Molecular Identification and Characterization of Two Groups of Phytoplasma and Candidatus Liberibacter Asiaticus in Single or Mixed Infection of Citrus maxima on Hainan Island of China

SIMPLE SUMMARY: Based on the 16S rRNA and β-operon gene fragments, two subgroups of phytoplasma—CmPII-hn belonging to 16SrII-V and CmPXXXII-hn belonging to 16SrXXXII-D—and Candidatus Liberibacter asiaticus CmLas-hn were detected separately in 12, 2 and 6 out of 54 citrus samples of Citrus maxima, an important economic crop in Hainan Island, China, infected with Huanglongbing. Among the detection results, mixed infection of 16SrII-V subgroup phytoplasma and Candidatus Liberibacter asiaticus was identified in four samples, accounting for 7.4%. The CmPII-hn strain was in a cluster belonging to the 16SrII-V subgroup, with a 99% bootstrap value. The CmPXXXII-hn strain, Trema tomentosa witches’ broom phytoplasma, belonging to 16SrXXXII-D, and the other 16SrXXXII subgroup strains were in one cluster with a 99% bootstrap value. Sixteen variable loci were detected in the 16S rRNA genes of the tested 16SrXXXII group phytoplasma strains, of which two bases had an insertion/deletion. The CmLas-hn strain and Candidatus Liberibacter asiaticus were in one independent cluster with a 99% bootstrap value. In the study, Citrus maxima, showing yellowing and mottled leaves as disease symptoms, were found, which could have been infected separately by 16SrII-V and 16SrXXXII-D subgroup phytoplasmas or could have been subjected to mixed infection by 16SrII-V phytoplasmas and Candidatus Liberibacter asiaticus in China. ABSTRACT: The pathogens associated with citrus Huanglongbing symptoms, including yellowing and mottled leaves in Citrus maxima, an important economic crop on Hainan Island of China, were identified and characterized. In the study, detection, genetic variation and phylogenetic relationship analysis of the pathogens were performed based on 16S rRNA and β-operon gene fragments specific to phytoplasma and Candidatus Liberibacter asiaticus. The results indicated that the pathogens—such as phytoplasma strains of CmPII-hn belonging to the 16SrII-V subgroup and CmPXXXII-hn belonging to the 16SrXXXII-D subgroup, as well as Candidatus Liberibacter asiaticus strains CmLas-hn—were identified in the diseased plant samples, with numbers of 12, 2 and 6 out of 54, respectively. Among them, mixed infection with the 16SrII-V subgroup phytoplasma and Candidatus Liberibacter asiaticus was found in the study, accounting for 7.4% (four samples). The phytoplasma strains of CmPII-hn—Tephrosia purpurea witches’ broom, Melochia corchorifolia witches’ broom and Emilia sonchifolia witches’ broom—were clustered into one clade belonging to the 16SrII-V subgroup, with a 99% bootstrap value. The phytoplasma strains of CmPXXXII-hn and Trema tomentosa witches’ broom belonging to 16SrXXXII-D, and the other 16SrXXXII subgroup strains were clustered into one clade belonging to the 16SrXXXII group with a 99% bootstrap value. There were 16 variable loci in the 16S rRNA gene sequences of the tested 16SrXXXII group phytoplasma strains, of which two bases had an insertion/deletion. The strains of Candidatus Liberibacter asiaticus, identified in the study and the strains that had been deposited in GenBank, were in one independent cluster with a 99% bootstrap value. To our knowledge, this is the first report showing that Citrus maxima can be infected by 16SrII-V and16SrXXXII-D subgroup phytoplasmas in China. Moreover, this is also the first report in which the plants are co-infected by 16SrII-V subgroup phytoplasmas and Candidatus Liberibacter asiaticus. More comprehensive and detailed identification and characterization of the pathogens associated with the diseased symptoms in Citrus maxima on the island in China would be beneficial for epidemic monitoring and for the effective prevention and control of related plant diseases.