Abstract:

Objective: Based on the investigation of Trema levigata broom in Xinping county, Yuxi city, Yunnan province, this study aimed to understand the occurrence and damage degree of the disease, and further determine the pathogens and their classification status by molecular biological method. Method: The incidence of the disease was obtained by general survey method, and its damage degree was evaluated. The total DNA of the affected plants was extracted, and the 16S rDNA sequences were amplified by using nested-PCR from the total DNA with phytoplasma universal primer pair P1/P7 and R16F2n/R16R2, then the amplified fragments were cloned and sequenced. A sequence analysis was performed with iPhyClassifier, an online classification and identification tool of phytoplasma, and a phylogenetic tree based on the 16S rDNA sequence was constructed by using Megax software to determine the pathogens and their classification status. The homology of related sequences were compared by online analysis software MUSCLE. Result: The survey result indicated that T. levigata witches' broom approximately occurred in east longitude 101°35'-101°53' and north latitude 23°56'-24°20'. It was mainly in the dry-hot valley region at the low altitudes from 600 to 400 m with relatively high temperature, which also showed that high temperature was conducive to the occurrence of phytoplasma disease. In this region, the disease occurred for three consecutive years. The average incidence rate was 38.9% with moderate damage in July 2018. The 1 246 bp 16S rDNA sequence of T. levigata witches' broom phytoplasma was amplified by PCR amplification (strain: TLWBYN0, GenBank accession: MN513329). Analysis showed that TLWBYN01 shared 99.8% similarity with the reference strain of Candidatus Phytoplasma asteris (GenBank accession: M30790), thus the phytoplasma under study was a 'Candidatus Phytoplasma asteris' related strain and belonged to a member of 16S rI group. The virtual RFLP pattern derived from the F2nR2 fragment of TLWBYN01 was most similar to the reference pattern of the 16SrⅠ, subgroup X (GenBank accession: JF781308), with a pattern similarity coefficient of 0.98. The enzymatic profiling of 17 restriction enzymes showed that only MseI was different from the 16S rI-X reference strain. Thus, the phytoplasma was a variant of 16S rI-X. TLWBYN01 had 99.2% and 99.6% homology respectively with phytoplasma of papaya bunchy top (JF781308) and lagenaria siceraria yellow little leaf (LT594117、LT594118), two members of 16S rI-X subgroup. Conclusion: Trema levigata witches' broom from Xinping county of Yuxi city is a local common disease with moderate damage. But once being infected, the plant growth could be seriously affected. T. levigata witches' broom phytoplasma belongs to the member of 16S rI group and is a variant of 16S rI-X subgroup with being less reported so far, and T. levigata is a new host of phytoplasma. These findings are of great significance for further study of phytoplasma. At the same time the plant is the pioneer tree species for greening and widely distributed, thus the investigation and study on the disease could provide a basis for prevention and control of forest diseases in this region.

Key words: phytoplasma, Trema levigata, witches'-broom, 16S rDNA, molecular identification