| Because the excessive use of chemical fertilizer will produce many environmental problems,the sustainable development strategy of using biological fertilizer instead of chemical fertilizer has attracted extensive attention.Plant growth promoting rhizobacteria(PGPR)can improve soil fertility,regulate soil microbial community structure,and inhibit the growth of harmful organisms by secreting siderophores,auxin,ACC deaminase,hydrolase,nitrogen fixation and phosphorus solubilization,so as to promote plant growth and improve plant stress resistance,reduce the environmental pollution caused by the use of pesticides and fertilizers in production.In this study,six PGPR strains were identified,and the growthpromoting antagonism experiment was used to verify the growth promoting and antagonistic properties of the six PGPR strains.The effects of single inoculation with PGPR and combined inoculation with Suillus luteus on annual and biennial Pinus sylvestris var.mongolica,soil physical and chemical properties and soil microbial community structure were studied,construct a "P.sylvestris var.mongolica-PGPR-ectomycorrhizal fungus" system and compare the growth promotion effects of six PGPR strains,and the strains with good effects were selected for the experiments of resistance to Damping-off and drought resistance of P.sylvestris var.mongolica.The species composition and community structure of rhizosphere soil microorganisms were studied by high throughput sequencing technology.The effects of PGPR on the abundance of beneficial bacteria and antagonists were compared.Combined with the effects of PGPR on plant biomass,root structure,enzyme activity and soil nutrient enzyme activity,the effect and mechanism of PGPR on the resistance of P.sylvestris var.mongolica to Damping-off were studied.Combined with the in vitro growth dynamics analysis of PGPR under water stress,pot experiment and soil micro ecological environment results of high throughput sequencing,the effect and mechanism of PGPR on improving drought resistance of P.sylvestris var.mongolica were studied.Genome sequencing technology was used to understand the mechanism of PGPR strain promoting the growth and improving the disease resistance and drought resistance of P.sylvestris var.mongolica at the molecular level.The main results are as follows:(1)Six PGPR strains were all gram-negative bacteria,the comparison and identification revealed that the 6 strains were Pseudomonas mandelli A36,Serratia plymuthica A13,Pseudomonas koreensis A20,Pseudomonas chlororaphis A54,Acinetobacter lwofii A07,Pseudomonas extremaustralis A39;Six PGPR strains all had the ability to fix nitrogen and dissolve phosphorus,secrete indole acetic acid(IAA)and siderophores.Among them,strains A54,A07 and A3 9 showed strong growth promoting characteristics,and these three strains had the ability to produce hydrolases.Except for strain A36,the other 5 PGPR have antagonistic effects on Rhizoctonia solani,and the inhibition rate can reach 45.30%-69.33%.Strains A54,A07 and A39 showed strong antagonistic characteristics.(2)Whether annual or biennial plants,the combined inoculation treatment of PGPR and S.luteus increased the seedling biomass more.Similarly,compared with the control,the compound inoculation significantly increased the nutrient content and enzyme activity of plants and soil.A54,A07 and A39 strains had more prominent growth promoting ability in the 6 PGPR strains.The high throughput sequencing results showed that compared with the control,the inoculation of strain A3 9 significantly increased the specific OTU of bacteria and fungi,and the strain A13 significantly increased the community diversity of bacteria and fungi.Although other treatment groups did not significantly increase the community diversity,there were significant differences in dominant microflora and fungi among treatment groups.Almost all soil physical and chemical factors were positively correlated with inoculation treatment group,and also correlated with dominant bacteria genera and fungi genera.(3)Strains A54,A07 and A39 had a significant inhibitory effect on Damping-off,and the highest control effect was 64.7%.Compared with infected plants,inoculation with PGPR significantly increased plant biomass,root index,nutrient content and enzyme activity,soil nutrient content and enzyme activity.It was found that plant biomass was positively correlated with soil physical and chemical factors,while the incidence rate of Damping-off was negatively correlated with soil physical and chemical factors of PGPR treatment.The results of high throughput sequencing analysis showed that compared with infected plants,PGPR inoculation increased the total number of OTUs and unique OTUs in the sample,and significantly increased the relative abundance of beneficial bacteria and antagonistic bacteria in soil samples.The relative abundance of beneficial bacteria and antagonists bacteria was significantly negatively correlated with the incidence of Damping-off,indicating that PGPR helps P.sylvestris var.mongolica seedlings resist Rhizoctonia solani disease by regulating plants,soil physical and chemical factors and soil micro-ecological environment.(4)Strains A54,A07 and A39 had drought tolerance and ACC deaminase activity.Under any drought stress mode,PGPR inoculation could significantly increase plant biomass,soil nutrient enzyme activity,accumulate osmotic adjustment substances,increase antioxidant enzyme activity,and reduce membrane lipid peroxidation products.The results of high throughput sequencing showed that drought stress reduced the number of OTU and the relative abundance of soil bacteria,while PGPR inoculation significantly increased some bacterial genera(Serratia,Pseudomonas,etc.),and these bacteria were reported as drought resistant bacteria,it showed that PGPR helps P.sylvestris var.mongolica resist drought stress by producing ACC deaminase,increasing plant enzyme activity and the abundance of drought resistant bacteria in the soil.(5)The genome sequencing analysis of A54,A07 and A39 strains was completed.The genome sizes of A54,A07 and A39 were 6489442 bp,5969322 bp and 6834719 bp respectively,and 5745,5791 and 6190 coding genes were obtained respectively,and 78,88 and 85 genes related to antibiotics were obtained respectively,and 6 enzymes related to carbohydrates were obtained,and 5 virulence factors were obtained,the three PGPR strains contained a large number of genes to reduce pathogenicity.The functional genes related to growth promotion and stress resistance of PGPR strain were screened from the genome sequencing results.There were 12 metabolic pathways related to phosphorus dissolution,the functional genes included pyruvate kinase,malate synthase,acetate kinase,citrate synthase,shikimate kinase,etc.The core enzymes related to nitrogen fixation included nitrogenase and vanadium-dependent nitrogenase,as well as nitrate reductase,nitrite reductase,Nrt,NrtABCD and other functional genes.There was a complete indole acetamide pathway in the tryptophan metabolic pathway related to IAA,which could generate IAA through monooxygenase and amidase.There were two metabolic pathways related to the production of siderophores.Functional genes included protoporphyrin/coproporphyrin ferrochelatase,iron(?)transport system substrate-binding protein,iron(?)transport system permease protein and iron(?)transport system ATP-binding protein.In summary,this study selected out PGPR that can promote the growth of P.sylvestris var.mongolica and improve the disease resistance and drought resistance of P.sylvestris var.mongolica.The constructed "P.sylvestris var.mongolica-PGPR-ectomycorrhizal fungus"growth promoting system has certain innovation in the utilization of rhizosphere microorganisms.The use of soil microbial high-throughput sequencing technology and genome sequencing technology reveals the molecular mechanism of PGPR to promote the growth of P.sylvestris var.mongolica,improve disease and drought resistance,provides basic data reference for how plant rhizosphere growth promoting bacteria promote the growth and stress resistance of P.sylvestris var.mongolica,and represents a promising sustainable scheme to replace chemical fertilizer.The main innovations of this paper:(1)The genome sequencing and analysis of Acinetobacter lwofii and Pseudomonas extremeaustralis were reported for the first time,and the genome circle map was constructed.(2)The constructed "P.sylvestris var.mongolica-PGPR-exomycorrhizal fungus"interaction system has a certain degree of innovation in the utilization of rhizosphere microorganisms.(3)Simultaneously use soil microbial high-throughput sequencing and bacterial genome sequencing technologies to reveal the potential molecular mechanisms and functional genes that PGPR promotes the growth of P.sylvestris var.mongolica seedlings and improves the resistance of P.sylvestris var.mongolica. |
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