Development of efficient methods and identification of barcodes for discriminating Grapevine flavescence dorée sensu-stricto from other related phytoplasmas and investigation of potential correlation between taxonomic identity and grapevine, alders and hazelnut plant hosts

This research project focused on ways to distinguish Grapevine flavescence dorée sensu-stricto (GFD) from other related phytoplasmas and to investigate a possible relationship between taxonomic identity and the host plants grapevine, alder, and hazelnut.

Samples of hazelnuts were collected in different regions of Europe to investigate the presence and prevalence of phytoplasmas. Phytoplasma 16SrV was detected in asymptomatic wild hazelnuts in France, Germany, and Italy and in symptomatic cultivated hazelnuts in Slovenia and central Italy. In addition, other phytoplasma species, such as 'Candidatus Phytoplasma fragariae' and 16SrIX, were identified. To investigate possible insect vectors of 16SrV phytoplasma associated with hazelnuts, an extensive search was conducted, and samples of leafhoppers were collected. It was found that infection rates of leafhoppers collected on hazelnuts were significantly lower than those previously found on alders. Special attention was paid to Orientus ishidae, and preliminary transmission experiments were conducted to better understand their role.

Another objective of the study was to determine the diversity of 16SrV phytoplasma strains. Different genotypes were detected in grapevine, hazelnut, and alder in different countries. Some genotypes were found in both grapevine and hazelnut, suggesting a possible host crossover. In addition, new genotypes were discovered in hazelnuts and alders, expanding our knowledge of phytoplasma strains. 

In an effort to improve diagnostic methods, several tests were evaluated to distinguish GFD phytoplasmas from other 16SrV phytoplasmas, and three tests (two nested-PCRs followed by nucleotide sequence analysis and one real-time PCR) were extensively validated in a test performance study. In addition, a prototype HTS-bioinformatics pipeline based on the original EDNA concept has been developed but needs further testing and validation.

The results of this project contribute to a more complete understanding of the epidemiological cycle of 16SrV phytoplasmas and may help in the development of effective strategies to control the disease. Further research in this area will provide valuable insights into the complex interactions between phytoplasmas, hosts, and vectors.