Diversity Of Phytoplasma Disease And Molecular Detection Of Phytoplasma Associated With Arecanut Yellow Leaf In Hainan Province

Phytoplasmas, formerly called mycoplasma-like organisms, were cell wall-less pathogenic prokaryotes and resided in the sieve cell of plant phloem tissue, which caused diseases in hundreds of economic plant species worldwide. The report here investigated and studied phytoplasma diseases in Hainan systematically. We identified pathogens of 16 phytoplasma disease on molecular level and cleared the taxonomy of these pathogens which helped us to aware of the diversity of phytoplasma diseases in Hainan. Real-time PCR was done to Arecanut yellow leaf disease which caused serious damages to Arecanut production, the second pillar production only to natural rubber in Hainan. A set of simple, rapid and reliable detection system for arecanut yellow leaf disease was established to monitor the epidemic and control of the disease. This research made a comprehensive and systemic investigation on phytoplasma diseases in Hainan for the first time. Using phytoplasma universal primer pairs R16mF2/R16mR1 and R16F2n/ R16R2, phytoplasmas were detected in 50 of 61 plant samples from 18 counties in Hainan province by polymerase chain reaction (PCR). Based on sequences comparison of Blast program in GenBank, construction of phylogenetic tree and analysis of iPhyClassifier, 16 phytoplasma diseases have been identified. They were Stylosanthes witches'-broom, Stylosanthes yellows, Crotalaria witches'-broom, Peanut witches'-broom, Periwinkle little leaf, Eryngium foetidum witches'-broom, Praxelis clematidea Witches'-Broom, Common parthenium witches'-broom, Gynura crepidioides phyllody, Desmodium ovalifolium witches'-broom, Bamboo witches'-broom and Chinaberry witches'-broom. These pathogens classified into 6 subgroup as follow, 16SrI-B subgroup, 16SrI-B** subgroup, 16Srâ… -G subgroup, 16Srâ… -S subgroup, 16SrII-A subgroup, 16Sr II-A** subgroup, 16Sr II-M subgroup and 16Sr V-H subgroup, among which 16Srâ… -G subgroup, 16Srâ… -S subgroup, 16Sr II-M subgroup and 16Sr V-H subgroup was proposed as novel strains, highly enriched the diversity of phytoplasma diseases. The 16 phytoplasma diseases identified here distributed in 18 counties in Hainan. Peanut witches'-broom(16Sr II)and Aster yellows(16Srâ… )were the main strains while Elm yellows(16Srâ…¤)sporadically. 16Sr II phytoplasma were detected in all the legumina and compositae samples Based on a systematic investigation into arecanut yellow leaf disease in Hainan, the disease has been epidemic in Tunchang, Ding'an, Qionghai, Wanning, Lingshui, Baoting, Sanya, et al. in Hainan and caused serious consequences. Infected area of arecanut in the province has reached over 3 million mu. Early infected orchards have already destroyed, and crop failure. In seriously diseased orchards, the incidence of illness up to 90%, cut 78% -80%, or even never produced. Incidence has already reached 10-30% in some orchards. The disease is spreading rapidly in Hainan.We identified the phytoplasma caused arecanut yellow leaf disease in Hainan belongs to Aster yellows group at the molecular level for the first time and generated a new subgroup 16Srâ… -G.. . On the basis The phytoplasma was temporarily called Arecanut yellow leaf (AYL) of the pathogen identified, a TaqMan MGB probe was designed for detection of arecanut yellow leaf disease according to the conservative region of AYL phytoplasma 16S rDNA gene. A real-time fluorescence PCR detection system for arecanut yellow leaf disease was established. The detecting accuracy for unexpanded inflorescence and unexpanded leaf came up to 100% and 80%, respectively. The detecting accuracy came up to 26.7%, 46.7 and 60% respectively for 2nd leaf, 3rd leaf and 5rd leaf.