Screening,Identification Of Plant Growth-promoting Rhizotrophic Bacteria And Application Of Microbial Consortium

The application of pesticides and industrial chemical fertilizers is an important measure to prevent diseases and increase production of crops in agricultural production.As a result,serious environmental pollution has been caused,which has attracted wide attention.A large number of studies have shown that biological fertilizers have the characteristics of long-term,pollution-free,low cost and wide application.The plant rhizosphere growth-promoting bacteria(PGPR)contained in biological fertilizers can effectively colonize in the plant rhizosphere,improve soil fertility through their metabolic activities,inhibit the growth of plant pathogens,and provide nutrients for plant growth,which can make up for the shortcomings of pesticides and chemical fertilizers,and reduce the shortcomings of chemical fertilizers and agricultures and use of drugs.At present,PGPR has become one of the hotspots in the research of bio-fertilizer.It is of great significance for enriching the resources of PGPR strains and developing bio-fertilizer to search for high-efficient and broad-spectrum PGPR with phosphorus-soluble,nitrogen-fixing,secreting plant growth promoters or inhibiting the growth of plant pathogens.In this study,the rhizosphere soil of rice-wheat rotation crop near a phosphorus chemical plant in Hubei Province was used as the material,and the phosphorus solubility of the strain was used as the basis for screening,in order to screen the PGRR,and the multi-functional characteristics and application of the strain were studied.Specifically as follows:1.Screening,Identification and Genome-wide Information Analysis of High-efficient Phosphorus-soluble Strains(1)Screening of strains:After enrichment culture,plate screening and shaking flask screening,three strains of high-efficiency phosphorus-solubilizing bacteria(MEM07,MEM35,MEM40)were screened.Among them,the phosphorus solubility of MEM40 was261.35 mg/L,so strain MEM40 was selected for further study.(2)Identification of morphology,physiology,biochemistry and 16S rDNABased on the analysis of cell morphology,physiology,biochemistry and 16S rDNA sequence,the strain MEM40 was identified as Rahnella aquatilis and its preservation number was CCTCC NO.M 2018463.(3)Genomic sequencing of MEM40 strain:Gen Bank login number:CP036490.1.The results showed that the genome size of MEM40 strain was 5,596,168 bp,with 5,021,016 BP circular chromosome DNA,525,842 BP circular plasmid 1 and 49,310 BP circular plasmid 2.The number of gene islands in MEM40 genome is 11,including dissimilatory nitrate reduction pathway and multiple secondary metabolite gene clusters.2.The multifunctional characteristics of the strain were discussed.(1)Determination of phosphorus dissolving effect of MEM40 strain:Activated MEM40strain solution was inoculated into liquid calcium phosphate,magnesium phosphate and phosphate rock powder media according to 2%inoculation amount,and its phosphorus dissolving effect was determined.The results showed that the soluble phosphorus content of MEM40 reached the maximum at 32H in liquid calcium phosphate medium,and the soluble phosphorus content was 299.55 mg/L.When MEM40 was inoculated in liquid magnesium phosphate medium and phosphate rock powder medium,the soluble phosphorus content reached the maximum at 32H and 24h,respectively,411.56 mg/L and 289.42 mg/L.(2)Determination of IAA-producing ability of MEM40 strain:The strain MEM40 was inoculated into L-tryptophan liquid medium to determine its IAA-producing ability.The results showed that the strain MEM40 had the ability to produce IAA.After 24 hours of culture,the amount of IAA in the solution reached the maximum,which was 44.71 mg/L.(3)Determination of nitrogen fixation ability of MEM40 strain:The strain MEM40 was inoculated into the improved Ahl nitrogen-free liquid medium to determine its nitrogen fixation ability.The results showed that the total nitrogen content in the solution could reach23.48 mg/L after 15 days of incubation.(4)Determination of the ability of MEM40 strain to plant pathogenic bacteria:Plate confrontation method was used to study the inhibition of MEM40 on plant pathogenic fungi.The results showed that strain MEM40 could effectively inhibit the growth of rice blast fungus and wheat scab fungus mycelium,but had no significant inhibitory effect on Strawberry gray mould fungus and peanut leaf mould fungus.3.Composition of Compound Microflora MEMF03The antagonistic analysis of strain MEM40 with multifunctional strain MEL01(CCTCC NO.M2015778)and strain CICC NO.20024(named MEM02)purchased from China Industrial Microbial Preservation and Management Center showed that there was no inhibition among the strains.Three strains of bacteria were compounded according to the ratio of 1:1:1 to construct a multi-functional microbial consortium(MEMF03).4.The multifunctional characteristics and application of the complex flora MEMF03were discussed.(1)Measurement of environmental tolerance of MEM40 strain and compound microflora MEMF03:The environmental tolerance of MEM40 and microbial consortium MEMF03 was studied by shaking flask test.The results showed that MEM40 had higher tolerance to environmental factors(temperature,pH and salt concentration).The compound microflora MEMF03 had stronger tolerance to environment,significantly higher tolerance than MEM40.The growth of MEM40 and MEMF03 showed a trend of first increasing and then decreasing with the increase of temperature and pH.The optimum pH for growth of MEM40 and MEMF03 was 7.0 and 6.0 respectively.The optimum temperature for growth of MEM40 and MEMF03 was 28 C.The growth of MEM40 was obviously inhibited when the salt concentration was higher than 1%.In contrast,the growth of MEMF03 was in good condition when the salt concentration was lower than 4%,while the salt concentration was higher.At4%,the absorbance OD₆₀₀nm of MEMF03 decreases greatly and gradually less than 1.0,but it can still grow,and the growth is significantly higher than that of MEM40.(2)Determination of the multifunctional characteristics of the microbial consortium MEMF03:The multifunctional characteristics of the microbial consortium MEMF03 were studied.The results showed that the microbial consortium MEMF03 had the functions of phosphorus solubilization,nitrogen fixation,IAA production,chitinase production and inhibition of the growth of plant pathogenic fungi.Specifically as follows:(1)Soluble phosphorus content of microbial consortium MEMF03 can reach 184.79 mg/L after 24hours'incubation in liquid medium of calcium phosphate,and its ability of dissolving phosphorus can be increased by 19.22%.(2)IAA production:After 24 hours'incubation in liquid medium of L-tryptophan,IAA content of microbial consortium MEMF03 can reach21.88 mg/L,which decreaset,but still has the ability of high IAA production;(3)Nitrogen fixation;Function:After 15 days of incubation in modified Ahl nitrogen-free liquid medium,the total nitrogen content in the solution was 29.126 mg/L,and the nitrogen fixation ability was increased by 24.06%.(4)Chitinase production:After 48 hours of incubation in chitin fermentation medium,the activity of chitinase in solution could reach 15.79 U/mL,and the function of MEM40 was increased by MEMF03.(5)Inhibiting the growth of plant pathogenic fungi:Plate confrontation experiment showed that the compound flora MEMF03 could significantly inhibit the growth of rice blast fungus,wheat scab fungus,peanut leaf mould fungus and strawberry gray mould fungus,and the inhibition ability and inhibition range were significantly improved compared with strain MEM40.(3)Determination of promoting effect of MEM40 strain and compound microflora MEMF03:Taking pakchoi and rice as research objects,the promoting effect of MEM40 and MEMF03 on plant growth was studied.The results showed that the fresh weight,root length,stem diameter and plant height of Chinese cabbage increased by 23.53%,23.64%,24.00%and 32.13%respectively after applying MEM40 fermentation broth,and the fresh weight,root length,stem diameter and plant height of Chinese cabbage increased by 37.47%,65.81%,48.00%and 73.85%respectively after applying MEMF03 fermentation broth11.29%,34.11%,19.35%,31.57%.In rice pot experiment,the tiller number,plant height,fresh weight and dry weight of rice increased by 11.21%,11.36%,15.25%and 29.11%respectively after applying MEM40fermentation broth,and the tiller number,plant height,fresh weight and dry weight increased by 23.36%,29.67%,52.34%,61.47%respectively after applying MEM40 fermentation broth,which increased by 10.92%,16.44%,32.18%and 25.07%compared with applying MEM40.In conclusion,MEM40 is a multifunctional strain with phosphorus solubilization(calcium phosphate,magnesium phosphate,phosphate rock powder),nitrogen fixation,IAA production and inhibition of plant pathogenic fungi.It has strong tolerance to the environment and can effectively promote the growth of pakchoi and rice.It was constructed into a complex flora(MEMF03)by compounding with Burkholderia Gladiolus(MEL01)and Azotobacter(MEM02).Its multi-functional range and effect have been improved,the environmental tolerance has been further enhanced,and the growth-promoting ability of pakchoi and rice has also been enhanced after mixing.Therefore,the multi-functional strain MEM40 and the mixed multi-functional complex flora MEMF03 have great potential in promoting plant growth and achieving green and sustainable development of ecological agriculture.