Multigene differentiation of 'Candidatus Phytoplasma solani' strains from different geographic origins and diverse host species.

The knowledge of genetic variability in phytoplasmas is a fundamental resource for the study of their epidemiology and a valuable support to improve the monitoring and the management of their associated diseases. Multigene analyses have been used widely to characterize phytoplasmas strains providing relevant information about their epidemiology. ‘Candidatus Phytoplasma solani’ is associated with “bois noir” (BN) in grapevines and yellowing in many other cultivated and wild plant species such as tomato, pepper, bindweed, causing serious damages in many parts of the world. The epidemiology of the diseases associated with this phytoplasma is very complex considering its wide host range distribution, the interactions with both host plant and insect vectors, and the diffusion of the bacterium through infected plant propagation material. The genetic variability of 'Ca. P. solani' strains, in different host species and in several geographic areas was widely studied by genotyping selected non-ribosomal genes. The polymerase chain reaction coupled with restriction fragment length polymorphism analyses and sequencing was applied to samples collected during more than 20 years in Italy, Portugal, Hungary and Serbia in a multigene analysis on vmp1, stamp and tuf genes. A total of 116 'Ca. P. solani' strains was examined and allowed the differentiation of 26 genetic variants confirming the differential variability of the studied genes. In particular, the vmp1 and stamp genes showed the presence of 14 and 5 RFLP profiles, respectively, while the tuf gene grouped all tested strains into two profiles. In the vmp1 gene sequences it was registered the largest variability; the V3-RsaI RFLP profile was the most present in the Italian grapevine samples tested. A large part of the samples tested were collected in Italy and Serbia; in the comparison between the two geographic areas only one genetic variant was found in common, confirming the local distribution of the strains. Considering the time frame of the sample collection and the geographical distribution of the variants identified, it appears that populations of this phytoplasma are generally less variable on local scale leading in some case to the emergence of epidemic strains. In particular, the grapevine samples collected during 2020 in Tuscany revealed the predominance of tuf type-a strain and showed the same variant in all the samples, indicating the possible emergence of an epidemic BN strain in that region. In addition, the identification of two stamp variants (St5 and St10) that have shown specific epidemiological characteristics linked to different virulence in the field indicates the need to continue to study and to monitor the disease with specific molecular analyses considering that the strains with different virulence can lead to epidemic infections with significant economic damages. The presence of mixed infections with different phytoplasmas indicate another important element to consider for the ‘Ca. P. solani’-associated diseases epidemiology. These results confirm that except for tuf gene there is no specificity of ‘Ca. P. solani’ variants linked to different host species and different epidemiological cycles. Their differentiation can be explained by geographic distribution of host and insect vectors, year of infection and epidemic outbreaks registered during the time. Strains diversity combined with epidemiological data are useful also to identify sources of inoculum, new host species and to monitor the spreading of the phytoplasma both locally and on a larger environmental scale for focused management purposes.