Study on dynamic content change of Candidatus Liberibacter asiaticus in Citrus

Abstract: 【Objective】Citrus Huanglongbing (HLB) is one of the most serious diseases of citrus around the world. In China, HLB is caused by Candidatus Liberibacter asiaticus(Las), a gram-negative, phloem-restricted and psyllid-transmitted bacteria. The most effective control methods in China contain planting healthy citrus seedlings, controlling psyllids, and eliminating infected trees. To provide feasible evaluation methods for elimination of infected trees and prolong the economic life of citrus trees, we explored the epidemics of citrus HLB in Ganzhou area by monitoring the growth pattern of Las in infected citrus trees.【Methods】The monitoring was carried out in an orchard where citrus trees were planted for years in Ganzhou area of Jiangxi province, and the psyllids were under strict control. Twenty-five trees were selected for monitoring and were divided into five levels according to the yellowing symptom degrees. From August 2017 to October 2018, leaves were collected from each selected tree. The midrib of collected leaves was cut out by a razor blade, which was washed with 75% ethyl alcohol in advance,and 100 mg of midrib was ground by FastPrep-24 5 G. Total DNA was extracted from the equivalent of midrib tissue by using AxyPrepTMMultisource Genomic DNA Miniprep Kit. The specific primers HLBas/HLBr targeting the 16 S rDNA of Las, and probe HLBp, were employed in the TaqMan qPCR detection system. To evaluate the sensitivity of this detection method, the plasmid containing the amplicon sequence of HLBas/HLBr was constructed by Beijing Genomics Institute, which was prepared at a gradient diluted 10 times by DEPC water to generate a standard curve for quantification of Las. The Las in foliage samples collected from August 2017 to October 2018 was detected and analyzed using Excel2019. Collecting the climate data from weather website, the rainy days and mean daily maximum temperatures for each month were obtained and their correlations with the Las contents were analyzed using SPSS 22.0.【Results】The plasmid pUC57-Las containing the amplicon sequence of HLBas/HLBr was obtained and verified by PCR and sequencing. The standard curves constructed by testing the pUC57-Las diluted in water had an efficiency of 98.1% with the correlation coefficient of 0.999. The unit DNA concentration was converted to template copy number, by the linear formula Y=-3.369 lg(CN) +44.422(Y: CT, CN: template copy number/DNA concentration). The sensitivity of the qPCR used in this study was 1.67 fg · L^-1. In the monitoring research, the annual dynamic of Las contents in citrus leaves showed two peaks from August to November 2017 and May to July 2018, and two valley periods from April to May 2018 and July to August 2018. In the trees that were ranked as 1, 2, 3, 4 and 5 disease levels, the contents of Las in the north branches was higher than that in the south, whereas in trees showing symptom of 4 th level, the content of Las in the north branches was lower than that in the south. Comparing with Las contents of different disease levels, Las contents in the most severe symptoms of 5 th disease level was lower than the other area, and looking from peaks, the level was higher and the las content was lower, so the severity of the symptoms was contrary to Las content. The daily maximum temperature had no correlation with Las contents, with coefficient of association of-0.12 and no significance(p > 0.05). Similarly, number of rainy days also had no correlation with Las contents, with coefficient of association of-0.071 and no significance(p > 0.05).【Conclusion】We quantitatively monitored the growth and distribution pattern of Las in citrus trees in Ganzhou area by employing a TaqMan qPCR detection method and construction of a plasmid containing the HLBas/HLBr amplicon sequence.The monitoring spans from August 2017 to October 2018 found that the Las in the infected trees showed a certain pattern of increase and decline, which was close to the previous research that the Las contents dramatically increased in October. There was no correlation with two climatic factors, mean maximum temperature and rainy days in each month, and the data showed temperature and rainy days were not the factors affecting HLB in orchard. The orchard was a good place for the experiment because of strict control of psyllid, so the psyllid influence can be excluded. The psyllids were insect media of Las and its population dynamics should be included in this monitoring research. During the whole study spanning from 2017 to 2018, however, the orchard, where the monitoring was carried out,strict control of psyllids was applied. Therefore, no psyllids were observed in our survey. Above all, the results will be helpful to understand the Las content outbreak period and influence factors of HLB in the orchard. In addition to the control of HLB, our results will provide a basis for evaluating the prevention and control effects of HLB, it may reduce the control cost and citrus growers' loss.