Early phytoplasma detection on table grapes showing yellowing symptoms in Apulia

Grapevine yellows (GYs) is one of the most damaging phytoplasma-associated diseases that causes severe yield losses and is worldwide distributed in grapevine growing areas. In Europe, one of the most widespread GYs is “bois noir” (BN), associated with ‘Candidatus Phytoplasma solani’ phytoplasmas (16SrXII-A) and already reported in several areas and in almost all Italian regions, where its presence is constantly increasing. In the frame of the mandatory program of surveillance for the quarantine phytoplasma disease “flavescence dorée”, a survey for grapevine yellows is constantly carried out in Apulia region and during the last decades identification and molecular characterization of BN phytoplasmas was achieved in symptomatic grapevine samples collected from the diverse provinces. In the recent years there is an increasing cultivation of new table-grape varieties, with low labour cost, disease resistant, more attractive for the market, like seedless rounded berries with crunchy pulp, sweet taste with light fruity aroma. Among the new seedless table grape varieties, Apulian farmers is investing heavily in the Autumn Pearl cultivar. During a survey conducted in spring 2023 on 3-5 years old Autumn Pearl located in four organic vineyards in Castellaneta (Taranto province) asymmetrical, reddening and downward curling of leaves with redness of the main veins, abortion of inflorescences and shrivelling of berries, necrosis of the rachises and tendrils were observed. Moreover, at the beginning of autumn, shortening of the distal internodes, flattening and cracking of the leaves and sometimes green islands and poor lignification of the branches, limited to the most productive plants, were registered in the same vineyards. The percentage of symptomatic vines was different according to the year of plantation and generally the most severely affected plants were distributed mostly in aggregates. Molecular analyses conducted from leaf samples collected in late summer 2023 led to the identification of BN phytoplasmas in all the symptomatic samples tested. In order to set up an early and reliable diagnostic method for phytoplasma detection, a survey was conducted in early May 2024 and samples of leaves and rachis with light phytoplasma symptoms were collected from the same infected vineyards previously analysed. DNA was then extracted from a number of berries (5-8) with a commercial kit DNeasy Mericon Food Kit (Qiagen) and from leaf veins with a CTAB- based protocol. Nested-PCR with phytoplasma universal primers followed by RFLP and sequencing analyses allowed the identification of BN phytoplasmas from the different plant organs, in both single and mixed infections with other phytoplasmas that are currently under molecular characterization. The results obtained in this work highlighted the need of a punctual and continuous monitoring to avoid the introduction of infected plant material and of a good, reliable and early diagnostic method for phytoplasma identification, in order to prevent the eventual spread of diseases with effect on the production and health of vineyards.