Molecular delineation of a phytoplasma representative of the novel 16SrVII-G subgroup found in citrus trees with huanglongbing symptoms

Leaf symptoms of chlorosis and blotchy mottling, typically associated with agents of huanglongbing, were found in commercial citrus orchard located in the state of São Paulo, the main citrus producer region in Brazil. Affected trees tested negative for bacteria ‘ Ca . Liberibacter americanus’ and ‘ Ca . Liberibacter asiaticus’. So, the total DNA was submitted to nested PCR employing the primer pairs P1/Tint followed by R16F2n/R16R2. Genomic fragments of 1.2 kb were amplified, sequenced and analyzed by computer-simulated RFLP, which confirmed the presence of a 16SrVII phytoplasma in symptomatic plants. Collective restriction patterns allowed distinguish the detected phytoplasma from all other members of the 16SrVII group using enzymes Bfa I and Mse I. In addition, the citrus phytoplasma showed a similarity coefficient lower than 0.97, when compared with all representative strains of subgroups within of the 16SrVII group, characterizing, therefore, a new subgroup. In agreement, the phylogenetic tree displayed that this strain emerges from a distinct branch in relation to those 16SrVII phytoplasmas currently described. Based on these features, the strain identified in the present study may be delineated as a new phytoplasma representative of the novel 16SrVII-G subgroup. Moreover, the results implicate citrus as a new host for a phytoplasma belonging to the group 16SrVII.