Multilocus sequence analysis for detection of finer genetic variation and phylogenetic interrelatedness in 16SrI group phytoplasma strains infecting different plants in China

The group 16SrI phytoplasmas are associated with severe diseases of many cash and ecological plants throughout the world. To date, the genetic variation and population structure of very closely related phytoplasma strains are still not fully understood in China. In this study, a multilocus sequence analysis (MLSA) scheme was developed using ten housekeeping genes (rp, tuf, secA, secY, ipt, dnaK, fusA, gyrB, pyrG and rpoB) fragments compared with 16S rDNA to analyze 18 phytoplasma strains infecting chinaberry, lettuce, mulberry, paulownia and periwinkle from ten provinces in China. The nucleotide site polymorphisms resolved all strains into 15 sequence types (STs), demonstrating extensive genetic diversity among the 16SrI group strain population. All the strains, classified in 16SrI-B and-D subgroup by 16S rDNA analysis, clustered into one clade and clearly differentiated into discrete subclades by phylogenetic analysis of the concatenated gene sequences. The 10 chinaberry witches’ broom (CWB) strains that were most closely related to two mulberry dwarf (MD) and hardly distinguished with 16S rDNA, were definitely split into four distinct clusters and 8 STs apparently congruent to their geographical locations. Two lettuce yellows (LY) strains in Sanming, Fujian province, China were more closely related to the onion yellows OY-M strain in Japan than the periwinkle virescence (PeV) and paulownia witches’ broom (PaWB) strains in China. The levels of variation in dnaK gene were higher than those in 16S rDNA and other genes tested. This MLSA is a promising approach for phytoplasma differentiation as well as for in depth examination of strain diversity and evolution of various 16Sr groups or subgroups.