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Flavescence dorée (FD) is a major grapevine disease in Europe, despite the quarantine status of its causal agent [FD phytoplasma (FDp)] and the mandatory monitoring and vector control practices. As alarming FD epidemic outbreaks continue to appear in Tuscany (Central Italy), a 4-year survey was carried out in the main wine-growing areas of the region, where FD presence was investigated in both primary and secondary FDp hosts and vectors, i.e., Vitis vinifera (VV), Clematis vitalba (CV), Alnus glutinosa (AG), Scaphoideus titanus (ST), and Dictyophara europaea (DE). This work i) confirmed FD diffusion in almost the whole of Tuscany and even with an increased occurrence rate (approximately 50% of the samples tested positive); ii) highlighted a complex FDp ecology also in the Tuscan vineyard agroecosystem, as FDp was reported not only in VV and ST but also in secondary vectors (DE) and hosts (AG and CV); iii) reported nine FDp strains (three of which were novel) belonging to all the three methionine aminopeptidase (map) clusters, i.e., map-FD1, map-FD2, and map-FD3, with the map-FD3/M51 genotype mostly reported in not only VV but also DE and CV; and iv) further confirmed a complex FDp ecology in the vineyard agroecosystem, also by phylogenetic analyses carried out at both Italian and European levels, which also showed some relations between the Tuscan FDp strains and those reported in the Balkan and the French scenarios. We believe that the outcomes reported here will be useful in preventing and controlling the spread of harmful FD.

Tomato brown rugose fruit virus (ToBRFV) represents an emerging viral threat to the productivity of tomato and pepper protected cultivation worldwide. This virus has got the status of quarantine organism in the European Union (EU) countries. In particular, tomato and pepper seeds will need to be free of ToBRFV before entering the EU and before coming on the market. Thus, lab tests are needed. Here, we develop and validate a one-step reverse transcription LAMP platform for the detection of ToBRFV in tomato and pepper leaves, by real-time assay [reverse transcription loop-mediated isothermal amplification (RT-LAMP)] and visual screening (visual RT-LAMP). Moreover, these methods can also be applied successfully for ToBRFV detection in tomato and pepper seeds. The diagnostic specificity and sensitivity of both RT-LAMP and visual RT-LAMP are both 100%, with a detection limit of nearly 2.25 fg/μl, showing the same sensitivity as RT-qPCR Sybr Green, but 100 times more sensitive than end-point RT-PCR diagnostic methods. In artificially contaminated seeds, the proposed LAMP assays detected ToBRFV in 100% of contaminated seed lots, for up to 0.025–0.033% contamination rates in tomato and pepper, respectively. Our results demonstrate that the proposed LAMP assays are simple, inexpensive, and sensitive enough for the detection of ToBRFV, especially in seed health testing. Hence, these methods have great potential application in the routine detection of ToBRFV, both in seeds and plants, reducing the risk of epidemics.