Research On Pathogens Diversity Of Wheat Blue Dwarf Disease

It's very difficult for diversity study of phytoplasma because of phytolasma's uncultured characteristic. Nowadays research mainly on diversity of phytoplasmas in plants. Wheat blue dwarf (WBD) is the first and unique phytoplasma disease in wheat transmitted by Psammotettix striatus L. in China. WBD is the main epidemic disease in arid and semiarid wheat regions in northwest of China. A great loss would be caused in the production process of winter wheat once it outbreak. Whether there are other phytoplasmas existed in wheat was unknown. What we should do was to find out the answer. Based on the ideas above, we collected 8 samples from Hancheng,Heyang, Shaanxi and Dingxi, Gansu. Using universal primer pairs we cloned conserved sequences of 16S rRNA and ribosomal protein (rp) genes. Combined with RFLP analysis we identified and classified the phytoplasmas in winter wheat and got results as follow:1. We cloned 16S rRNA partial sequences from 11 wheat samples of 3 regions using universal primer pairs R16F2m/R16R1m and R16F2n/ R16R2. The 11 sequences amplified from winter wheat was cluster into 3 branches. Homology of 4 sequences reach to 99.97% and their length were 1249 bp. 5 sequences were 1247 bp and they shared 99.98% homology. While two sequences displayed divergence with the other 9 sequences. BLAST analysis showed the phytoplasmas in winter wheat shared highest homology with 16Srâ… group phytoplasmas which revealed they had closest relationship.2. With universal 16Srâ… specific rp gene primer pairs rpF1/rpR1 and rp(â… )F1/ rp(â… )R1, 3 sequences were cloned from the same total DNAs of 3 types of 16S rRNA genes. The 3 sequences showed high homology, and the homology between each other reach to 99%. The sequences length were 1214bp, 1212bp and 1212bp, respectively. Using BLAST tool in NCBI, we knew the sequences shared highest homology with 16Srâ… group phytoplasmas which was consensus with analysis of 16S rRNA gene. So we made a conclusion that the phytoplasmas in winter wheat belonged to 16Srâ… group.3. Virtual restriction analysis tools iPhyclassifier and were introduced to analyse gene and rp gene sequences. Using iPhyclassifier to analyse 16S rRNA gene, we found that 3 kinds of band-type were manifested with Mseâ… and Hhaâ… . One type was identity with 16Srâ… -B subgroup, another was the same as 16Srâ… -C subgroup, while one kind cannot match anyone described before. Tool pDRAW32 analysed the restriction enzyme sites of the 3 rp gene fragments and discovered that 3 sequence have the same restriction enzyme sites with Mseâ… , Aluâ… and Tsp509â… , which was agree with analysis of rp gene sequences.4. According to virtual restriction analysis, restriction enzymes Mseâ… and Hhaâ… were chosen to digest the 3 types fragment sequences. Restriction fragment length polymorphism analysis confirmed that the virtual restriction analysis was right. 3 kinds of digestions manifested after digested with restriction enzymes Mseâ… and Hhaâ… . Compared with the classical band-type, they were classified into 3 subgroups of 16Srâ… , 16Srâ… -B, 16Srâ… -C and a new subgroup we named as 16Srâ… -S.5. Phylogenetic tree was constructed with Mega 4.0 using 3 16S rRNA partial sequences represent the 3 type phytoplasmas and 22 other phytoplasma strains representing 14 primary phytoplasma groups according to the classification scheme of Lee (1998). We can see that the 3 phytoplasmas was cluster 3 different branchs. One clustered one branch with Tomato big bud, 16Srâ… -B; one came together with Clover phyllody (CPh); while another itself in a branch. The results further confirmed the conclusion before.The trial here present us the diversity of phytoplasmas in winter wheat of three regions. It's lay a solid foundation for further study on phytoplasma disease in winter wheat and provide new ideas on phytoplasma disease control.