Primary Study On Species And Intraspecific Differentiations Of HLB Pathogens In Eight Provinces Of China

Citrus huanglongbing(HLB)is a destructive citrus disease caused by Candidatus Liberibacter, distributed widely in Asia,Africa and America.It has caused tremendous economic loss to the Chinese citrus industry in Guangdong,Guangxi,Fujian and Taiwan,etc.Three different species of HLB bacteria are currently known,Candidatus Liberibacter asiaticus, Ca.L.africanus and Ca.L.americanus.Abroad correlative researches demonstrate the phenomena of differentiations in bacteria of Ca.L.asiaticus.HLB was first discovered in China.However,the species of HLB bacteria and differentiations in bacteria of Ca.L.asiaticus in China have not been deeply studied.Identifying the species of HLB bacteria and condition of differentiations in bacteria of Ca.L.asiaticus in China are very important for the detection,control and further research of HLB of China.Different molecular detection techniques were established and perfected in this research. And these techniques were used to study the species of HLB bacteria of eight provinces in China such as Guangdong,Guangxi,Fujian,Jiangxi,Zhejiang,Hunan,Yunnan and Guizhou.Restriction endonuclease fingerprinting-single strand conformation polymorphism(REF-SSCP)technique and sequence analysis were applied to observe the condition of differentiations in bacteria of Ca.L. asiaticus in China.Bacteria of Ca.L.americanus in China was confirmed by sequencing.The following results are achieved:1.Nested PCR and semi-nested PCR for detection of Ca.L.americanus were developed in this study.The amplified bands were 545bp and 549bp respectively.It is the first time in the world to set up these two PCR protocols specifically for detection of Ca.L.americanus.2.In 330 samples from eight provinces of China which had HLB,96 samples were detected to contain bacteria of Ca.L.asiaticus,1 sample from Hunan was detected to contain bacteria of Ca. L.americanus.No sample was detected to contain bacteria of Ca.L.africanus.3.Some symptomatic samples could not be detected to contain any one of the three liberibacters.4.It is the fisrt time to use REF-SSCP technique to detect nucleotide mutations of 16S rDNA of Ca. L.asiaticus in international wide.This gene region of Ca.L.asiaticus in China was very conservative.No obvious difference had been found in this gene region of 96 samples.5.By sequence analysis of 16S rDNA,16S/23S ribosomal intergenic region and ribosomal protein genes ofβoperon amplified from Ca.L.asiaticus agents of eights provinces,it was found that the sequence identities were all very high in this three regions,for 99.6%-100%,99.5%-100% and 99.6%-100%,respectively.It could be concluded that the condition of differentiations in bacteria of Ca.L.asiaticus in China might be non-existed or not distinct.6.549bp fragment from 16S rDNA of Ca.L.americanus agent from China was cloned,sequenced, and compared to Ca.L.asiaticus(L22532),Ca.L.africanus(L22533),and Ca.L.americanus (AY742824)published in Gen Bank.The similarities were 94.0%,94.3%and 99.6%, respectively,suggesting that this one agent from Hunan should be Ca.L.americanus.It is the first time in the world to find Ca.L.americanus in China.