Analyzing The Bacterial Diversity Of Endophytic Bacteria In Diaphorina Citri With Candidatus Liberibacter Asiaticus And Functional Verification

Huanglongbing (HLB) is a deadly diseases of citrus. There is no efficient meansof prevention and cure. Once the plant disease, they must be eradicated and burn away.The pathogenic bacteria, Candidatus Liberibacter asiaticus (Ca.L.asiaticus), a phloemlimited, alpha proteobacteria, can’t be cultured in vitro. Diaphorina citri belongs to theinsecta, Homoptera, Psyllidae, mainly parasitizes citrus and Murraya paniculata.Diaphorina citri is main media of HLB and is home to multiple of bacterial endophyte.In order to analyze the endosymbiotic bacteria in Diaphorina citri which mayaffect the pathogenesis of the Candidatus Liberibacter asiaticus, we compared thebacterial diversity of endophytic bacteria in different samples of Diaphorina citri bytraditional isolating culture method and PCR-DGGE, and verified the result by q-PCR.32times isolated culture of endosymbiotic bacterium in Diaphorina citri withoutCa.L.asiaticus, we found total14kinds of bacteria, belonging to10genuses, such asBacillus sp.(3), Erwinia sp.(1), Klebsiella sp.(1), Staphylococcus sp.(2), Arthrobactersp.(1), Pantoea sp.(2), Pectobacterium sp.(1), Salmonella sp.(1), Streptomyces sp.(1),Massilia brevitalea sp.(1).4kinds of endosymbiotic bacterium in Diaphorina citri withCa.L.asiaticus had all been found in Diaphorina citri without Ca.L.asiaticus. Theybelong to Bacillus sp., Klebsiella sp., Pectobacterium sp. Isolation frequency of Dc-11,a kind of Bacillus aerophilus, in Diaphorina citri with or without Ca.L.asiaticus bothare100%, which indicated it’s resident flora in Diaphorina citri.As the electrophoretogram of16S rDNA PCR-DGGE reveals, there are significantdifferences between Diaphorina citri with Ca.L.asiaticus and without Ca.L.asiaticus inthe same tissues; but no significant differences were found in different tissues from thesame Diaphorina citri. Predominan bands No.10(Wolbachia sp.),No.12(Wolbachiapipientis), No.13(Syncytium endosymbiont of Diaphorina citri), No.14(Unculturedbacterium), No.19(Serratia marcescens), No.21and No.22(both areStenotrophomonas maltophilia) are steadily stay in Diaphorina citri with or withoutCa.L.asiaticus. Enterobacter sp. is one of the dominant bacteria endosymbioticbacterium of abdomen of Diaphorina citri with Ca.L.asiaticus. Wolbachia(band No.1,No.10, No.12) also parasitize in Diaphorina citri. The result also indicates thatcompany of Ca.L.asiaticus might change the population and quantity of endosymbioticbacteria. Comparing the results of traditional isolating culture method and PCR-DGGE, wefound obvious differences of kinds of dominant bacteria population, so we choseBacillus sp., Salmonella sp., Enterobacter sp. for q-PCR in8Diaphorina citri samples.Bacillus sp. is both detected in traditional isolating culture method and PCR-DGGE.Salmonella sp. is isolated in Diaphorina citri without Ca.L.asiaticus while not detectedin PCR-DGGE. Enterobacter sp. is only appearing in PCR-DGGE. We use total amountof bacteria as a amount of contrast in order to get relative amount of the3chosenbacteria respectively. Then map using-Lg value of relative amount of the3chosenbacteria respectively after LSD test. Comparing relative amount of the3chosen bacteriaand relative amount of Ca.L.asiaticus in every Diaphorina citri sample beforecomparing similarity of the same bacteria in different samples in order to analyze theinteraction of the3chosen bacteria and Ca.L.asiaticus.Showing the results of q-PCR test of the3chosen bacteria and Ca.L.asiaticus andanalyzing:(1) Ca.L.asiaticus Relative amount of Diaphorina citri sample No.1--No.4gets to0.2%-3.56%, while order of magnitudes of No.5--No.8gets to10-6-10-7.Diaphorina citri sample No.1--No.4test positive of Ca.L.asiaticus but sampleNo.5--No.8test negative.(2)Enterobacter sp. Order of magnitude of relative amountof Diaphorina citri sample No.1--No.4is5-8more than sample No.1--No.4, whichmeans relative amount of Enterobacter sp. in Diaphorina citri with Ca.L.asiaticus is103times more than in Diaphorina citri without Ca.L.asiaticus in these8samples. SoEnterobacter sp. and Ca.L.asiaticus are positively correlated relationship. Relativeamount of Ca.L.asiaticus and Enterobacter sp. are both very low, getting to10-5-10-7,sowe can regard them as test negative after refer to specialty of q-PCR.(3)Salmonella sp.Relative amount of Salmonella sp. are all low in every sample, order of magnitudesgetting10-4-10-7.(4)Bacillus sp. Relative amount of Salmonella sp. are also low inevery sample, and have little difference in different sample. This result has explainedthe different of traditional isolating culture method and PCR-DGGE, and verifiedreliability of traditional isolating culture method and PCR-DGGE before.Comprehensive the above results, this study got the following conclusions:(1)Company of Ca.L.asiaticus changed the variety and structure of endosymbioticsymbiotic bacteria.(2) Bacterial species of culturable bacteria in Diaphorina citriwithout Ca.L.asiaticus are more than in Diaphorina citri with Ca.L.asiaticus.PCR-DGGE revealed that there are significant differences between Diaphorina citriwith Ca.L.asiaticus and without Ca.L.asiaticus. So company of Ca.L.asiaticus changed the species and quantity of endosymbiotic symbiotic bacteria.(3) Suspected residentflora Bacillus aerophilus was isolated frequently in Diaphorina citri.(4) There is someinteraction between Enterobacter sp. and Ca.L.asiaticus, but the interaction mechanismremain to further investigate.