Molecular Detection And Identification Of Mulberry Dwarf, Jujube Witches'-broom, Bamboo Witches'-broom And Three New Phytoplasma Diseases In Shandong Province

In this study, Mulberry dwarf phytoplasma, Jujube witches'-broom and Bamboo witches'-broom in Shandong province were identified using molecular biotechnology. In addition, three new phytoplasma diseases on Amaranth, Liriodendron and Vitex negundo conducted a preliminary detection and identification.Infected tissues of mulberry yellow dwarf disease samples collected from Tai'an in Shandong province were assayed by polymerase chain reaction (PCR), a 1.5 kb fragment of 16S rRNA gene, a 800bp fragment of elongation factor gene and a 1.2 kb fragment of ribosomal protein gene was obtained and sequenced respectively. The sequences of 16S rRNA gene, tuf gene and rp gene were exported to pDRAW32 software for in silico restriction digestion and virtual gel plotting. Combining the results of phylogenetic analysis, we classified Mulberry dwarf phytoplasma into subgroups 16SrI-B, tufI-B and rpI-B.Through PCR analysis, phytoplasmas were found in all the witches'-broom jujube samples collected from eleven different regions in Shandong province. Combining the analytical results of virtual RFLP pattern of 16S rRNA gene, rp gene and the phylogenetic analysis, we classified Jujube witches'-broom into subgroup 16SrV-B. In addition, electron microscopy examination of the ultra-thin sections of the stem of Tai'an phytoplasma-infected jujube sample showed sieve tube elements with typical phytoplasmas.Bamboo plants exhibiting typical witches'-broom symptoms were observed in Jinan, Rizhao and Tai'an in Shandong Province. Using universal primers for phytoplasma 16S rRNA gene, PCR amplification was conducted to detect phytoplasmas from these bamboo samples. A specific fragment was obtained and we sequenced it. RFLP and phylogenetic pattern showed that the phytoplasma associated with these three bamboo witches'-broom isolates belonged to subgroup 16SrI-B. This was consistent with the previously study result, and also showed that phytoplasma strains associated with bamboo witches'-broom were common exist.Polymerase chain reaction (PCR) or nested-PCR analysis showed that special fragment of phytoplasma 16S rRNA gene were obtained respectively from abnormal tissues of three plant, Amaranth, Liriodendron and Vitex. And the fragment of ribosomal protein gene was obtained from Amaranth tissue. RFLP and phylogenetic analysis showed that the phytoplasma associated with amaranth chlorisis belongs to subgroup 16SrI-B and rpI-B, the phytoplasma associated with liriodendron yellow belongs to 16SrI-B subgroup of Aster yellows phytoplasma group and the phytoplasma associated with vitex negundo yellow falls into Elm yellow group (16SrV).