Genetic Diversity of Flavescence Dorée Phytoplasmas at the Vineyard Scale

Abstract

To study the role of wild areas around the vineyards in the epidemiology of flavescence dorée (FD) and track the origin of new foci, two phytoplasma genetic markers, dnaK and malG, were developed for FD phytoplasma (FDp) characterization. The two genes and the vmpA locus were used to genetically characterize FDp populations at seven agroecosystems of a wine-growing Italian region. Vitis vinifera, “gone-wild” V. vinifera and rootstocks, Clematis spp., and Scaphoideus titanus adults were sampled within and outside the vineyards. A range of genotypes infecting the different hosts of the FDp epidemiological cycle was found. Type FD-C isolates were fairly homogeneous compared to type FD-D ones. Most of the FD-D variability was correlated with the malG sequence, and a duplication of this locus was demonstrated for this strain. Coinfection with FD-C and FD-D strains was rare, suggesting possible competition between the two. Similar levels of FDp genetic variation recorded for grapevines or leafhoppers of cultivated and wild areas and co-occurrence of many FDp genotypes inside and outside the vineyards supported the idea of the importance of wild or abandoned Vitis plants and associated S. titanus insects in the epidemiology of the disease. Genetic profiles of FDp found in Clematis were never found in the other hosts, indicating that this species does not take part in the disease cycle in the area. Due to the robustness of analyses using dnaK for discriminating between FD-C and FD-D strains and the high variability of malG sequences, these are efficient markers to study FDp populations and epidemiology at a small geographical scale. IMPORTANCE Flavescence dorée, a threatening disease of grapevine caused by FD phytoplasma (FDp), is distributed within the most important wine-producing areas of Europe and has severe effects on both vineyard productivity and landscape management. FDp is a quarantine pest in Europe, and despite the efforts to contain the pathogen, the disease is still spreading. In this work, new genetic markers for the fine genetic characterization of FDp at local scale are presented. Our findings improve the knowledge of FDp epidemiological cycle and offer the possibility of tracking the route of the FDp infection. In particular, due to its high genetic variability, one of the newly developed markers could be sufficient to track the origin of new infection foci, either from the wild areas or from nurseries.

When a Palearctic bacterium meets a Nearctic insect vector: Genetic and ecological insights into the emergence of the grapevine Flavescence dorée epidemics in Europe

“…It was also the most prevalent genotype detected in D. europaea and C. vitalba collected in Serbia and Montenegro [43]. M51 was not detected in Italy during the present survey but was previously detected in Tuscany [23] and later on in Switzerland [44] and northwestern Italy [42]. On one hand, our data show evidence that M51 may have emerged from C. vitalba in Balkans and subsequently propagated westward to Italy where a secondary diversification into M12, M6 and M3 may have occurred possibly as a result of the presence of established S. titanus populations that migrated from France.…”

“…In early 2000s, M50 represented 15% of the FD cases in France [24], and in this study M50 was detected in FD outbreaks of Tuscany. M50 was also sporadically detected in Croatia [40], Switzerland [41] and in C. vitalba of Eastern Italian region of Veneto [23] and in north-western Italy [42], but was absent in the current alder and grapevine samples from Serbia. As shown by MLSA, the M50s detected in France, Veneto and Croatia were genetically different [23,40], and we therefore cannot exclude multiple independent emergences of M50 from alders.…”

Evidence suggesting interactions between immunodominant membrane protein Imp of Flavescence dorée phytoplasma and protein extracts from distantly related insect species

“…The isolate was obtained from infective S. titanus adults that were collected in 2015 from an experimental vineyard in Piedmont and allowed to feed on V. faba in the laboratory. This FD phytoplasma isolate was genetically identified as member of 16SrV‐D subgroup based on DNA sequence analysis (Rossi et al ). FD‐Dp was then routinely maintained under controlled conditions with sequential transmissions from broad beans to broad beans by the experimental vector E. variegatus , continuously reared under laboratory conditions on oat.…”

Potential role of the alien planthopper Ricania speculum as vector of Flavescence dorée phytoplasma

“…The isolate was obtained from infective S. titanus adults that were collected in 2015 from an experimental vineyard located at the CREA research institute (Asti, Piedmont) and allowed to feed on V. faba in the laboratory. This FDp isolate was genetically identified as D strain on dnaK gene (MH547710.1) and mixed profile on malG gene (malG_1 MH547713, malG_3 MH547715, malG_6 MH547718, malG_16 MH547728, malG_34 MH547746), according to (Rossi et al 2019). FDp was then routinely maintained under controlled conditions by sequential transmissions from V. faba to V. faba by the experimental vector E. variegatus, continuously reared under laboratory conditions on Avena sativa (oats) as described in Rashidi et al (2014).…”

“…DNAKf/DNAKr primers, flanking FDp dnaK gene portion (MH547710.1), were used to detect FDp presence in R. speculum samples, by conventional PCR under standard reaction conditions with an annealing temperature of 55°C (Rossi et al 2019). The corresponding dnaK PCR products obtained from positive samples were purified with DNA Clean & Concentrator kit (Zymo Research) and Sanger sequenced by BMR Genomics (Padova, Italia), to confirm the identity of FDp strain.…”