Occurrence of 'Candidatus Phytoplasma asteris'-Related Strains infecting Chinaberry (Melia azedarach) Showing Chlorotic Leaf Symptoms on Hainan Island of China.

Abstract

Chinaberry (Melia azedarach), belonging to the family of Meliaceae, is an ornamental tree distributes across southern of China. In the autumn of 2021, In an area of 400 acres located in Wanning city of Hainan Province, a tropical island in China, with coordinates of 110°28'42.72″E, 19°2'9.96″N, about 20 % (100) of the chinaberry trees showed disease symptoms included chlorotic leaves. The disease symptoms were consistent with infections by a phloem-limited prokaryotic pathogen phytoplasma. The samples of six symptomatic and three asymptomatic were collected for pathogen detection. To identify the pathogen, total nucleic acids were extracted from 0.10 g fresh leaf tissues from the diseased and healthy plant using CTAB DNA extraction method based on Doyle and Doyle. Three primer pairs of R16mF2/R16mR1, secAfor1/secArev3 and fTuf1/rTuf1 were used for specific identification of phytoplasma conserved gene fragments of 16S rDNA, secA and tuf, PCR amplification. Target PCR bands were amplified from the DNA of six diseased chinaberry samples, but not from the DNA of the healthy samples. The products of amplified were cloned and sequenced by Biotechnology (Shanghai) Co., Ltd. (Guangzhou, China). The phytoplasma gene sequences of 16S rRNA, secA and tuf were obtained and all the sequences were identical with the length of 1336 bp, 710 bp and 955 bp, respectively. Representative sequence data for strain MaCL-hn were deposited in Genbank under accession Nos. OR438638 (16S rDNA), OR513089 (secA) and OR860415 (tuf). The phytoplasma strain identified in the study was described as chinaberry chlorotic leaf (MaCL) phytoplasma, MaCL-hn strain. BLAST search based on 16S rRNA genes showed that 43 strains in 16SrI group 'Candidatus Phytoplasma asteris' showed 100% similarity with the 16SRNA sequence of MaCL-hn. BLAST search based on secA genes showed that 9 strains in the phytoplasma group showed 100% similarity with the 16SRNA sequence of MaCL-hn. BLAST search based on tuf genes showed that 21 strains in the phytoplasma group showed 100% similarity with the 16SRNA sequence of MaCL-hn. RFLP analysis based on iPhyClassifier indicated that the MaCL-hn strain was a member of 16SrI-B subgroup with a similarity coefficient 1.00 to the reference phytoplasma strain (AP006628). Phylogenetic tree was constructed based on 16S rRNA by MEGA 11.0 using neighbor-joining (NJ) method with 1000 bootstrap value. The results showed that the MaCL-hn strains were clustered into one clade with 16SrI group 'Ca. Phytoplasma asteris' related strains with 99 % bootstrap value. Multilocus sequence analysis (MLSA) based on the concatenated sequences with the length of 3001 bp including the sequences of 16S rRNA, secA and tuf showed that the MaCL-hn strains were clustered into one clade with the phytoplasma strains in the group with 100 % bootstrap value. To our knowledge, this is the first report that chinaberry can be infected by 'Ca. Phytoplasma asteris'-related strains belonging to 16SrI-B subgroup on Hainan Island of China. This finding in the study will contribute to the epidemic monitoring and the preventive management of the phytoplasmas and their related diseases.