Screening Of High-efficiency Functional Bacteria In Rhizosphere Soil Of Dalbergia Odorifera And Sandalwood And Research The Biological Compound Fertilizer

Dalbergia odorifera is the fabaceae butterfly flower plantsand Sandalwood is the semiparasitic plant,both of them are valuable annatto timber and important medicinal species.But they grow slowly and the wild resources is endangered.In order to protect their cultivated germplasm resources,the experiments of large area planting shows that the effect of Dalbergia odoriferaand Sandalwood mixed cultivation is good.But there has many problems such as they grow poor and have long rearing period in Seedling phase,the rapidly available N,K and dissolved inorganic phosphorus in soil is low.In order to solve the problems,the High-efficiency functional bacteria were isolated from the rhizosphere soil of Dalbergia odorifera and Sandalwood.In order to develop biological compound fertilizer,we optimize the culture medium composition and liquid fermentation conditions.We use the bran as the adsorption carrier of functional microflora and rotten chicken manure as the main organic matte,and add a variety of pharmaceutical aids.The results as follow:(1)Screening of efficient functional bacteria in rhizosphere soil of Dalbergia Odorifera and SandalwoodThe high-efficiency azotobacter,phosphate-solubilizing bacteria and potassium bacteria were screened from Dalbergia Odorifera and Sandalwood rhizosphere soil by selective medium.We determine the activity of azotobacter by acetylene reduction method and total nitrogen colorimetric method.Of the isolate strains,3strains had higher nitrogen fixing capacity,with the range of nitrogen live above 400 nmol.h-1.mL-1,and the amount of nitrogen in more than 40 mg/L.The results showed that the nitrogenase live and the amount of nitrogen of strains have no significant linear relationship.We screen phosphate solubilizing bacteria by the molybdenum antimony resistance colorimetric method and molybdenum antimony scandium colorimetric method.The D/d value of 11 strains was greater than 2.0,and the inorganic phosphoru ssolubility of strains JT-P25,JT-P15 were 233.27 μg/mL,and 196.47 ug/mL.The inorganic phosphorus solubility of strains JT-P25 and JT-P15 were19.22 μg/mL and 25.05 μg/mL.The soluble potassium content of 23 strains were more than 48 ug/mL by atomic absorption spectrometry.The soluble potassium content of JT-K21 was 132.68 μg/mL.Salkowski colorimetry measure was used to determine IAA-producing abilities of strains.Among them stain JT-N301,JT-N304 and JT-N305 had the IAA-producing capacity above 35.0 μg/mL.Four stains of the phosphate-solubilizing strains had higher IAA-producing capacity above 25ug/mL,among them stain JT-P25 had the highest IAA-producing capacity of 32.14μg/mL.The results showed that no significant correlation was observed amongst IAA secretion and final pH of the culture medium.After comprehensive consideration,we screen out strains JT-N301,JT-N304,JT-N305,JT-P15,JT-P25 and JT-K21 to develop biological compound fertilizer.Based on morphology,physiological and biochemical property and 16S rDNA sequence analysis,JT-N301,JT-N304,JT-N305,JT-P15,JT-P25 and JT-K21 were preliminarily identified as Burkholderia anthina,Enterobacter ludwigii and Burkholderia seminalis,Bacillus pumilus,Bacillus subtilis and Pseudomonas putida,respectively.(2)Fermentation optimization experiment of soil efficient functional bacteria.The growth curve of strains showed that both of them were in plateau when the train time from 24h to36 h.And we can sample and inoculate at this time.The results showed that the six strains between each other has no antagonistic.The research of strains mixed culture showed that the nitrogenase activity of azotobacter was 498.66 nmol.h-1.mL-1,and the inorganic phosphorus solubility of phosphate solubilizing bacterias was 256.44 μg/mL.In order to get the maximum cell concentration and the highest activity of microflora,we determine the optimum components of medium by carbon and nitrogen source of single factor experiment and the orthogonal experiment of medium.We determine the optimal fermentation conditions of microflora by liquid fermentation experiments.The optimal medium of azotobacter is glucoselO g/L,calcium carbonate 5.0 g/L,ammonium sulfate 0.2 g/L,seven water magnesium sulfate 0.2g/L,potassium dihydrogen phosphate 0.2 g/L,0.2 g/L of dihydrate calcium sulphate,sodium chloride 0.2 g/L and deionized water.The optimal liquid fermentation conditions of Azotobacter is temperature 34 ℃,pH 7.4,25%of loading volume,inoculum concentration 10%and 160 r/min.The optimal medium of phosphate-solubilizing bacteria is glucose 8 g/L,calcium phosphate 3 g/L,ammonium sulfate 0.5 g/L,yeast extract 0.3 g/L,potassium chloride 0.2 g/L,sodium chloride 0.1 g/L,seven water magnesium sulfate 0.1 g/L,manganese sulfate 0.004 g/L,ferrous sulfate 0.002 g/L and deionized water.The optimal liquid fermentation conditions of phosphate-solubilizing bacteria is temperature 34 ℃,pH 7.0,25%of loading volume,inoculum concentration 10%and the speed of 160 r/min.The optimal medium of potassium bacteria is sucrose 10 g,calcium phosphate 5.0 g/L,disodium hydrogen phosphate 0.2 g/L,ammonium sulfate 0.2 g/L,seven water magnesium sulfate 0.3 g/L,sodium chloride 0.2 g/L,calcium sulphate dihydrate 0.2 g/L,potassium feldspar powder 3 g/L and deionized water.The optimal liquid fermentation conditions of potassium bacteria is temperature 31℃,pH 7.0,30%of loading volume,inoculum concentration 10%and the speed of 160 r/min.(3)The research of biological compound fertilizerWe make fermented liquid of functional microflora adsorbed on bran,use rotten chicken manure as the main organic matte and add a variety of pharmaceutical aids to develop biological compound fertilizer.We design seven experiments with different proportion,and determine the indicators after ues the biological compound fertilizer.The results showed that the optimum treatment group is EG2.Compared with before used the biological compound fertilizer,the seedling height and ground diameter of dalbergiae cocobolo EG2 group have increased by 82.01%and 37.79%,and these of Sandalwood EG2 group have increased by 93.31%and 39.63%.Compared with CK group,organic matter,microbial tota,available N,available P and available K in soil of dalbergiae cocobolo EG2 group have increased by 89.61%,57.62%,41.16%,117.07%and 45.28%,respectively.And these of Sandalwood EG2 have increased by79.91%,60.4%,39.66%,116.88%and 43.88%,respectively.Through the test,We confirm the optimum proportion of biological compound fertilizer.At the first time of fermentation,the optimum proportion is dry chicken manure 100%,sterile water 100%,brown sugar 0.2%,coconut bran 20%and calcium superphosphate 5%,cassava dregs 1%and chicken manure starter 2 ml/Kg.At secondary fermentation,the optimum proportion is 60%of the initial fermentation rotten material,40%of the chemical compound fertilizer(N:P:K = 1:1:1)and the bran of functional bacteria liquid containing 4%.