一株高效溶磷伯克霍尔德菌的筛选鉴定及对马尾松幼苗的促生作用

采用标准稀释平板法从马尾松根际土中分离溶磷细菌,利用钼锑抗比色法测定溶磷细菌的溶磷特性;通过分析溶磷菌的溶磷能力与发酵液pH的关系,以及液相色谱-质谱 (HPLC-MS)联用对发酵液中有机酸的测定,探究其溶磷机制;通过对接种溶磷菌马尾松盆栽苗生长、生理、土壤养分和土壤酶活性的测定,明确溶磷菌对马尾松生长和生理的影响。结果表明: 由马尾松根际土壤中共筛选到溶磷细菌16株,其中菌株WJ27溶磷效果最优,液体培养5 d时的溶磷量达411.98 mg·L^-1。经过表型观察、生理生化鉴定和系统发育树分析,发现菌株WJ27属于伯克霍尔德菌属;其对不同磷源的溶磷特性存在差异,溶磷能力依次为: Ca₃(PO₄)₂(220.85 mg·L^-1)＞AlPO₄(182.33 mg·L^-1)＞FePO₄·2H₂O(129.19 mg·L^-1)＞CaHPO₄·2H₂O (115.23 mg·L^-1)。胞外有机酸测定结果表明,该菌株通过分泌柠檬酸、丙二酸等有机酸降低发酵液中pH,进而发挥溶磷作用;盆栽试验结果表明,接种菌株WJ27对马尾松幼苗生长、生理、土壤养分和土壤酶活性有积极作用。与对照相比,接种WJ27的马尾松的苗高、主根长、侧根数量、地上部(茎、枝、叶)鲜重、干重和根系鲜重、干重分别增加了14.3%、36.9%、56.1%、44.7%、60.0%、158.3%和100.0%;叶绿素b、总叶绿素、地上部可溶性蛋白和可溶性糖、根系活力和根系可溶性蛋白分别增加了145.8%、45.2%、206.3%、59.4%、80.5%和260.0%;根系超氧化物歧化酶、过氧化物酶和地上部过氧化氢酶的活性分别提高了71.2%、197.5%和36.6%;根际土壤中速效氮、速效钾、速效磷含量和土壤脲酶、过氧化氢酶、磷酸酶活性分别较对照土壤显著增加18.1%、17.0%、11.9%和34.3%、45.5%、62.6%。说明接种WJ27可以改善土壤养分和土壤酶活性,进而促进马尾松幼苗的生长。

Screening and identification of an efficient phosphate-solubilizing Burkholderia sp. and its growth-promoting effect on Pinus massoniana seedling

We isolated phosphate solubilizing bacteria (PSB) from the rhizosphere soil of P. massoniana by the standard dilution plating technique, and determined the phosphate solubilizing characteristics of PSB by the Molybdenum antimony resistance colorimetric method. We explored the mechanism of phosphorus dissolution through analysis of the relationship between the phosphate-solubilizing ability of PSB and the pH of the fermentation broth and the determination of organic acids in the fermentation broth by HPLC-MS. The effects of PSB on the growth and physiology of P. massoniana were clarified by measuring the growth, physiology, soil nutrients, and soil enzyme activity of potted P. massoniana seedlings inoculated with PSB. The results showed that 16 strains of PSB were screened from the rhizosphere of P. massoniana. Among those strains, WJ27 had the best effect of solubilizing phosphorus, with the amount of phosphate solubilized reaching 411.98 mg·L^-1 after five days of liquid culture. The strain was identified as Burkholderia sp. by phenotype observation, physiological and biochemical identification and phylogenetic tree analysis. The highest phosphate solubilizing capacity in four phosphorus sources mediums was Ca₃(PO₄)₂(220.85 mg·L^-1) > AlPO₄(182.33 mg·L^-1) > FePO₄·2H₂O (129.19 mg·L^-1) > CaHPO₄·2H₂O (115.23 mg·L^-1). WJ27 could reduce the pH of fermentation broth by secreting organic acids, such as citric acid and malonic acid, which contribute to phosphorus solubilization. Results from pot experiments showed that WJ27 had positive effects on the growth, physiology, soil nutrients, and soil enzyme activities of P. massoniana seedlings. Compared with the control, seedling height, main root length, total lateral root number, shoot (stem, leaf and branch) fresh weight, shoot dry weight, root fresh weight and root dry weight of P. massoniana inoculated with WJ27 increased by 14.3%, 36.9%, 56.1%, 44.7%, 60.0%, 158.3% and 100.0%, respectively. Chlorophyll b, total chlorophyll, shoot soluble protein, shoot soluble sugar, root activity, and root soluble protein increased by 145.8%, 45.2%, 206.3%, 59.4%, 80.5% and 260.0%, respectively. The SOD and POD acti-vities of root, and CAT activities of shoot of P. massoniana seedlings increased by 71.2%, 197.5% and 36.6%, respectively. Contents of available N, available K, available P, urease, catalase and phosphatase increased by 18.1%, 17.0%, 11.9%, 34.3%, 45.5%, and 62.6%, respectively. Burkholderia sp. WJ27 could improve soil nutrient concentrations and enzyme activities and thus facilitate the growth of P. massoniana seedlings.