| Citrus Huanglongbing(HLB)is one of the most destructive diseases of citrus industry and seriously threats the economic benefits of citrus for more than 100 years in China.The pathogen(Candidatus Liberibacter asiaticus,CLas)is a kind of gram-negative bacteria of Candidatus Liberibacter spp and could be transmitted by its insect vector,Diaphorina citri Kuwayama,in a persistent propagative manner in the planting areas.Outer membrane protein(OMP)is an important component of the bacteria and has the species specificity.Selecting OMP to analyze CLas diversity is beneficial to study the pathogenicity and epidemiological mechanism of CLas.And it is of great significance to prevent and cure citrus HLB.However,the infection mechanisms of CLas in vector citrus psyllid remain unknown.In this study,the outer membrane protein(OMP)genes of CLas isolates from five different HLB diseased areas in Fujian province have been analyzed by DNAman and MEGA.The results showed that the OMP genes of CLas have no species differences,and neither nucleotide insertions nor deletions.But there exists slightly differences in nucleotide sequences.To sum up,CLas of 5 diseased areas have a genetic diversity within species,and CLas in Nanping region is more different from other places.In order to analyze the infection pathway of CLas in citrus psyllid,we detected the carrier rate of CLas by PCR.The results showed that the carrier rate of CLas is less than 6%when the citrus psyllid fed on diseased citrus less than one day.When the citrus psyllid fed on diseased citrus from 2 days to 7 days,the citrus psyllid carrier CLas rate is significantly increased(P<0.05).when more than 7 days,the citrus psyllid carrier CLas rate hold steady at 50%.To study the infection mechanism of CLas in citrus psyllid by fluorescence in situ hybridization(FISH),we tried two different fixed methods to label the CLas in the alimentary canal of citrus psyllid,including Carnoy’s Fluid and paraformaldehyde(PFA).We found that PFA fixed method with a mild condition,the background of the probe residue after labeling was small and actin could be specifically marked.So we selected PFA fixed method to study the infection mechanisms of CLas in insect vector citrus psyllid.In this paper,we investigated the infection route of CLas in the digestive system of citrus psyllid by FISH.The results showed that CLas reached the midgut through the stylet to anterior intestine and the filter chamber after citrus psyllid acquised the CLas from HLB disease plants.At 2-6 days post first access to diseased plants(padp),CLas infected the midgut to set up the primay infection site.From then on,CLas was begin to spread along with the muscles of the midgut to other organs or tissues.At 8 days padp,the CLas could be observed in the midgut by immunofluorescence microsopy and detected in hemolymph of citrus psyllid by PCR.At 12 days padp,CLas were observed in the midgut and salivary glands.At 15 days padp,CLas systematically infected the whole intestinal tract.At 18 days padp,CLas infected salivary glands systematically.These all suggest that CLas have completed the infection cycle in its insect vector.In conclusion,this study analyzed and demonstrated the genetic diversity of CLas from different citrus HLB diseases areas in Fujian province.We explored the infection pathway in digestive system of citrus psyllid by FISH assay and observed the morphology and structure of CLas using electron microscopy.Our results figured out the infection mechanisms of CLas in insect vector citrus psyllid,which will shed a light on the research about the interaction between the CLas and its insect vector... |
