Molecular characterization and detection of 16SrIII group phytoplasma associated with Huanglongbing symptoms.

When Huanglongbing (HLB) was found in Brazil in 2004, 'Candidatus Liberibacter americanus' (Lam) was infecting most of the trees while 'Ca. L. asiaticus' (Las) was present in minor proportion. Currently, 'Ca. L. asiaticus' is the predominant bacterium associated with HLB in citrus trees in São Paulo (SP) and Minas Gerais (MG) States, the major citrus growing regions in Brazil. A phytoplasma from 16Sr group IX was associated with HLB symptoms in Brazil in 2007, in plants free of Liberibacter. In this report, HLB-samples testing negative for Las, Lam and 16SrIX phytoplasma, were infected with 16SrIII phytoplasmas. Co-infection with Las and 16SrIII was also found. The 16S rRNA gene sequences from 22 samples were obtained and sequenced, confirming that 16Sr group III phytoplasma is associated with HLB symptoms in SP and MG States. Ten single nucleotide polymorphisms (SNPs) were found in the 1,427 bp 16SrDNA sequences from 16SrIII phytoplasmas from citrus, while none was detected in 16SrDNA sequences among 16SrIX phytoplasma from citrus. Ribosomal protein (rp) rpsSrplVrpsC gene sequences were amplified with 16SrIII group specific primers and sequenced from a subset of nine samples and assembled into three groups based on eight SNPs. SNPs in 16S rRNA and rp gene sequences are common in 16SrIII phytoplasmas from other hosts and this phytoplasma group is widespread in South America. 16SrIII phytoplasmas highly related are commonly found in Melia azedarach, a widespread tree in Brazil and Argentina. The finding of a new phytoplasma associated with HLB symptoms, belonging to the 16SrIII group reinforces the need to develop diagnostic tools to assess HLB associated microbiome.