Molecular Diversity and Evolutionary Relatedness of Paulownia Witches’-Broom Phytoplasma in Different Geographical Distributions in China

SIMPLE SUMMARY: Paulownia witches’-broom (PaWB) is a severe and widespread disease caused by an obligate pathogen phytoplasma that causes substantial economic losses and ecological damage in China. As such, in this study, we first obtained detailed insight into the molecular diversity of PaWB phytoplasmas populations from different geographical distributions with multilocus sequence typing (MLST). There was comparatively high genetic diversity among the PaWB strains. Mutations in tuf and rpoB were driven by some positive selection pressure in the PaWB phytoplasma populations, and nine housekeeping genes, except for tuf, followed the neutral evolutionary model. However, the recombination among PaWB phytoplasma sequence types (STs) of each housekeeping gene, except for dnaK, was absent. The results of phylogenetic analysis revealed that the strains were clustered into two representative lineages of PaWB phytoplasma with obvious geographical differentiation. The predominant ST1 strains of CC1 were distributed in four geographical populations. The special PaWB phytoplasma CC2 was mainly distributed in Jiangxi and Fujian provinces in the south of the Yangtze River. The ST diversity of the northwest China population was the highest, including a singleton from Xi’an Shaanxi province, which was closely related with CC2. ABSTRACT: To reveal the distribution and transmission pathway of Paulownia witches’-broom (PaWB) disease, which is caused by phytoplasmas related to genetic variation, and the adaptability to the hosts and environments of the pathogenic population in different geographical regions in China, in this study, we used ten housekeeping gene fragments, including rp, fusA, secY, tuf, secA, dnaK, rpoB, pyrG, gyrB, and ipt, for multilocus sequence typing (MLST). A total of 142 PaWB phytoplasma strains were collected from 18 provinces or municipalities. The results showed that the genetic diversity was comparatively higher among the PaWB phytoplasma strains, and substantially different from that of the other 16SrI subgroup strains. The number of gene variation sites for different housekeeping genes in the PaWB phytoplasma strains ranged from 1 to 14 SNPs. Among them, rpoB (1.47%) and dnaK (1.12%) had higher genetic variation, and rp (0.20%) had the least genetic variation. The tuf and rpoB genes showed the fixation of positively selected beneficial mutations in the PaWB phytoplasma populations, and all housekeeping genes except tuf followed the neutral evolutionary model. We found an absence of recombination among PaWB phytoplasma sequence types (STs) for each housekeeping gene except dnaK, and no evidence for such recombination events for concatenated sequences of PaWB phytoplasma strains. The 22 sequence types were identified among the concatenated sequences of seven housekeeping genes (rp, fusA, secY, secA, tuf, dnaK, and rpoB) from 105 representative strains. We analyzed all 22 STs by goeBURST algorithm, forming two clonal complexes (CCs) and three singletons. Among them, ST1, as the primary founder of CC1, had the widest geographical distribution, accounting for 72.38% of all strains, with a high frequency of shared sequence type. The results of phylogenetic analysis of the concatenated sequences further revealed that the 105 strains were clustered into two representative lineages of PaWB phytoplasma, with obvious geographical differentiation. The ST1 strains of highly homogeneous lineage-1 were a widespread and predominant population in diseased areas. Lineage-2 contained strains from Jiangxi, Fujian, and Shaanxi provinces, highlighting the close genetic relatedness of the strains in these regions, which was also consistent with the results of most single-gene phylogenetic analysis of each gene. We also found that the variability in the northwest China population was higher than in other geographical populations; the range of genetic differentiation between the south of the Yangtze River population and the Huang-huai-hai Plain (or southwest China) population was relatively large. The achieved diversity and evolution data, as well as the MLST technique, are helpful for epidemiological studies and guiding PaWB disease control decisions.