Molecular Detection And Identification Of Two Phytoplasmal Diseases

Phytoplasma is a plant pathogenic prokaryotes that can cause many kinds of plant diseases. By use of molecular biology techniques, the JWB phytoplasma in some parts of China and poplar yellows pathogen in henan province were detected and identified.The main results read as follows:1) By use of PCR, the 16S rDNA,elongation factor Tu (tuf gene) and ribosomal protein (rp) gene of phytoplasma associated with JWB in Beijing and Hebei districts were amplified separately with universal primer pairs R16F2n/R16R2, fTufu/rTufu and rp(v)F1A/rp(v)R1A. We obtained 16S rDNA gene of 1.2 kb, tuf gene of 0.8 kb and rp gene of 1.2 kb from the diseased samples. The results of sequencing and homologous comparison with other phytoplasmas showed that JWB in Beijing shared a high identity with other JWB phytoplasmas in 16S rDNA gene; JWB in Beijing shared 92.84% similarity with Flavescence doree (FD) phytoplasma (Candidatus Phytoplasma vitis) in tuf gene. The tuf gene of JWB in Beijing shared a low identity of 57.29% with JWB tuf gene in Shaanxi district which had been already reported.The homology analysis for sequences of rp gene showed that a high identity with members of the 16SrV group phytoplasmas, with homology rate higher than 96 %. It shared mostly identity of 99.83% to the 16SrV group of JWB strain Taishan and Hemp fiber witches'-broom phytoplasma (HFWB).2) By use of universal primers P1/P7 and R16F2n/R16R2 for 16S rDNA gene, a specific fragment of 1.2 kb in length was amplified with nested PCR from poplar showing typical phytoplasma symptom of yellowing in henan province. By use of universal primers pairs rpF1/rpR1 and rp(â… )F1/rp(â… )R1 for rp genes, a specific fragment of 1.2 kb was amplified with nested PCR from poplar diseased sample.The results of sequencing and homologous comparison with other phytoplasmas showed that the phytoplasma strain is one of the members of 16Sr I group.According to the PCR with JWB phytoplasmas in Beijing and Hebei districts, we discovered the JWB tuf gene in Beijing and Hebei had a high difference between with other districts. The results provided the basis for the classification in subgroup of the 16SrV group phytoplasmas. By use of universal primers for dectection of phytoplasma, DNA fragments were obtained from the total DNA of poplar yellows sample.The results indicated existence of phytoplasm aassociated with poplar in Henan province. This is the first report of phytoplasma associated with poplar yellows disease in China.