Identification and characterization of a phytoplasma associated with black locust yellow disease in two provinces of Iran

A phytoplasma disease was associated with black locust trees showing yellows, a devastating disease found in different geographical areas of Iran. Infected trees showed yellowing, reduced leaf size, short internodes and proliferation of buds, dieback and decline. The disease was transmitted to healthy black locust and periwinkle plants in greenhouse. DNA extractions were made from midrib of leaf tissues. Nested PCR assays with R16mF2/R16mR1 universal phytoplasma primers detected a 1.4 kb product and an 850 bp product was obtained using Fu5/Ru3 primers in double nested PCR. Phytoplasmas were detected from all symptomatic black locust trees as well as inoculated black locust and periwinkle plants. Analysis of 16S rRNA gene sequences showed that phytoplasmas associated with black locust yellows (BLYp) were related to 'Candidatus Phytoplasma phoenicium' (group 16SrIX). Moreover, four 16S rDNA genetic isolates (BLYp-a to -d), distinguished by 1-10 single nucleotide polymorphisms (SNPs), were identified in BLYp population. Virtual RFLP analysis determined that BLYp-a represents a new tentative subgroup within the 16SrIX group; BLYp-b, BLYp-c and BLYp-d were assigned to subgroup 16SrIX-C. Actual RFLP profiles derived from EcoRI, RsaI, HinfI and TaqI enzymes confirmed virtual RFLP subgroup characterization. Phylogenetic analysis showed that BLYp isolates shared a recent common ancestor with some 16SrIX-C phytoplasma strains. Moreover, comparisons of 16S rRNA and SecY gene nucleotide sequence raised the question as to whether BLYp and other 16SrIX-C phytoplasmas identified in Iran are identical or closely related strains. To the best of our knowledge, this is the first report of 16SrIX phytoplasmas infecting black locust.