Molecular Detection And Identification Of Phytoplasmas Associated With Mulberry Dwarf, Jujube Witches'-broom And Other Three Phytoplasmas In Shandong Province

In this study, phytoplasmas associated with Mulberry dwarf, Jujube witches'-broom, Amaranth chlorosis, Chinaberry witches'-broom and Sophora witches'-broom were detected and identified using molecular biotechnology.A fragment of about 1.4 kb was amplified in DNA samples extracted from plant tissue infected by mulberry dwarf phytoplasma in Shandong province. The fragment was 1361bp containing 1242bp of secY gene which encodes 414 amino acids. Phylogenetic analysis, homology comparison and computer simulated RFLP analysis with nine enzymes indicated that mulberry dwarf phytoplasma belongs to secYI-B subgroup. 842bp of secA gene was obtained and sequenced which encodes 280 amino acids, analysis of secA gene indicated that mulberry dwarf phytoplasma belongs to 16SrI-B subgroup identical to the results of analysis based on 16S rRNA gene.The secY gene of Jujube witches'broom phytoplasma (JWB) collected from eleven areas in Shandong Province was amplified using FD9f/r primers, the fragment was 1421bp. The sequence of Jujube witches'broom phytoplasma was compared with the related sequences of phytoplasma in NCBI. The results of phylogenetic analysis and virtual RFLP showed that jujube witches'broom phytoplasma belonged to subgroup secYV-C, having the closest relationship with CLY5 (secYV-B) and PY-In (secYV-N). The secA gene was 836bp encoding 277 amino acids, the results showed that Jujube witches'broom phytoplasma belongs to 16SrV group, the same as the result reported before.Amaranthus viridis showing symptoms of typical chlorosis was collected and detected by polymerase chain reaction (PCR), a 800bp fragment of elongation factor gene, a 1.4 kb fragment of secY gene, and a 0.8 kb fragment of secA gene was obtained and sequenced respectively. Combining the results of homology comparison, phylogenetic analysis and computer simulated RFLP analysis, we classified amaranth chlorosis phytoplasma (AmC) into subgroups tufI-B and secYI-B and it shared high homology with mulberry dwarf phytoplasma.Chinaberry witches'broom phytoplasma (CWB) collected from Shandong Province was detected and identified. Five different type universal primer pairs of phytoplasma were used to amplify 1.4 kb fragment of 16S rRNA, 1.2 kb fragment of rp gene, 0.8 kb fragment of tuf gene, 1.4 kb fragment of secY gene and 0.8 kb fragment of secA gene. The PCR-amplified products were cloned and sequenced. The sequences of chinaberry witches'broom phytoplasma were compared with the related sequences of phytoplasma in NCBI. The results of phylogenetic analysis and virtual RFLP showed that chinaberry witches'broom phytoplasma belonged to subgroup 16SrI-B,rpI-B,tufI-B and secYI-L.Sequencing of PCR products and electron microscopy were used to investigate the possible association of phytoplasma with sophora witches'-broom (SWB). All results indicated the presence of phytoplasma in the symptomatic sophora plants. Sequence analysis of 16S rRNA gene, rp gene, secY gene and secA gene confirmed that the phytoplasma associated with SWB belongs to subgroup 16SrV-B, rpV-C and secYV-C.