| Cunninghamia lanceolata is a subtropical conifer species, as an endemic fast-growing conifer species of china, it is grown in more than16provinces and its lumber yield is more than25%of commercial timber in china. However, repeated pure artificial continuous cropping of cunninghamia lanceolata has caused severe decline in soil fertility and its productivity. Though the structure of forest land and the loss of nutritional elements can be effectively reduced by constructing mixed forest with species arrangement, improvement in soil fertilities is limited by merely optimizing forest stand, and soil is still lack of N,P elements, which severely restricts sustainable development of plantation. In this study, by filtering azotobacter and phosphate-solubilizing bacteriaon of cunninghamia lanceolata, efficient strains of nitrogen fixation and phosphate solubilizing bacteria were obtained. Moreover, their IAA secretion and physiological and biochemical characteristics were investigated, and by optimizing fermentation conditions and their proper proportion, forming compound bacterium fertilizers to be used by cunninghamia lanceolata seedlings to test fertilizer efficiency.(1) The rhizosphere Azotobacter of cunninghaimia lanceolata was screened with Ashby culture medium and its nitrogenase activity was determined with acetylene reduction assay.16strains showing stronger nitrogen fixing ability were found. According to the ratio of D/d (which D stood for transparent circle diameter while d for colony size),12strains organic phosphorus and inorganic phosphorus bacteria of more than1.6from cunninghaimia lanceolata were screened with the Meng Jinna organic medium and PKO culture medium. The IAA secretion of the screened nitrogen-fixing bacteria, phosphorus dissolving bacteria were determined with Salkowski colorimetry. The results showed that the nitrogenase activity of5strains in16strains of bacteria were more than150nmol·mL-1·h-1, NGJ-4rank the highest with264.7nmol·mL-1·h-1, followed by NGX-5237.4nmol·mL-1·h-1. Most of the isolates sttains can produce ghrelin, the secretion ability of6strains was strong, and the IAA concentrations were greater than20mg/L-1. NGX-535.8mg/L-1,NGJ-332.1mg/L-1, NGJ-427.8mg/L-1, NGJ-827.7mg/L-1, respectively. The organic phosphorus dissolving bacteria with higher capacity were Py16, PylOand Py3, and the IAA concentrations of those strains were7.131mg/100mL,5.907mg/100mL and6.514mg/100mL, respectively. For inorganic phosphorus dissolving bacteria, the Pw1O, Pw6, Pw20showed higher capacity, the corresponding value was23.2mg/100mL,18.59 mg/100mL,17.26mg/100mL, respectively. The organic phosphorus bacteria showed promising IAA secretion capability are Py18, Py16and Py3(respectively38.80mg/L,37.29mg/L and35.79mg/L), while inorganic phosphorus bacteria were Pw6, Pw8, Pw21(45.340mg/L,39.340mg/L,27.480mg/L).In conclusion, the Azotobacter strains(NGJ-4, NGX-5and NGJ-8) organic phosphorus degrading bacterium strains (Py16, Py3) and inorganic phosphorus bacterium strain (Pw6) have great development potentials in the multifunctional microbial fertilizer development.(2) The shaking flask fermentation condition of nitrogen-fixing bacteria and phosphorus-dissolving bacteria were optimized, through the single factor and orthogonalizing tests. The results showed that the optimal fermentation condition of nitrogen-fixing bacteria were glucose12g, sodium chloride0.2g, calcium carbonate5g, potassium dihydrogen phosphate0.2g, magnesium sulfate0.2g calcium sulfate0.2g, water1000mL, pH7.2, at the temperature of33℃While the optimum condition for inorganic phosphobacteria were glucose lOg, calcium phosphate3g, ammonium sulfate,0.5g, sodium chloride0.1g, magnesium sulfate0.1g, potassium chloride0.2g, manganese sulfate0.004g, ferrous sulfate0.002g, yeast extract,0.6g, water1000ml, pH7.2, at the temperature of30℃. The optimal fermentation condition of organic phosphobacteria were glucose10g, ammonium sulfate0.5g, sodium chloride0.3g, potassium chloride0.3g, ferrous sulfate0.03g, manganese sulfate0.03g, calcium carbonate5g, yeast extract0.4g, lecithin0.1g, water1000ml, pH7.2, at the temperature of35℃. The optimal incubating conditions were at160rpm with10%inoculation quantity and60mL liquid in250mL Erlenmeyer flask. Under these conditions the enzyme activity of nitrogen-fixing bacteria and the phosphate-solubilizing capacity of phosphobacteria were maximum.(3) According to the optimum fermentation conditions, nitrogen-fixing bacteria, organic phosphorus bacteria and inorganic phosphorus bacteria were diluted with sterile water.the compound in different proportions, ash and chitin as the carrier applied to the0.5-year-old Cunninghamia lanceolata seedlings of17,the month growthrate of33.9%～90.5%, so got the best composite biological bacterial fertilizer formula: nitrogen-fixing bacteria, organic phosphorus bacteria and inorganic phosphorus bacteria were diluted into the mass percent concentration was15%, by volume ratio1.5:1:1proportion, solid carrier in accordance with ash20%,50%of the fermentation medium,30%of the mixed bacteria. |
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