| The whitefly Bemisia tabaci is a species complex composed of numerous biotypes,which also has been reported as a major agricultural pest due to its broad hosts,destructive damage,like directly feeding,honey dew secreted induced plant mildew stains,and transmission of plant viruses.In this study,Rickettsia negative B.tabaci biotype B and Rickettsia positive B.tabaci biotype B have been used as research objects,we used PCR to identify the endosymbionts infecting B.tabaci biotype B populations in South China,and real-time quantitative PCR to study their infection concentrations in various stages of whitefly development,and test the Rickettsia content changes in plant tissues.We looked at spatial localization of the Rickettsia in whitefies and plants phloem tissues with fluorescence in situ hybridization(FISH)and transmission electron microscope(TEM),respectively.We also analyzed the Rickettsia effects in plant defense responses by using Digital gene expression spectrum technology(DGE).Finally,we determined the nutrition level of the plants and whiteflies biological characteristics effected by Rickettsia.The main results were as follows: 1.Infection dynamics of endosymbionts and Rickettsia localization patterns in whitefliesWe found that field population of B.tabaci biotype B were infected with at least one primary endosymbiont(Portiera)and three secondary endosymbionts(Rickettsia,Hamiltonella,Hemipteriphilus).The infection percentages of these secondary endosymbionts in B.tabaci adults were 32.6 %,93.8 %,and 97.9 %,respectively.The Real-time fluorescent quantitative PCR results showed that relative titer amounts of Portiera,Hamiltonella and Rickettsia all generally increased with the development of whiteflies.The dynamics of Hamiltonella were similar to Portiera,which titer increased mainly in 2 instar and 4 instar nymphy periods.The titer of Rickettsia increased mainly in whiteflies eclosion process.The titers of Portiera have no differences between male and female,but titers of Hamiltonella and Rickettsia in female adults were significantly higher than male adults.In the periods of egg to 3 instar nymphy,the titers of Portiera were highest,followed by Hamiltonella,Rickettsia titers were lowest.In the periods of 4 instar nymphy and adults,the titers of Portiera have no differences compared with Hamiltonella,but still higher than the titers of Rickettsia.FISH detection results showed that the Rickettsia were limited in the central eggs and nymph bacteriocytes,or scattered in eggs and nympha body not including bacteriocytes,the former were termed as “confined” pattern,the latter were termed as “scattered” pattern.In adults,Rickettsia localized in the foot,wax glands,female colleterial glands,and bacteriocytes which scattered in the abdomen of the female adults.2.The titer dynamics and location of Rickettsia in plantsWe identified the Rickettsia in cotton,tomato and cowpea plants.Conventional PCR results showed that the Rickettsia can be transmitted to cotton,tomato and cowpea plants by Rickettsia infected whiteflies,the Rickettsia were alive in plants.TEM observations showed that the Rickettsia localized at the plants phloem sieve tube,and only one Rickettsia out of every plant cell.The Real-time quantitative PCR results showed that the titers of Rickettsia in plants were increased with the increase of whiteflies feeding time,but reduce after reaching peak;the feeding time of the highest titers of Rickettsia in cotton plants were 28 d,in plants tomato were 21 d,in plants cowpea were 14d;the whiteflies were removed after feeding 7 d,the titer of Rickettsia in cotton leaves were decrease with time,in tomato leaves were slightly increase after decrease,in cowpea leaves decrease firstly then remain unchanged.3.Rickettsia involved in regulating plant defense responsesWe analysed the effects of Rickettsia to cotton and tomato defense reactions using DGE.Results showed that there were 60 in cotton and 85 in tomato differentially expressed genes caused by Rickettsia.These differentially expressed genes including protein kinases,protease inhibitors,transcription factors,metabolites synthase,and pathogen-related genes,etc,which mainly enrichment of in Plant hormone signal transduction pathways(Plant hormone signal transduction),Plant pathogen interaction pathways(Plant-pathogen interaction),the MAPK signaling pathways(MAPK signaling pathway-Plant),secondary metabolites synthesis(Biosynthesis of secondary metabolites)pathways.In cotton plants,the defenses based on initial immune response,JA signaling pathways and secondary metabolites were suppressed,while the the defenses based on SA defense responses signal transduction pathways were increased.In tomato plants,the defenses based on initial immune response and SA signal transduction pathways were increased,while the defense reaction based on JA signaling pathways,secondary metabolites,and stress response to mechanical damage were suppressed.4.The effects of Rickettsia on plant nutrition metabolismThe results of soluble sugar content determination showed that the soluble sugar content were increased in cotton and tomato leaves after Rickettsia positive whiteflies feeding compared with Rickettsia negative whiteflies feeding,no difference in cowpea plant leaves.The results of free amino acid content determination showed that the content of total free amino acid were increased in cotton and tomato leaves after Rickettsia positive whiteflies feeding compared with the Rickettsia negative whiteflies feeding,while decreased in cowpea;the essential amino acid in cotton,cowpea,tomato leaves were all increased,too.5.The effects of Rickettsia horizontal transmission on whiteflies biological characteristicsThe results showed that the spawning,female ratio in Rickettsia positive whiteflies were significantly higher than Rickettsia negative whiteflies,the developmental duration were significantly shorter than Rickettsia negative whiteflies.When Rickettsia exist in plants,the spawning of Rickettsia negative whiteflies were significantly increase. |
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