Molecular Identification Of Four Phytoplasma Diseases In Hainan Province And Periwinkle Phytoplasma Disease Analysis Of Related Genes

The Phytoplasma disease is a worldwide disease, which have caused devastating losses in economic crop in China. Plant species are rich in Hainan and temperature is suitable in Hainan.Hainan area is the appropriate district of phytoplasma diseases. Some Phytoplasma diseases have been seriously harm the development of Hainan's industry, Such as Areca industry.In this paper, the molecular identification of four phytoplasma diseases in Hainan Province and analysis of related genes.The results are as follows:Using phytoplasma universal primer pairs R16mF2/R16mR1and R16F2n/R16R2, Phytoplasmas are detected in4plant samples from23plant samples in Hainan province by nested polymerase chain reaction (nested-PCR). They are Hot pepper little leaf phytoplasma (HoPLL), Pepper yellow disease phytoplasma (PePY), Heterocarpum yellow disease phytoplasma (DeHY), Cassava witches'-broom disease phytoplasma (CasWY). HoPLL, CasWB and DeHY is1248bp.PepY is1246bp.They have a nucleotide content of46.95%-47.60%C+G. To construct phylogenetic tree of16S rDNA and to analysis by iPhyClassifier program, the result is that these pathogens classified into two subgroups as follow:HoPLL, CasWB and DeHY is16Srâ…¡-A subgroup and PepY is16SrI-B subgroup.One complete circle plasmid designated as pPLLHn-1from Periwinkle little leaf phytoplasma is determined. The plasmid is comprised of4102bp (GenBank accession number: JN835187) and have a nucleotide content of24.82%G+C and encode five ORFs. It is predicted that ORF2and ORF4of the pPLLHn-1plasmid contain two hydrophobic transmembrane domains, respectively, and to localize at membrane.Signal peptide values of ORF3is0.948. Homologous comparison show that Cop homology between ORF3and the Cop of EcOYW1is97%. According to the characteristics of secreted proteins, ORF3protein is concluded a secreted protein. The study also found that the SSB protein is more conservative than the RepA protein and there is a very highly conserved functional motif in RepA protein.The secA and secY are cloned by PCR from the samples of Periwinkle little leaf phytoplasma strain Hainan (PLL-Hn) and Periwinkle yellows phytoplasma strain Hainan (PY-Hn),After sequenced,it is found that secA of PLL-Hn is2523bp(GenBank accession number: JQ911770) which coded556amino acids, secY of PLL-Hn is1255bp (GenBank accession number: JQ911769) which coded413amino acids, secA of PY-Hn is836bp (GenBank accession number: JQ911772) which coded277amino acids, secY of PY-Hn is1417bp(GenBank accession number: JQ911771) which included secY-1and secY-2coded299and104amino acids respectively,and these two genes secY-1and secY-2aren't overlapped. The homologic analysis shows that PLL-Hn belongs to16SrI-B subgroup and PY-Hn belongs to16SrV group. It is predicted that the secY protein of Periwinkle little leaf phytoplasma strain Hainan contain nine hydrophobic transmembrane domains. It is predicted that the secY protein of Periwinkle yellows phytoplasma strain Hainan contain five hydrophobic transmembrane domains.These proteins maybe localize at membrane and transport secreted proteins and then participate in the interaction with the host plant.