Comparison of European stone fruit yellows phytoplasma strains differing in virulence by multi-gene sequence analyses

Abstract

Twenty strains of the ESFY phytoplasma, which on the basis of graft-inoculation experiments greatly differ in aggressiveness, were examined by sequence analyses of several PCR-amplified non-ribosomal genes in order to identify molecular markers linked to virulence. These strains, which were maintained in P. insititia rootstock St. Julien GF 655/2 were indistinguishable with techniques for routine phytoplasma differentiation and characterization such as sequence and RFLP analyses of PCR-amplified rDNA. Also, the virulent ESFY strains maintained in periwinkle, namely GSFY1, GSFY2 and ESFY1, as well as an avirulent strain of the same phytoplasma, maintained in apricot, which was identified in recovered apricot trees in France and used there as a cross protecting agent, were included in the work for comparison. For PCR amplification, primers were designed from a number of genes distributed over the chromosome of the closely related apple proliferation phytoplasma strain AT. Visible PCR products were only obtained with primer pairs derived from the tuf gene which encodes the elongation factor Tu (EF-Tu), rpsC (<7>rps3) gene encoding the ribosomal protein S3, tlyC gene which encodes a hemolysin known as a membrane-damaging agent and important virulence factor of many bacteria, the imp and fol genes encoding an immunodominant membrane protein and an enzyme involved in the folate biosynthesis, respectively. Nucleotide sequence comparisons revealed that the highest genomic variability occurred within the imp gene sequence with dissimilarity values ranging from 0.2 to 4.6%. For the remaining genes, the strains examined proved to be identical or nearly identical. Within the tuf gene, an extra TaqI site known to occur in strain GSFY1 was not identified in other strains. The genetic differences observed among the strains examined are neither suitable markers for strain differentiation nor linked to pathological traits.

Keywords: European stone fruit yellows, strain virulence, 16SrX group, tlyC gene, Prunus spp.