荒漠植物根际土壤固氮微生物的筛选及其抗逆促生特性

doi:10.13560/j.cnki.biotech.bull.1985.2024-1242

摘要/Abstract

摘要：

目的对荒漠灌丛根际土壤固氮微生物多样性、抗逆性和促生特性进行研究，为挖掘荒漠微生物资源提供基础。方法采用分离培养方法及固氮酶基因nifH的PCR扩增，从4种荒漠灌丛根际土壤中分离筛选固氮微生物，通过菌落形态观察、菌株生理生化特征测定及16S rRNA基因序列分析，对固氮菌株进行鉴定；通过NaCl、NaOH和PEG6000胁迫检测菌株的盐碱耐受性和抗旱性，通过比色法测定菌株溶磷、产生IAA、铁载体和ACC脱氨酶等促生潜能。通过苜蓿盆栽接种实验验证菌株的促生效果。结果从4种灌丛根际土壤中共筛选获得固氮微生物61株，菌株多为革兰氏阴性，H₂O₂酶反应阳性；16S rRNA序列分析结果表明菌株隶属于38个属，其中假单胞菌属（Pseudomonas）、芽胞杆菌属（Bacillus）、固氮菌属（Azotobacter）和剑菌属（Ensifer）分离频率较高，柠条根际固氮微生物多样性最为丰富。所有菌株在pH 10的条件下可正常生长，大多数菌株可耐受5%的NaCl和15%的PEG6000，表现出良好的抗逆性。菌株的促生特性存在差异，17株具有溶磷能力，发酵液中有效磷增量最高达到135.84 mg/L；15株菌能够产铁载体；8株具有产ACC脱氨酶活性，酶活最高为10.63 U/mg；19株具有分泌IAA的能力，IAA产量在3.41-56.93 mg/L之间，有6个菌株同时具备3种以上促生潜能，其中SDQ-1和MC-20菌株综合性能良好。盆栽接种实验表明，接种处理能显著促进苜蓿幼苗生长，复合菌剂组F促生效果最显著，与未接种对照CK相比，幼苗株高、鲜重、根长和根面积分别显著提高了55.88%、147.53%、292.65%和306.38%。结论宁夏荒漠灌丛根际土壤中固氮微生物多样性极其丰富，多数菌株具有良好的抗逆性和多种促生潜能，具有进一步开发利用的价值。

关键词: 荒漠植物, 固氮微生物, 抗逆性, 促生功能, 多样性

Abstract:

Objective Studies on the diversity, resistance and growth-promotion characteristics of nitrogen-fixing microorganisms in the rhizosphere soil of desert shrubs may provide a basis for exploring desert microbial resources. Method Nitrogen-fixing microorganisms were isolated and screened from the rhizosphere soil of four desert scrubs using isolation and culture methods and PCR amplification of the nitrogen-fixing enzyme gene nifH. Nitrogen-fixing strains were identified through observation of colony morphology, determination of physiological and biochemical characteristics, and analysis of the 16S rRNA gene sequences. The tolerance to salt alkali and the resistances of the strains to drought were tested by NaCl, NaOH and PEG6000 stresses. The growth-promoting characteristics of strains such as phosphorus-solubilizing ability, IAA production, siderophore production, and ACC deaminase were detected by colorimetric method. The growth-promoting effects of the strains were verified through the pot inoculation experiment of alfalfa. Result A total of 61 strains of nitrogen-fixing microorganisms were screened from the rhizosphere soil of four shrubs. Most of the strains were gram-negative bacteria, and the H₂O₂ enzyme reaction were positive. The results of 16S rRNA sequence analysis showed that the strains belonged to 38 genera, among which Pseudomonas, Bacillus, Azotobacter and Ensifer had higher isolation frequency, and the diversity of nitrogen-fixing microorganisms in the rhizosphere of Caragana korshinskii was the most abundant. All strains grew normally under the condition of pH10, and most strains tolerated 5% NaCl and 15% PEG6000, showing strong stress resistance. The growth-promoting characteristics of the strains varied, total 17 strains had the ability of dissolving phosphorus, and these strains' maximum amount of phosphorus to be dissolved was 135.84 mg/L; total 15 strains produced siderophores. Eight strains had ACC deaminase activity, the highest enzyme activity was 10.63 U/mg; nineteen strains had the ability to secrete IAA, and these strains' IAA production was between 3.41-56.93 mg/L. There were 6 strains with more than 3 kinds of growth-promoting potential. Among them, the SDQ-1 and MC-20 strains had good comprehensive performance. The alfalfa pot inoculation experiment showed that the inoculation treatment significantly promoted the growth of alfalfa seedlings, and the growth-promoting effect of compound microbial agent group F was the most significant. Compared with the control CK, the seedling height, fresh weight, root length and root area significantly increased by 55.88%, 147.53%, 292.65% and 306.38%, respectively. Conclusion The nitrogen-fixing microbial diversity in the rhizosphere soil of the desert shrub in Ningxia is extremely abundant, and most of the strains have good stress resistance and multiple growth-promoting potentials, which have the value of further development and utilization.

Key words: desert plants, nitrogen-fixing microorganisms, resistance, growth-promoting function, diversity

张钧杰, 刘爽, 胡明珠, 石雪瑞, 代金霞. 荒漠植物根际土壤固氮微生物的筛选及其抗逆促生特性[J]. 生物技术通报, 2025, 41(6): 317-326.

ZHANG Jun-jie, LIU Shuang, HU Ming-zhu, SHI Xue-rui, DAI Jin-xia. Screening of Nitrogen-fixing Microorganisms in Rhizosphere Soil of Desert Plants and Their Stress-resistant and Growth-promoting Characteristics[J]. Biotechnology Bulletin, 2025, 41(6): 317-326.

图/表 8

表1 固氮菌株筛选结果

Table 1 Screening results of nitrogen-fixing bacterial strains

土壤样品 Soil sample	联合固氮培养基 Combined nitrogen-fixing culture medium	固氮类芽胞杆菌培养基 Nitrogen-fixing Bacillus culture medium	无氮培养基 Nitrogen-free culture medium	nifH基因阳性菌株 nifH gene-positive strain
NT	23	12	9	31
SDQ	12	15	9	13
MTC	12	9	12	13
MC	11	9	11	4

图1 部分固氮菌株的菌落形态A：联合固氮培养基；B：固氮类芽胞杆菌培养基；C：无氮培养基

Fig. 1 Colony morphology of some nitrogen-fixing strainsA: Combined nitrogen-fixing culture medium. B: Nitrogen-fixing Bacillus culture medium. C: Nitrogen-free culture medium

图2 固氮微生物在4种灌丛土壤中的分布情况

Fig. 2 Distribution of nitrogen-fixing microorganisms in rhizosphere soils of four shrubs

表2 菌株抗逆性筛选结果

Table2 Screening results of strain stress resistance

培养条件 Cultivation conditions		正常生长菌数 Normal growth count
NaCl	3%	61
	5%	52
	7%	35
	9%	13
PEG6000	15%	52
	25%	18
	35%	13
pH	9	61
	10	61
	11	33
	12	4

图3 部分菌株的促生活性图中小写字母表示不同菌株之间促生能力差异达到显著水平（P<0.05）；实验均设有3个重复，下同

Fig. 3 Growth-promoting activities of some strainsThe lowercase letters in the figure indicate significant difference in growth-promoting ability among different strains (P<0.05).There are 3 replicates in each experiment, the same below

图4 不同处理的盆栽苜蓿长势图CK：未接种对照组；D1、D2：分别为SDQ-1和MC-20单一菌剂接种组；F：复合接种组

Fig. 4 Growth of potted alfalfa under different treatmentsCK: The uninoculated control group; D1 and D2: single inoculation groups of SDQ-1and MC-20, respectively; F: the compound inoculation group

图5 不同处理苜蓿根系相关指标CK：未接种对照组根系扫描数据；D1、D2：分别为SDQ-1和MC-20单一菌剂接种组根系扫描数据；F：复合接种组根系扫描数据。下同

Fig. 5 Root-related indexes of alfalfa under different treatmentsCK: Control group root scanning data; D1, D2: the root scan data for the SDQ-1 and MC-20 single inoculant groups were obtained; F: root scanning data of the composite inoculation group. the same below

图6 不同处理苜蓿根系扫描图

Fig. 6 Scans of alfalfa roots with different treatments

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