| Huanglongbing is a devastating disease that poses a great threat to citrus industry worldwide.Diaphorina citri is not only a key pest on citrus,but also an important insect vector of Huanglongbing.At present,the abuse of chemical pesticides leads to severe insect resistance problem and destruction of ecological environment.Biological control becomes the future trend of green plant protection against the D.citri mediated infection.In this study,the diversity of culturable endophytic bacteria in D.citri was analyzed by isolation and identification.In addition,the community structure and compositions of endophytic bacteria in D.citri prior and after infection with Candidatus Liberibacter asiaticus were compared.Furthermore,entomogenous fungi with strong virulence against D.citri were screened.Moreover,the transcriptome changes of D.citri at different time points after Beauveria bassiana infection were analyzed,and the immune-related genes of D.citri induced by the fungal infection were identified.The main results are as follows:(1)The diversity of culturable endophytic bacteria of D.citri in 8 regions of Guangdong Province was determined,and 114 strains of endophytic bacteria of D.citri were isolated and preserved,which were classified as 15 genera of 3 phyla.The culturable endophytic species of D.citri from different regions were similar with minor difference in diversity.The dominant bacteria of D.citri were identified,including Bacillus,Pseudomonas,Pantoea,etc.A total of 59 Bacillus isolates,14 Pseudomonas isolates,and 10 Pantoea isolates were obtained,which accounted for 51.75%,12.28% and 8.77% of the total endophytic bacteria isolated from D.citri,respectively.Cluster analysis showed that 59 strains of Bacillus bacteria belonged to 4 major species,namely Bacillus megaterium,Bacillus pumilus,Bacillus cereus and Bacillus gibsonii.(2)The composition structure and changes of the endophytic bacteria of D.citri infected with Ca.L.asiaticus were determined.Infestation of Ca.L.asiaticus markedly changed the microbial structure and composition of D.citri,and the total number and types of endophytic bacteria were greatly reduced.After infection with Ca.L.asiaticus,the relative proportions of eight dominant phyla such as Actinobacteria,Firmicutes,Acidobacteria and Verrucomicrobia in D.citri were significantly reduced,with only the Proteobacteria was increased significantly in relative proportion.At the genus level,the relative abundance of endophytic bacteria of D.citri before and after infection with Ca.L.asiaticus was also changed significantly.The relative abundance of most dominant bacteria were decreased,except the relative abundance of Wolbachia was increased from 1.81 to 2.14% and no difference was found in the abundance of Carsonella(34.90% vs.34.84%)before and after infection.(3)A total of 57 fungi were isolated from D.citri,and among them,10 fungal isolates were able to infect D.citri with two showing strong lethal activity on D.citri.These two isolates were identified as Paecilomyces variotii and B.bassiana,respectively.The spore fluid immersion method was used to compare the virulence of both fungal strains to D.citri adults,and a time-dose-mortality model was used to estimate lethal doses and the lethal period of exposure to each fungus.Both P.variotii and B.bassiana were highly pathogenic to D.citri adults,causing about 81% and 96% mortality of D.citri 7 days after inoculation,respectively.The two fungal strains should therefore be considered as potential biological control agents for the control and prevention of citrus Huanglongbing.To our knowledge,this is the first report in China that P.variotii infects D.citri.(4)The Illumina high-throughput sequencing platform was used to analyze the transcriptomes of infected and uninfected D.citri 24,48 and 72 h after treatment.Differentially expressed genes and their functions,classifications and signaling pathways were analyzed using bioinformatic tools.A total of 971,1671 and 752 differentially expressed genes were obtained from the transcriptome data of CK vs.S24 h,CK vs.S48 h,and CK vs.S72 h,respectively.Among them,405,614 and 542 DEGs were significantly differentially enriched in 56,83 and 60 GO terms,respectively.KEGG pathway analysis indicated that 98,333,and 247 expressed genes were significantly different enriched in 10,27,and 25 metabolic pathways,respectively.Differentially expressed genes are mainly enriched in related pathways,such as energy metabolism,ion transport,transcription and translation regulation,reproduction and development regulation,and immune defence response,and most encode potential genes related to immune recognition and regulation.(5)Through bioinformatics methods such as gene sequence alignment,domain analysis and phylogenetic tree construction,the immune related genes in D.citri were screened.A total of 80 immune-related genes were identified,including 21 immune recognition genes.12 signal transduction genes,39 modulator genes and 8 effector genes.q RT-PCR analysis was used to verify the expression changes of 15 immune-related genes over time.The results suggest that these genes may play an important role in the process of D.citri against B.bassiana infection. |
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