| Phenacoccus solenopsis Tinsley is a crucial agricultural pest.It has strong adaptability and a wide range of hosts,which has severe threats to agricultural.At present,the adaptation mechanism of P.solenopsis to host plants is still unclear.The saliva of herbivorous contains a large number of symbiotic bacteria.Symbiotic bacteria of saliva play an important role in the regulation of plant-insect interactions.Earlier studies have found that P.solenopsis feeding can inhibit plant defense response,but the function of P.solenopsis salivary symbiotic bacteria in regulating plant defense response is still unknown.In this study,the composition and abundance of symbiotic bacteria in the saliva of P.solenopsis were determined.Difference in feeding behavior of P.solenopsis between antibiotic treatment and control were analyzed.The defense responses of cotton induced by antibiotic treated and untreated-P.solenopsis were measured.The role of salivary symbiotic bacteria in the interaction between P.solenopsis and cotton was investigated.Then the salivary symbiotic bacteria were isolated and identified.After P.solenopsis were reinoculated with symbiotic bacteria,the feeding behavior of P.solenopsis and the changes in cotton defense response were measured again.By using label-free proteomics analysis,the composition of proteins in the saliva of antibiotic treated and untreated-P.solenopsis were investigated.The main results of the study are as follows:1 Community composition and diversity of salivary symbiotic bacteria in P.solenopsisThe 16 S r RNA gene amplicon high-throughput sequencing was performed on the collected saliva of at least 30,000 P.solenopsis,and the community composition and diversity of the saliva bacteria in P.solenopsis were investigated.The main phylums in saliva are Proteobacteria and Firmicutes,which account for 66.34% and 24.28%,respectively.The five most abundant genera and their proportions are Enterobacter 31.57%,Ochrobactrum 6.66%,Pelomonas5.13%,Erwinia 4.49% and Acinetobacter 4.05%.After antibiotic(100 μg/m L penicillin G sodium + 200 μg/m L streptomycin)treatment,the abundance of Enterobacter bacteria was reduced to 0.04%,and there were significant differences in the pielou index between groups,PG50 had the lowest species uniformity.NMDS analysis showed significant differences in the composition between control and antibiotic treatment,indicated that the salivary composition of P.solenopsis was changed after the antibiotic treatment.2 Effect of antibiotic treatment on the feeding and growth of P.solenopsisThe feeding behavior was measured by EPG after P.solenopsis was treated with antibiotic for 48 hours.The effect of antibiotic treatment on the growth period,reproduction ability and survival rate of P.solenopsis were measured.After antibiotic treatment,the feeding time of P.solenopsis on cotton was disturbed.Compared with control,E1 duration was reduced by 4.00%,and E2 duration was reduced by 14.34%,indicated that feeding time in phloem was significantly reduced,and the duration of C increased by 14.99%,indicated that the duration of stylet probing was significantly increased.Compared with the control,P.solenopsis treated with antibiotic for 48 h had a significant increase in the first instar period,but there was no significant difference in generation period,egg production and survival rate,showed that the short-term antibiotic treatment had no significant effect on the growth and fecundity of P.solenopsis.3 Effect of antibiotic treatment on cotton defense responseExpression of cotton defense genes,accumulation of JA and SA in cotton after antibiotic treated P.solenopsis were measured.The result showed that P.solenopsis inhibit the expression of the cotton JA pathway-related genes LOX1,OPR3 and Jaz1,and the expression of the cotton SA pathway genes PR1,PAL and 4CL were induced.The accumulation of JA in cotton after 12,24 and 48 h of P.solenopsis infection was significantly lower than control,and the SA accumulation was significantly higher than control after P.solenopsis infection for 4,6,12 and48 h,indicating that the SA accumulated in cotton plants but the JA accumulation was decreased after P.solenopsis infection.After antibiotic treated P.solenopsis infection,the cotton JA pathway-related genes LOX1,OPR3 and Jaz1 were significantly up-regulated,there was no significant difference in expression of SA pathway related genes PR1,PAL and 4CL at 24 h and48h.JA accumulation in cotton leaf increased and was significantly higher than control at 6 h,and there was no significant difference in accumulation of SA compared with control,indicating that antibiotic treated P.solenopsis infection activated the JA defense pathway of cotton.4 Isolation and identification of the salivary symbiotic bacteria of P.solenopsisSalivary bacteria were separated and cultured,and the isolated strains were identified by using 16 S r RNA gene sequencing.Enterobacteriaceae,Bacillus,Micrococcus,Staphylococcus and Stenotrophomonas strains were cultivated and identified.The phylogenetic analysis results show that the isolated Enterobacteriaceae strains and Stenotrophomonas strains are divided into one group,indicated that the two have closely genetic relationship,and the isolated Bacillus bacteria are homologous to the reported Bacillus cereus,evolution distance is 0.0032.5 Functional verification of the salivary symbiotic bacteria of P.solenopsisAfter P.solenopsis were reinoculated with symbiotic bacteria,the feeding behavior of P.solenopsis was measured by EPG.The expression of cotton defense genes and JA/SA accumulation in cotton after P.solenopsis infection were also measured.The feeding duration of P.solenopsis of reinoculation with Enterobacteriaceae and Stenotrophomonas strain increased significantly,and E wave duration increased by 95.59 min and 88.81 min,respectively.Compared with the control,there was no significant difference between reinoculation of other bacteria.Expression of SA pathway related genes PR1,PAL and 4CL were induced and the expression of JA pathway related genes Lox1,OPR3 and Jaz1 were suppressed again after P.solenopsis reinoculation with Enterobacteriaceae and Stenotrophomonas bacteria.At the same time,the JA accumulation in cotton leaf was significantly reduced,but there is no significant difference in SA accumulation,indicating that Enterobacteriaceae and Stenotrophomonas bacteria play an important role in the interactions between P.solenopsis and cotton.The distribution of Enterobacteriaceae and Stenotrophomonas bacteria in the P.solenopsis was detected by fluorescence in situ hybridization(FISH).The results showed that Enterobacteriaceae and Stenotrophomonas bacteria have strong fluorescence in the clypeus and the tissues nearby the mouthparts of P.solenopsis,suggesting that these two bacteria may be secreted into plants with saliva.6 Effect of antibiotic treatment on the protein composition of P.solenopsis salivaLabel-free quantification proteomics was used to analyzed the salivary protein composition of antibiotic treated and untreated-P.solenopsis.A total of 35 salivary proteins from P.solenopsis were identified.There were18 and 19 proteins were identified by comparing Uni Prot with the database of Pseudococcidae and salivary bacteria,respectively,mainly including tubulin,chitinase,Dicer3,ATP synthase,heat shock protein,ribosomal protein,histones,cytochrome P450 enzymes,etc.,which were involved in carbohydrate transport and metabolism,post-translational modification,and molecular chaperones.The difference in protein composition in the saliva between antibiotic treated and untreated-P.solenopsis was analyzed.Compared with the control,the content of 16 kinds of protein decreased significantly after antibiotic treatment,including heat shock protein 70,40 S ribosomal protein S27 a,heat shock protein 70-4,histones H3,ATP synthase α subunit fragment,60 S ribosomal protein L35,ATP synthase β subunit fragment,ribosome-recycling factor,Proteasome-associated ATPase,60 S ribosomal protein L40,30 S ribosomal protein S14,beta-actin,sulfite reductase [NADPH]hemoprotein beta-component,ESAT-6-like protein Esx B,V-type proton ATPase subunit E and heat shock protein.However,the content of proteasome-associated ATPase was significantly increased compared with the control.The results showed antibiotic treatment altered the composition of salivary protein in P.solenopsis,and the content of various proteins were significantly reduced. |
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