Screening And Evaluation Of PGPR Resources From 6 Gansu Native Grass And Research On Growth Promoting Mechanism

Soil is the foundation of life and the foundation of agriculture.The severity of soil degradation in domestic farmland has threatened national agriculture and food security.As a large agricultural province,Gansu faces the same problems as the whole country,such as soil degradation,environmental pollution and agricultural product safety caused by improper use of chemical fertilizers and pesticides.One of the ideas to solve this problem is to replace a certain proportion of chemical fertilizers and pesticides with microbial fertilizer,and the basis of microbial fertilizer research is to have a rich and diverse resource of growth-promoting bacteria,PGPR resources are one of biological resources and an important part of the national resource security strategy.The geographical environment in Gansu is very complex,with plateaus,mountains,glaciers,river valleys,grasslands and deserts in the region.This leads to fragile ecosystems on the one hand,and rich biological species resources on the other.As a local dominant plant,native grass not only maintains the balance of the natural ecosystem,but also provides products or raw materials for agricultural production.The rhizosphere of native grasses is inhabiting to many kinds of microorganisms,which is a natural treasure house of bacteria resources,need to be explored,studied,protected and utilized by researchers.In this study,rhizosphere microorganisms of native grasses,Onobrychis viciifolia,Polygonum viviparum,Koeleria cristata,Angelica sinensis,Notopterygium incisum,and Allium mongolicum,were used as the research object.Use selective medium to isolate plant growth-promoting rhizobacteria(PGPR);screen excellent PGPR through research on the growth-promoting properties of phosphorus dissolution,nitrogen fixation and secretion of IAA;identify and preserve the strains with excellent growth-promoting properties.Through the inoculation experiment,gene cloning and whole genome sequencing(WGS)analysis to study mechanism of symbiosis and nitrogen fixation;through the detection of organic acids and the expression of pqq gene to study the mechanism of phosphate solubilization;the bio-control mechanism of Bacillus is studied by cloning lipopeptide genes.The results and conclusions were as follows:(1)Among the nitrogen-fixing bacteria,the nitrogenase activity of strains MSN-1,MSN-4 and MSN-6 isolated from the rhizosphere of Allium mongolicum,was significantly better than that of the strains from the other plants(p<0.01),which were 57.07,60.82 and64.41nmol C₂H₄·h^-1m L^-1,respectively;the strian LD-1 isolated from the root nodule of sainfoin,the root nodule nitrogenase activity is 6.42umol C₂H₄·g-1·h-1;the strain HD-4 can form nodules with sainfoin,but no nitrogenase activity was detected in the nodules.Among the phosphate solubilizing bacteria,16 strains have outstanding ability to dissolve inorganic phosphorus,with a phosphate dissolving amount of 224.69?420.33?g/m L,the strain LHP-1 is derived from the rhizosphere of Onobrychis viciifolia;and the strains TZP-2,TZP-3,TZP-7,TZP-8 and TZP-12 are derived from the rhizosphere of Polygonum viviparum;the strains TQP-1?TQP-2 and TQP3 from the rhizosphere of Koeleria cristata,MDP-4,MDP-7 and MDP-8 are isolated from the rhizosphere of Angelica sinensis,WQP-1.WQP-5,WQP-6 and WQP-7 were isolated from the rhizosphere of Notopterygium incisum;there are 24 strains with outstanding ability to dissolve organic phosphorus(phosphate index>200%),WQM-13 and WQM-13 isolated from Notopterygium incisum with a phosphorus dissolving index of 411%and 434%are the best.The strains with outstanding ability to secrete IAA are TQP-3 from Notopterygium incisum and MSN-6 from Allium mongolicum,the amounts of IAA secreted are 26.17 and 27.67?g/ml,which are significantly different from other strains(p<0.01).(2)Three strains with outstanding nitrogen fixation traits isolated in this study were identified as Ensifer meliloti;the strain LD-1,which nodulates and fixes nitrogen with Onobrychis viciifolia,was identified as Rhizobium yanglingense,the nodulation and non-nitrogen-fixing strain HD-4 was identified as Mesorhizobium robiniae.The 16 strains with outstanding ability to dissolve inorganic phosphorus were identified as Pseudomonas mediterranea,Pseudomonas grimontii,Pseudomonas simiae,Pseudomonas brassicacearum,Pseudomonas laurylsulfatiphila,Pseudomonas frederiksbergensis,Pseudomonas thivervalensis and Acinetobacter calcoaceticus.The two most prominently dissolving organic phosphorus strains,WQM-11 and WQM-13,were identified as Falsirhodobacter deserti.The secreted IAA prominent MSN-6 and TQP-3 are respectively identified as E.meliloti and Pseudomonas sp..The 6 biocontrol strains were identified as belonging to Bacillus atrophaeus,Bacillus altitudinis,Bacillus subtilis and Bacillus siamensis.(3)Cloning the nodulation factor genes(nod A,nod B,nod C and nod D)and nitrogenase genes(nif H,nif D and nif K)of 12 rhizobia isolated from sainfoin root nodule,the results showed the strain R.yanglingense LD-1 that can nodulate with sainfoin and detect the nitrogenase activity has all the nodulation factor genes and nitrogenase genes;the nif D gene was not detected from the strain M.robiniae HD-4,and the inoculation experiment showed M.robiniae HD-4 could form the root nodule without capability of nitrogen fixation with sainfoin;nodulation factor and nitrogenase genes were not detected on the remaining 10rhizobia,and the nodulation test was negative.Strain R.yanglingense LD-1 whole genome sequencing analysis showed that plasmid 4 has 92.3%symbiotic and nitrogen-fixing genes,the plasmid 4 is a symbiotic plasmid.(4)13 species of Pseudomonas(Pseudomonas graminis,Pseudomonas putida,Pseudomonas simiae,Pseudomonas mandelii,Pseudomonas chlororaphis,Pseudomonas arsenicoxydans,Pseudomonas grimontii,Pseudomonas mediterranea,Pseudomonas thivervalensis,Pseudomonas frederiksbergensis,Pseudomonas antarctica,Pseudomonas helmanticensis and Pseudomonas paralactis)and 1 species of Acinetobacter(Acinetobacter calcoaceticus)dissolve inorganic phosphorus by secreting one or more of gluconic acid,oxalic acid,malic acid,lactic acid,acetic acid or citric acid.The secretion of organic acids and the relative expression of pqq C gene of the 12 species of pseudomonas(Pseudomonas graminis,Pseudomonas putida,Pseudomonas simiae,Pseudomonas mandelii,Pseudomonas chlororaphis,Pseudomonas arsenicoxydans,Pseudomonas grimontii,Pseudomonas mediterranea,Pseudomonas thivervalensis,Pseudomonas frederiksbergensis,Pseudomonas antarctica and Pseudomonas helmanticensis)increased with the decrease of p H value of the culture medium,and the pqq C gene was always up-regulated during the period of p H decrease.(5)Antibacterial experiments showed that 6 strains(Bacillus atrophaeus TZF1,Bacillus altitudinis TZF2,Bacillus subtilis TQF1?TQF3 and Bacillus siamensis TQF4)all showed antagonistic effects on Helminthosporium tritici-vulgaris B1,Alternaria solani B2,Fusarium oxysporum B3,Rhizoctonia solani B4,Sclerotinia sclerotiorum B5 and Bipolaris maydis B6,among which the strain TZF1 also has resistance to crown gall pathogen R.radiobacter BJ-2 of Cerasus humilis.Iturin gene related to antagonistic fungi was cloned from all 6 strains of biocontrol bacillus.Strain TZF1 cloned the surfactin gene,surfactin gene was not found on the other 5 strains.Bacillomycin D gene was found on the strains TZF1 and TQF1.Fengycin gene was not cloned in all strains of 6 Bacillus.Therefore,iturin is the molecular genetics basis of these 6 bio-control bacillus antagonizing 6 kinds of pathogenic fungi,and the surfactin gene is the inherent reason that B.atrophaeus TZF1antagonizes the pathogenic bacteria R.radiobacter BJ-2.