Identification of PSB in soybean rhizosphere and secretion of organic acids in the process of phosphate solubilizing
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摘要:目的
明确吉林省地区大豆Glycine max根际溶磷菌的种类及溶磷特点。
方法利用溶磷圈法筛选溶磷菌，16S rDNA序列测定和Vitek2生理生化系统对菌株进行分析鉴定。测定菌株生长量、溶磷量、培养基pH变化与有机酸的产生。
结果从大豆根际土壤中分离获得4株溶磷菌株WJ1、WJ3、WJ5和WJ6。4个菌株分别属于假单胞菌属Pseudomonas sp.、肠杆菌属Enterobacter sp.、苍白杆菌属Ochrobacterum sp.和克雷伯菌属 Klebsiella sp.。4株溶磷菌96 h内最大溶磷量分别为558、478、596和586 μg·mL-1。甲基红试验和培养物pH测定结果表明：4个菌株在溶磷过程中可使培养物的pH下降，当pH小于4时，会明显阻碍菌株的生长。pH为5时，WJ1和WJ3的生长受到轻微的影响，WJ5和WJ6能正常生长。GC-MS对菌株在溶磷过程中产生的有机化合物的分析表明，4菌株均分泌多种有机酸，其中α-酮戊二酸在WJ1、WJ3和WJ6菌株溶磷过程中大量产生。
结论4菌株具有较好的溶磷能力，溶磷过程中分泌有机酸种类不完全相同，有机酸的分泌造成培养基的pH降低，影响菌体生长，而菌体数量决定各菌株的溶磷量。
Abstract:ObjectiveIn order to determine the species and characteristics of phosphate solubilizing bacteria (PSB) in soybean, Glycine max, rhizosphere soil in Jilin Province.
MethodPSB were screened based on the formation of a halo zone of dissolving phosphate, and were identified by 16S rDNA gene sequencing and Vitek 2 system. Bacterial growth rates, soluble phosphorus concentrations, pH changes of the media and the types and amounts of secreted organic acids were determined.
ResultWJ1, WJ3, WJ5 and WJ6 were isolated from soybean rhizosphere soil, and were identified as Pseudomonas sp., Enterobacter sp., Ochrobacterum sp. and Klebsiella sp. respectively. The maximum soluble phosphorus concentrations within 96 h of inoculating four PSB were 558, 478, 596 and 586 μg·mL-1 respectively. The methyl red tests and pH measurements indicated that media pH all decreased during the phosphate solubilizing process of four PSB. Bacterial growth was limited when pH was below 4. Growth of WJ1 and WJ3 were slightly affected at pH 5, whereas WJ5 and WJ6 grew normally. Various organic compounds secreted from four PSB were analyzed by GC-MS, and among them α-ketoglutaric acid was identified as an abundant compound secreted by WJ1, WJ3 and WJ6.
ConclusionAll four PSB can effectively solubilize phosphate. The types of organic acids produced by the four PSB are not exactly the same, and the secretion of organic acids causes medium pH to decline, which inhibit the bacterial growth. The number of the bacteria determinines the amount of dissolved phosphorus.
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Keywords:
- Glycine max /
- phosphate solubilizing bacteria /
- pH /
- organic acid /
- phosphate solubilizing
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图 1 不同溶磷菌在接菌96 h内溶磷量与生长曲线的变化
Figure 1. The growth of different PSB and the amounts of P released by the PSB within 96 h of inoculation
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图 2 不同溶磷菌的培养基在接菌18 h内的甲基红显色结果
Figure 2. The methyl red tests of acidification levels of media within 18 h of inoculating different PSB
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图 3 不同溶磷菌在12 h内对改良NBRIP和LB培养基pH的影响
Figure 3. Changes in pH of modified NBRIP media and LB media within 12 h of incubating different PSB
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图 4 不同溶磷菌在不同初始pH LB培养基中的生长曲线
Figure 4. The growth curves of different PSB in LB media with different initial pH
表 1 不同溶磷菌溶磷24 h培养物中的有机化合物相对含量1)
Table 1 Relative contents of organic compounds secreted from different PSB during 24 h of culturation
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