Seasonal variation and detection frequency of Candidatus Liberibacter asiaticus in Binchuan, Yunnan province China

Abstract Huanglongbing (HLB) is one of the most serious diseases affecting citrus trees worldwide. The causal agent of HLB is the nonculturable endophytic bacterium Candidatus Liberibacter asiaticus ( C las) and Diaphorina citri (Asian citrus psyllid, ACP) its insect vector. In this study, we investigated the seasonal variations in the population dynamics of C las and assessed the possible genetic variations among the identified isolates. We conducted a disease survey in tangerine citrus groves affected with HLB in Binchuan county, Yunnan province, China, on a monthly basis over a period of one year. The collected leaf samples were analyzed every month via nested PCR, targeting the 16S rRNA and the β-operon ribosomal protein-encoding genes of C las. The abundance of the HLB-causing pathogen changes several times, with high temperature in July 2017 yielding low pathogen abundance, followed by a high peak in December and January 2017-18. The number of bacteria inside each tree was inconsistent, showing drastic variations every month. C las-negatively affected the endophytic microbial communities inside the citrus trees. There could be a direct relationship between the low number of endophytes and the high number of pathogens present inside each tree. Moreover, the genetic diversity of C las can be used to elucidate the pattern and epidemiology of this pathogen in different regions. The obtained sequences were phylogenetically related to other sequences from China. A comparison of C las gene sequences revealed little variation among the isolates circulating in China. The primers used in this study did not show cross-reactivity with other plant pathogens or with endophytes isolated from citrus trees. In addition, no mismatches were observed when an in silico analysis was performed. In summary, this is the first report of the seasonal variation of HLB-causing pathogen in tangerine citrus varieties in China. Environmental influences and endophyte acquisition during different months help us understand the C las population during different seasons, and to improve pathogen detection. The monthly fluctuations in pathogen abundance could aid in the design of proper management strategies in the field.