Screening and Optimization of High-efficiency Phosphate-solubilizing Bacteria and Wheat Growth Promotion in Slightly Alkaline Soil

doi:10.13560/j.cnki.biotech.bull.1985.2025-1452

Abstract

Abstract:

Objective To address the severe phosphorus (P) immobilization and low plant availability in slightly alkaline soils, this study aimed to screen high-efficiency and stress-tolerant phosphate-solubilizing bacteria (PSB), optimize their culture conditions, clarify the P-solubilizing mechanism, and verify their effects on soil P activation and wheat growth promotion. Method A total of 26 candidate strains from 7 genera were used as test materials. High-efficiency PSB were isolated via primary screening by the plate transparent zone method (using inorganic P selective medium, with phosphorus-solubilizing index as the evaluation criterion) and secondary screening by the liquid culture method (soluble P content determined by molybdenum antimony anti-spectrophotometry). Single-factor experiments and response surface methodology (RSM) were conducted to optimize the cluture conditions. High-performance liquid chromatography (HPLC) was used to identify organic acid components, and Pearson correlation analysis was performed to reveal the relationship between organic acid profiles and P-solubilizing efficiency. Wheat pot experiments were carried out to evaluate soil application effects. Result An efficient PSB strain was identified as Enterobacter asburiae N4129-2AT. This strain maintained stable P-solubilizing capacity under low temperature (15 ℃) and high alkalinity (pH 10). After RSM optimization, the optimal culture conditions were confirmed to be 35 ℃, initial pH 6, and an inoculum size of 7.2%. Under these conditions, the soluble P content in fermentation broth reached 823.471 mg/L within 72 h, which was 23.7% higher than that before optimization, and the culture cycle was shortened by 96 h. E. asburiae N4129-2AT stably secreted six types of small-molecule organic acids. The core P-solubilizing mechanism was acidification mediated by organic acids, and the key factor improving P-solubilizing efficiency was organic acids strongly associated with P solubilization rather than their content proportion; total organic acid content only played an auxiliary role. In wheat pot experiments, all PSB treatments significantly increased soil available P content and wheat growth parameters compared with the control. The medium-dose optimized whole bacterial broth treatment showed the best performance: soil available P content increased by 376.76%, wheat the root dry weight increased by 157.4%, and root-shoot ratio decreased by 22.69%. A highly significant positive correlation was observed between soil available P and wheat growth parameters. Conclusion E.asburiae N4129-2AT possesses excellent stress tolerance (low temperature and high alkalinity) and high P-solubilizing efficiency. Its P-solubilizing mechanism, dominated by strongly associated organic acids through targeted optimization of organic acid profiles, breaks the traditional cognition that total organic acid content determines P-solubilizing capacity, providing a theoretical reference for the improvement of similar strains. This strain and its optimized culture conditions offer reliable technical support for P activation in slightly alkaline soils and high-yield wheat production.

Key words: phosphate-solubilizing bacteria, strain screening, response surface methodology, HPLC, phosphorus activation, wheat growth promotion

LYU Hong-zhen, XU Li-li, ZHANG Lin, GAO Min, TAN Zhen-wei, WANG Ya-wen, ZHANG Xiu-ge⁵ WANG Shuo⁶ MA Ai-jun, WANG Cheng-min. Screening and Optimization of High-efficiency Phosphate-solubilizing Bacteria and Wheat Growth Promotion in Slightly Alkaline Soil[J]. Biotechnology Bulletin, 2026, 42(8): 263-275.

Figures/Tables 15

Table 1 Factors and levels for response surface analysis

水平编码 Level code	因素 Factors
水平编码 Level code	A温度 A Temperature (℃)	B pH B pH	C接种量 C Inoculum size (%)
-1	25	6	5
0	30	8	7
1	35	10	9

Table 2 Experimental design and response values for response surface methodology

编号 Number	A 温度 A Temperature （℃）	B pH	C 接种量 C Inoculum size （%）	可溶性磷含量 Soluble phosphorus content （mg/L）
1	25	6	7	641.026
2	35	6	7	813.609
3	25	10	7	433.925
4	35	10	7	510.355
5	25	8	5	599.112
6	35	8	5	608.974
7	25	8	9	493.097
8	35	8	9	692.801
9	30	6	5	714.990
10	30	10	5	436.390
11	30	6	9	653.353
12	30	10	9	483.235
13	30	8	7	641.026
14	30	8	7	643.491
15	30	8	7	663.215
16	30	8	7	658.284
17	30	8	7	663.215

Fig. 1 Dynamic changes in soluble phosphate content during the cultivation of different phosphate-solubilizing strains

Fig. 2 Hemolytic activity test (A) and phytopathogenicity assay (B) of strain N4129-2ATCK: blank control; T: inoculated with strain N4129-2AT

Fig. 3 Effects of single factors on P-solubilizing capacity of N4129-2AT

Table 3 Analysis of variance (ANOVA) table

来源 Source	平方和 Sum of squares	自由度 Degree of freedom	均方 Mean square	F值 F value	P值 P value
模型 Model	1.720E+05	9	19 113.08	117.05	<0.001
A-温度 Temperature	26 286.91	1	26 286.91	160.98	<0.001
B-pH	1.150E+05	1	1.150E+05	704.11	<0.001
C-接种量 Inoculum size	170.96	1	170.96	1.05	0.340 3
AB	2 311.37	1	2 311.37	14.15	0.007 1
AC	9 010.03	1	9 010.03	55.18	0.000 1
BC	2 942.05	1	2 942.05	18.02	0.003 8
A²	802.63	1	802.63	4.92	0.062 1
B²	6 841.92	1	6 841.92	41.90	0.000 3
C²	7 266.73	1	7 266.73	44.50	0.000 3
残差Residual	1 143.06	7	163.29
失拟项Lack of fit	676.23	3	225.41	1.93	0.266 2
纯误差Pure error	466.83	4	116.71
总和 Total	1.732E+05	16

Fig. 4 Response surface and contour plots for pairwise interactions of factors affecting P-solubilizing capacity of phosphate-solubilizing bacteria

Fig. 5 Dynamic changes in soluble phosphate content and pH during incubation of N4129-2AT under optimal conditions

Fig. 6 Correlation heatmap of organic acids, pH, and P-solubilization of N4129-2AT under different conditionsIni: Initial condition; Str: stress condition; Opt: optimal condition; P: soluble phosphorus content; TA: tartaric acid; FA: formic acid; MA: malic acid; LA: lactic acid; SA: succinic acid; AA: acetic acid. *P<0.05, **P<0.01, ***P<0.001, The same below

Fig. 7 Dynamic changes of total organic acids and pH under different culture conditions as well as the proportion of core organic acids

Fig. 8 Dynamic changes in content of core phosphate-solubilizing organic acids during the 7-day incubation period under different conditions

Fig. 9 Effects of different treatments on soil available P content and its correlation with pH

Fig. 10 Effects of different treatments on growth indicators of wheat seedlings

Table 4 Correlation between soil available phosphorus and wheat seedling growth indices

关联指标组合 Indicator combination	相关系数 Correlation coefficient	P-value
AP-株高 AP-Plant height	0.855^**	<0.001
AP-主根长 AP-Taproot length	0.752^**	<0.001
AP-地上部干重 AP-Shoot dry weight	0.965^**	<0.001
AP-地下部干重 AP-Root dry weight	0.665^**	<0.001
AP-根冠比 AP-Root-shoot ratio	-0.701^**	<0.001

Fig. 11 Correlation between soil available phosphorus and wheat shoot dry weight

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