Diversity Of Phytoplasma Strains And Associated With Diseases In Yunnan Province

Phytoplasmas are phytopathogenic prokaryotes that were formerly named mycoplasma-like organisms (MLOs), which lacking cell walls and inhibit phloem sieve elements in infected plants. Till now, phytoplasma cannot be cultured in vitro and have been associated with diseases in several hundred plant species.Use advanvantage of resourceful microorganisms in Yunnan Province, we aimed at 16SrRNA gene, which is a relatively conservative gene in the evolution process of prokaryotes and had a research on phytoplasma diseases in Yunnan. Main results or conclusion of this research are as follows:1,Many doubtful phytoplasma diseases, which were collected from about 30 county of 12 different regions in Yunnan Province according the symptomatic characteristic of phytoplasma disease, were detected and identified by nested-PCR asssy, in which the phytoplsma universal primers for 16SrRNA gene were used. As a result, the phytoplasma diseases occurred in Yunnan Province were confirmed and 15 phytoplasma diseases have been identified. They are cactus witches'-broom, bamboo witches'-broom, peach red (yellows), orange little leaf, Christmas cactus witches'-broom, paulownia witches'-broom, petunia dwarf, petunia stem flattening, sunshine tree witches'-broom, chinaberry witches'-broom, carnation yellows, Lucerne witches'-broom, lily flower virescence and lily stem flattening. Among these diseases, cactus witches'-broom phytoplsma disease is distributed widely in Yunnan Province, and there are 5 phytoplasma diseases, such as orange little leaf, Christmas cactus witches'-broom, petunia stem flattening, sunshine tree witches'-broom, carnation yellows, were the first time report in the world.2,The PCR amplified fragments were ligated into pGEM-T easy vector and then transformed into the component cell of E. coli, repectively. The combined plasmid were sequenced after identified by restrict enzyme digestion and PCR amplification. Based on the phylogenetic tree of 16SrDNA sequences construed by the minimum evolution method, the results showed that the phytoplasma strains of above-mentioned diseases are mainly associated with Ca. Phytoplasma asteris, Ca. Phytoplasma aurantifolia, Ca. Phytoplasma pruni and Ca. Phytoplsma ziziphi, which are the accepted phytoplasma species by the IRPCM phytoplasma working team, and these phytoplasma strains were classified into phytoplasma subgroup of 16SrI-B, 16SrII-C, 16SrIII-B and 16SrV-B. The results also confirmed the genetic relationships between these phytoplasma strains.3,The PCR-amplified 1.25kb 16SrDNA fragments of 29 cactus witches'-broom phytoplasma strains, which collected from 4 region in Yunnan Province, were digested in silico with 18 distinct restriction enzymes for RFLP analysis. The results indicated that only one diseased plant, which collected from Suan village of Kunming, was infected by a'Candidatus Phytoplasma asteris'-related phytoplasma and belongs to a member of 16SrI-B subgroup, whereas 28 diseased plants were infected by a phytoplasma of group 16SrII. The virtual RFLP patterns of 28 diseased plants were further analyses for genetic diversity. The results revealed that the genetic diversity of 28 cactus witches'-broom phytoplasma strains is 34.6%, and the similarity coefficient between two phytoplasma strains is from 0.83 to 1.00, the average is 0.965. All results indicated that the genetic diversity was existed amongst the cactus withces'-broom phytoplasma strain in Yunnan Province.4,A specific TaqMan probes were desiged and synthesized for real-time fluorescent PCR based on the 16SrRNA gene sequences of phytoplasma 16SrII group. A set of RTF-PCR reaction system and conditions were optimized to detect phytoplasmas of 16SrII group and this method was used to quantify the phytoplasma cells in the diseased cactus different tissues and diseased Christmas cactus. The results showed that the phytoplasma cells in the mature stem of cactus is 2.81×105 per ng total plant DNA and this titer is much more than those in the tissues of old stem, root and younger stem. Foutunately, the number of phytoplasma cells in the diseased Christmas cactus is the same as those in the mature stem of diseased cactus plant.5,15 diseased young tissues of phytoplasma diseases, which have identified with PCR assay before, were used to observed the ultrastructure using ultrathin section of electronic microscopy technique, the typical phytoplasmas bodies were observed only in the phloems of diseased plant of paulownia witches'-broom, cactus witches'-broom, chinaberry witches'-broom and Christmas cactus witches'-broom. The phytoplasmas appear as spherical, dumbbell and irregular branched filamentous pleiomorphic shapes. Most of phytoplasmas existed around the cell wall of sieve elements and a little are full of the space of all cells. Three types of development bodies, including developed, molder and proliferation and the phytoplasmas bodies are moving through the pores of sieve plate in the sieve elements also were examined.