First Report of Phytoplasma Infecting Pterocarpus indicus in China

Abstract

Pterocarpus indicus Willd., an adaptable and fast-growing landscape tree, is commonly cultivated in landscaping in southern China. In April 2018, a P. indicus tree with symptoms of flat branches and typical fasciation, which had the same symptoms of phytoplasma infection, was observed in Haikou, Hainan province, China. To determine the presence of phytoplasma, stem samples of symptomatic and asymptomatic P. indicus were subjected to total DNA extraction using the plant Genomic DNA Kit (Tiangen, Beijing, China), followed by nested polymerase chain reaction (PCR) detection of phytoplasma (Jomantiene et al. 1998; Lee et al. 1998). Briefly, the universal primer pair R16mF2/R16mR1 was employed for the initial PCR amplification of 16SrDNA fragment of phytoplasma and primer set R16F2n/R16R2 (Lee et al. 1998) for nested PCR amplification. As expected, the PCR products were generated from the samples of the symptomatic plant but not from the symptomless control samples. The anticipated amplicon was approximately 1.2 kb in size. The PCR products were gel purified using TIANgel Midi Purification Kit (Tiangen, Beijing, China) and cloned into pMD-18T vector (Takara Bio, Dalian, China). Three independent positive clones were subjected to Sanger sequencing. The obtained sequence was deposited into NCBI GenBank database with GenBank accession no. MH727702. A BLASTn search against the nonredundant nucleotide database revealed high sequence identities with the corresponding regions of phytoplasmas of aster yellows phytoplasma group (16SrI). A maximum of 99.7% nucleotide sequence identity with Malvastrum coromandelianum phyllody phytoplasma (GenBank accession no. MF490802) was observed. Moreover, an iPhyClassifier (Zhao et al. 2013) operation was used to perform sequence comparison and generate a virtual restriction fragment length polymorphism (RFLP) profile for the sequence derived from the symptomatic P. indicus sample, which showed the most similarity with that of the reference strain (GenBank accession no. AP006628) belonging to 16SrI group. However, this strain may represent a new subgroup because the similarity coefficient was only 0.91, which is within the values set for a new subgroup (Biswas et al. 2013). To our best knowledge, this is the first report of phytoplasma infecting P. indicus in China.