溶磷性大豆根瘤内生菌的筛选、抗性及系统发育和促生

Screening,resistance,phylogeny and growth promoting of phosphorus solubilizing bacteria isolated from soybean root nodules

对采自河南省不同地区的大豆根瘤进行内生菌分离纯化、溶磷性筛选试验。根据能否产生溶磷圈及溶磷圈直径(D)、菌落直径(d)和D/d值大小确定菌株溶磷能力,采用钼锑抗比色法测定培养液中有效磷含量;平板筛选法对筛选菌株进行耐盐性、耐酸碱、重金属等抗性测定,并对筛选菌株进行理化特性、16S r DNA、rec A序列和系统发育分析。结果表明,从分离纯化的324株内生菌中筛选出36株具有溶磷特性,其中20株有较强溶磷性。菌株DD291发酵液中可溶性磷含量最高(452 mg/L),发酵液p H与对照相比均有不同程度下降,最大降幅达2.92。大部分溶磷性内生菌具有较强耐盐碱性,对Pb2+、Cr6+和Cu2+有较高耐受性,对Ni2+和Hg2+抗性较弱。结合细胞形态、生理生化、16S r DNA、rec A序列和系统发育分析结果,菌株确定为Bacillus cereus,Enterobacter cancerogenus,E.cloacae和Pseudomonas putida。部分溶磷菌株对大豆的生长有促进作用,显示出潜在的应用前景。

Endophytes were isolated from soybean nodules collected from different regions of Henan province, and phosphorus dissolving experiments were performed on the purified strains. The ability of strains to dissolve phosphorus were primarily determined based on whether it can produce dissolved phosphorus halo, the diameter of the dissolved phosphorus halo （D）, the diameter of the colonies （d） and D/d value. The phosphate-solubilizing capacity of endophytes was assayed by molybdenum-antimony-D-iso-ascorbic-acid-colorimetry （MADAC） in inorganic phosphorus liquid culture. Resistances of the tested strains to such factors as salt, pH and heavy metals were measured through adopting plate screening method. Physiological and biochemical characteristics, 16S rDNA and recA sequencing, phylogenetic analysis and inoculation tests were determined for the strains. Results showed that 36 strains of the 324 isolated endophytes were able to dissolve phosphorus and 20 strains with stronger phosphate-solubilizing capacity（D/d=1.218-3.55）were selected for further studies. Phosphate-solubilizing capacity of strain DD291 during plate screening was maximum （D/d=3.55）. Soluble phosphorus content in fermented liquid increased 1-11.9 times compared with that of the control, and among them, such value of DD291was the highest （452 mg/L）, followed by that of DD284. However, all strains had different degree of pH decline compared with that of the control, and the biggest drop amounted to up to 2.92 units. Soluble phosphorus content increased, which held certain relevance to lower pH, but soluble phosphorus contents of strains with approximately close pH values were different. Most of the phosphorus dissolving endophytes had strong saline-alkaline tolerances. Some strains can tolerance and grow on plate containing maximum 8% salt concentration. Under the pH 11, strains DD283 and DD284 never grew, but DD291 grew well and its growth status was second only to that of control, and the rest 4 isolates （DD028, DD140, DD167 and DD186） survived. However, strains can grow under pH 7, second only to control. In the setting range of heavy metal concentration, only isolate DD283 of seven strains can tolerate five kinds of heavy metal simultaneously, whereas the remaining six strains can tolerate four different concentrations of heavy metals. In a whole, seven strains showed higher tolerances to heavy metal ions Pb^2＋, Cr^6＋ and Cu^2＋ under the tested concentrations, while their tolerances to Ni^2＋ and Hg2＋ were lower. Judged together by their cellular morphological characteristics, physiological and biochemical characteristics, 16S rDNA and recA sequencing results and phylogenetic analysis, these strains were identified as Bacillus cereus, Enterobacter cancerogenus, E. cloacae and Pseudomonas putida, respectively. Results of inoculation showed that shoot height and fresh weight of seedlings inoculated respectively endophytes and Rhizobium increased compared with those of controls. For Sinorhizobium fredii USDA205T, DD284, DD186 and DD167, seedling height significantly increased by 35.43%, 35.43%, 27.06% and 29.86%, respectively. Fresh weight of seedling inoculated with S. fredii USDA205T increased to the maximum extent （104%）, DD167 （58.46%） to the second. However, seedlings inoculated alone with soluble phosphorus endophytes did not nodule. Screening excellent strains with phosphorus-solubilizing ability, salinity tolerance and metal resistance from microenvironment, analyzing their phylogenetic positions and investigating their growth promotion effects on host soybean plants provide potential application prospects for the regulation and enhancement of host plants＇ growth environments, which are beneficial for soybean growth.