Screening,Identification And Optimization Of Culture Conditions Of Phosphate-Solubilizing Bacteria From Quercus Acutissima Rhizosphere Soil

With global climate change,the availability and maintenance of phosphorus in soils is also changing.Phosphorus is an essential nutrient for plant growth and development.Phosphorus is mainly in the form of organic or inorganic compounds in the soil and it's not easily absorbed and used by plants.Although the application of phosphorus fertilizers can improve the effective phosphorus content of the soil to a certain extent,the utilization rate of phosphorus fertilizers is very low,and the application of large amounts of phosphorus fertilizers can easily cause soil caking and environmental pollution.Phosphate solubilizing microorganism(PSM)can convert insoluble phosphorus into effective phosphorus in the soil,thus providing available phosphorus for plant growth and development.The purpose of this study is to find bacteria with the ability to solve phosphorus and use their abilities to increase the content of effective phosphorus in the soil,thus alleviating the problem of phosphorus deficiency in plants and present scientific evidence for the formulation of microbial fertilizers after optimizing the culture conditions,and provide a theoretical basis for the application of phosphate solubilizing microorganisms in real life.The main results are as follows.(1)Twenty-one bacterial strains capable of stable genetic phosphorus solubilization were screened from 30 inter-rooted soils of Quercus by primary screening in plates,followed by re-screening using the liquid culture method,and a total of 12 bacterial strains with phosphorus solubilization capacity above 70.00 mg/L were screened,with M3 and M49 having stronger phosphorus solubilization capacity,and their phosphorus solubilization indices in solid medium for inorganic phosphorus being 1.91 and 1.69 respectively The phosphorus solubilization indexes for M3 and M49 were 1.91 and 1.69 on inorganic phosphorus solid medium,respectively,and 190.67 mg/L and 151.84 mg/L in liquid medium,respectively.(2)By morphological observation,phy-biochemical tests and combined with 16S r DNA gene sequencing technology,the phosphate solubilizing bacteria M3 was identified as Burkholderia sp and the phosphate solubilizing strain M49 was Lactococcus sp.(3)Optimization of the growth aspects of the phosphate solubilizing bacteria was carried out for temperature,initial p H and loading volume respectively.The results showed that the growth of Burkholderia M3 was optimal at 38?,p H 6 and loading volume of 120 m L;the growth of Lactococcus M49 was optimal at 36?,initial p H 7 and loading volume of 80 m L.(4)To optimize the inorganic phosphorus medium for phosphorus solubilizing bacteria,firstly,the carbon source,nitrogen source,magnesium sulfate,initial p H,inoculum volume and shaking speed of Burkholderia M3 and Lactobacillus M49 were optimized,and then PB tests were conducted to screen out three factors that had significant effects on the phosphorus solubilizing ability of Burkholderia M3:carbon source,nitrogen source and magnesium sulfate;screened out the effects of carbon source,nitrogen source and the effect of carbon source,nitrogen source and initial p H on the phosphorus solubilization ability of Lactobacillus M49 was found to be more significant.(5)Based on the results of PB tests,the response surface optimization of Burkholderia M3 and Lactobacillus M49 with the strongest phosphorus solubilization capacity was carried out,and the optimized medium for Burkholderia M3 was finally obtained as follows:glucose12.109 g,yeast powder 0.082 g,Na Cl 0.500 g,KCl 0.500 g,Mg SO₄-7H₂O 0.533 g,Fe SO₄-7H₂O 0.030 g,and Fe SO₄-7H₂O 0.030 g.7H₂O 0.030 g,Mn SO₄-H₂O 0.030 g,Ca₃(PO₄)210.000 g,an initial p H of 6,an inoculum of 6%and a shaker speed of 200 r/min;the optimised medium for Lactococcus M49 was:sucrose 10.237 g,peptone 0.082 g/L,Na Cl 0.5g,KCl 0.5 g,Mg SO₄-7H₂O 0.500 g,Fe SO₄-7H₂O 0.030 g,and Fe SO₄-7H₂O 0.030 g.7H₂O0.030 g,Mn SO₄-H₂O 0.030 g,Ca₃(PO₄)2 10.000 g,an initial p H of 7,an inoculum of 6%and a shaker speed of 180 r/min.The optimized phosphorus solubilization of Burkholderia M3and Lactococcus M49 was 224.490 mg/L and 186.518 mg/L,respectively.The phosphorus solubilization was increased by 17.737%and 22.839%respectively compared to that without optimization.The model was then validated to be close to the actual measurements,which proved its value in optimizing the conditions for phosphorus solubilizing bacteria.(6)Due to the short test period,the fast-growing rockweed was chosen as the target for the greenhouse pot test of B.burgdorferi M3.The test results showed that the number of Burkholderia M3 colonized in the inter-rhizosphere of the rockweed treated with Burkholderia M3 stabilized after 10 days of inoculation with Burkholderia M3 and no longer decreased,and the concentration of Burkholderia M3 was about 7.7×10~4CFU/g at that time.After 25 days of incubation,the fresh and dry weights of rockweed inoculated with Burkholderia M3 were significantly higher and the differences were more significant.The effective phosphorus content in the inter-rhizosphere soil of rockweed inoculated with Burkholderia M3 increased with time and finally reached 52.13 mg/L compared to the control group not inoculated with Burkholderia M3.