Pcr Detection Of Citrus Huanglongbing And Investigation Of Disease Distribution In Hunan Province

Citrus Huanglongbing caused by Candidatus Liberibacter is one of the most devastating diseases of citrus industry, which was first reported in GuangDong and GuangXi regions of China. Hunan province belongs to the occasional citrus huanglongbing area. With global warming, citrus psyllids made easier to live through the winter in the north of Hunan province and the disease tends to expand in this area. In order to get the distribution of Citrus Huanglongbing and understand the genetic variation of this pathgen among different regions in Huann province,172citrus samples suspected to be Huanglongbing were collected form more than20counties in Hunan province and104samples were positive detected by PCR. In this study, the main results are as follows:1、Screening out the16S rDNA primers OI1/OI2and the ribosomal protein gene sequence primers TR/TL. It needs additional enzymes digestion to distinguish Candidatus liberibacter asiaticus and Candidatus liberibacter africanus with OI1/OI2primers, while the Candidatus liberibacter asiaticus could be detected by TR/TL with high specificity and stability. The agents of Hunan province shows a close relationship to Candidatus Liberibacter asiaticus M94319with the sequence similarity analysis.2、Through systematic survey, we were familiar with the distribution of citrus Huanglongbing in Hunan province. The survey results show that we can detect the pathogen from the samples from YueYang, the northest distinct of Hunan province, but no citrus psyllid had been found and it is considered that the disease was caused by infected seedling in this area. We proposed that TongDao、XinNing、DongKou、 WuGang、LongHui and JiangYong should be treated as new quarantine areas.3、Improved the citrus psyllid PCR detection method and updating the knowledge of its distribution in Hunan province. The result showed that the north boundary of citrus phyllid expanded to TongDao county in HuaiHua distinct in Hunan. 4、Based on target fragment cloning, sequencing, and sequence homology and cluster analysis, it is shown that the agent of citrus Huanglongbing in Hunan belongs to Candidatus Liberibacter asiaticus and the similarity of all the pathogens were more than99.1%and the pathogen has no significant difference between geographical distribution.