucleic acids extraction followed by CaPsol-specific identification by nested PCR amplification of stamp gene; (iii) sequencing and bioinformatic analyses (comparison with stamp sequence variants dataset available in literature) of stamp amplicons obtained from symptomatic grapevines and groundcover plants. Molecular analyses were carried out on 341, 55, and 108 samples collected during the field surveys from groundcover plants, C. arvensis and symptomatic grapevines, respectively. Nested PCR allowed identifying CaPsol in 7.6% of groundcover plants, 25.5% of C. arvensis plants, and 76.9% of grapevines. Within groundcover plants, only E. sativa, V. sativa, and P. fagopyrum were found CaPsol infected. Based on nucleotide sequence analysis of stamp amplicons, CaPsol strains harboring three stamp sequence variants were identified in grapevines: St5, St19, and St30. The variant St19 was found exclusively in the analyzed grapevines. The variants St5 and St30 were found also in CaPsol strains infecting E. sativa (St5), P. fagopyrum (St5), V. sativa (St5, St30), and C. arvensis (St5), suggesting their possible involvement in CaPsol transmission routes to grapevine. Interestingly, previous study demonstrated that H. obsoletus survival on V. sativa and C. arvensis are comparable, reinforcing its association to BN epidemiology. Results from this and further studies can indicate how to select the groundcover plants for green manure mixture, excluding the species putatively involved in BN diffusion.