<b>Aim</b>: Evaluation of the genetic variability of stolbur phytoplasma infecting grapevines, bindweeds and vegetables, collected in different central and southern Italian regions.<br></br> <b>Materials and Results</b>: Phytoplasma isolates belonging to stolbur subgroup 16SrXII-A were subjected to molecular characterization by polymerase chain reaction/ restriction fragment length polymorphism (PCR/RFLP), to investigate two different nonribosomal genes: <i>tuf</i> and <i>vmp1</i>. In grapevines, 32% of samples were infected by <i>tuf</i>-a type and 68% by <i>tuf</i>-b type, with different relative incidences in the regions surveyed. All herbaceous samples (bindweeds, tomato, tobacco, pepper, celery) were infected by <i>tuf</i>-b. The gene <i>vmp1</i> showed higher polymorphism in grapevines (nine profiles) than herbaceous plants (six) by RFLP analysis, in agreement with nucleotide sequences’ analysis and virtual digestions.<br></br> <b>Conclusions</b>: The phylogenetic analysis of <i>vmp1</i> gene sequences supports the RFLP data and demonstrates the accuracy of RFLP for preliminary assessments of genetic diversity of stolbur phytoplasmas and for screening different <i>vmp</i> types. Significance and Impact of the Study: Stolbur represents a serious phytosanitary problem in the areas under investigation, owing to heavy economic losses in infected grapevines and vegetables. Molecular information about the complex genotyping of the <i>vmp1</i> gene provides useful data towards a better understanding of stolbur epidemiology. Moreover, this study clarifies some different <i>vmp1</i> genotype classifications of stolbur, providing molecular data in comparison with previous investigations
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