菌株C8和B4的分离鉴定及其耐盐促生效果和机制

doi:10.13560/j.cnki.biotech.bull.1985.2022-1292

摘要/Abstract

摘要：

土壤盐渍化是影响农业生产的主要环境因素，合理使用根际促生菌是改良修复盐渍化土壤的有效途径。本研究从东营地区盐渍化土壤中分离筛选到两株耐盐促生菌株C8和B4，经形态学特征、生理生化特性、16S rDNA和gyrB基因序列分析，分别鉴定为氧化微杆菌（Microbacterium oxydans）和嗜麦芽窄食单胞菌（Stenotrophomonas maltophilia）。含盐LB培养基上检测结果显示，菌株C8耐6% NaCl，具有解钾、溶有机磷、溶无机磷和分泌生长素的功能；菌株B4耐8% NaCl，具有溶有机磷和分泌生长素的功能。C8和B4单独施用及配施对盐胁迫下番茄的促生作用及机制的试验结果表明，C8和B4单独施用及配施均显著促进盐胁迫下番茄种子萌发和幼苗生长，提高过氧化氢酶（CAT）和过氧化物酶（POD）活性，上调过氧化氢酶基因CAT1和CAT2表达量，增加植株K⁺含量，降低Na⁺含量和Na⁺/K⁺，上调液泡膜Na⁺/H⁺逆向转运蛋白基因NHX1和NHX3的表达量。C8和B4配施具有增效作用。以上结果表明，C8和B4通过调节抗氧化酶及Na⁺转运蛋白基因表达，提高植株抗氧化能力，维持体内离子稳态，提高植株耐盐性。

关键词: 氧化微杆菌株C8, 嗜麦芽窄食单胞菌株B4, 分离鉴定, 番茄, 耐盐促生作用

Abstract:

Soil salinization is one of the critical adverse environmental factors affecting agricultural crop growth and yield, and rational use of growth-promoting rhizotrophic bacteria is an effective way to improve and amend salinized soil. In the present study, two halotolerant bacterial strains C8 and B4 were isolated from saline soil in Dongying city, Shandong province. Through morphological observation, physiological and biochemical tests combined with 16S rDNA and gyrB gene sequence analysis, C8 and B4 were identified as Microbacterium oxydans and Stenotrophomonas maltophilia respectively. The results on the LB medium with NaCl showed that the C8 tolerated 6% NaCl, and had the abilities of dissolving potassium, organic and inorganic phosphorus as well as producing auxin. Strain B4 tolerated 8% NaCl, it was capable of solubilizing organic phosphorus and producing auxin. Using tomato as material, the effects and mechanism of sole inoculation with C8 or B4 and simultaneous inoculation of C8 and B4 on the seed germination and plant growth were investigated under salt stress. The results demonstrated that sole C8 or B4 inoculation and the simultaneous inoculation of C8 and B4 significantly promoted tomato seed germination and seedling growth, increased catalase（CAT）and peroxidase（POD）activities, and up-regulated the expressions of catalase gene CAT1 and CAT2. C8 and B4 application enhanced plant K⁺ content, but reduced Na⁺ content and Na⁺/ K⁺, and up-regulated the expression of vacuolar membrane Na⁺/H⁺ antiporter gene NHX1 and NHX3. Simultaneous application of C8 and B4 showed synergistic effect. The above results indicate that C8 and B4 enhance antioxidant capacity, maintain ion homeostasis in plants by regulating the expression of antioxidant enzyme and Na⁺ transporter genes, thus improve plant salt tolerance.

Key words: Microbacterium oxydans C8, Stenotrophomonas maltophilia B4, isolation and identification, tomato, salt tolerant and growth- promoting effect

车永梅, 郭艳苹, 刘广超, 叶青, 李雅华, 赵方贵, 刘新. 菌株C8和B4的分离鉴定及其耐盐促生效果和机制[J]. 生物技术通报, 2023, 39(5): 276-285.

CHE Yong-mei, GUO Yan-ping, LIU Guang-chao, YE Qing, LI Ya-hua, ZHAO Fang-gui, LIU Xin. Isolation and Identification of Bacterial Strain C8 and B4 and Their Halotolerant Growth-promoting Effects and Mechanisms[J]. Biotechnology Bulletin, 2023, 39(5): 276-285.

图/表 11

表1 定量PCR引物

Table 1 Primers used in quantitative PCR analysis

基因名称Primer	引物序列Primer sequence（5'-3'）
CAT1-FP	AAATGGGTTGAGTCTTTATCCGA
CAT1-RP	TCATTGATTTTTCACATTGTAGGCT
CAT2-FP	TTCTGCCCTTCTATTGTGGTTC
CAT2-RP	GTGATGAGCACACTTTGGAGC
SOS1-FP	TGGGAATCGGTTGTAGAC
SOS1-RP	GCCCAGCAAGTAAAAGCA
SOS2-FP	GGGAAGTATGAAGTTGGC
SOS2-RP	TCTCCTCCAGTGACAAAC
NHX1-FP	GATCACGCCTCTGTGGTTTCC
NHX1-RP	CAACAGTTCCAATAGCACCAAAA
NHX3-FP	CTCAAGAGTCACCACCAAGCA
NHX3-RP	CCAACCAAAACAAGACCCAACA

图1 不同盐浓度培养基中C8和B4的生长状态

Fig. 1 Growth states of C8 and B4 in medium with salt at different concentrations

表2 菌株C8和B4分解矿物钾、无机磷和有机磷及产生长素能力

Table 2 Abilities of bacterial strain C8 and B4 decomposing mineral potassium, inorganic and organic phosphorus as well as producing auxin

菌株编号 Strain No.	可溶钾含量Soluble potassium content/（μg·mL^-1）	无机磷含量Inorganic phosphorus content/（μg·mL^-1）	有机磷含量Organic phosphorus content/（μg·mL^-1）	生长素含量 IAA content/（μg·mL^-1）
C8	27.37±0.69	10.27±1.04	26.31±1.19	14.62
B4	0	0	29.38±2.03	11.39

图2 菌株C8和B4的菌落形态（A、B）及革兰氏染色（C、D）

Fig. 2 Colony morphologies（A, B）and Gram staining（C, D）of strains C8 and B4

表3 菌株C8和B4的生理生化特性

Table 3 Physiological and biochemical identification of strains C8 and B4

生理生化实验 Physiological and biochemical experiment	C8	生理生化实验 Physiological and biochemical experiment	B4
产IAA	+	产IAA	+
接触酶	+	接触酶	+
氧化酶	-	氧化酶	-
硝酸盐还原	-	硝酸盐还原	+
L-阿拉伯糖	-	L-阿拉伯糖	+
硫化氢	-	硫化氢	-
D-木糖	-	赖氨酸脱羧酶	+
蔗糖	+	明胶水解	+
葡萄糖	+	果糖	+

图3 菌株C8和B4基于16S rDNA（A、B）与gyrB（C、D）基因序列的系统发育树

Fig. 3 Phylogenetic tree of strain C8 and B4 based on 16S rDNA（A, B）and gyrB（C, D）gene sequences

表4 Effects of bacterial strains C8和B4 on the germinations of tomato seeds under salt stress

Table 4

Items	CK	C8	B4	C8+B4	NaCl	NaCl+C8	NaCl+B4	NaCl+C8+B4
发芽率 Germination rate/%	91.3±1.01a	93.4±1.75a	92.4±2.17a	94.9±1.95a	4.7±1.20e	37.8±1.70c	29.3±1.64d	75.1±2.39b
发芽势 Germination potential/%	90.2±2.14a	92.2±2.35a	91.7±1.07a	94.3±1.15a	3.5±1.17e	34.2±2.05c	19.2±2.33d	73.2±1.21b

图4 菌株C8和B4对盐胁迫下番茄植株生长的影响不同小写字母代表不同处理间差异显著（P<0.05），下同

Fig. 4 Effects of bacterial strain C8和B4 on the growths of tomato under salt stress The different lowercase letters indicated significant difference（P<0.05）between different treatments, the same below

图5 菌株C8和B4对盐胁迫下番茄植株活性氧代谢的影响

Fig. 5 Effects of bacterial strains C8和B4 on the reactive oxygen species metabolism of tomato under salt stress

图6 菌株C8和B4对盐胁迫下番茄植株Na+/K+平衡的影响

Fig. 6 Effects of bacterial strains C8和B4 on the Na+/K+ homeostasis of tomato under salt stress

图7 菌株C8和B4对盐胁迫下番茄植株Na+转运蛋白基因表达的影响

Fig. 7 Effects of bacterial strain C8 and B4 on the relative expressions of Na+ transporter genes of tomato under salt stress

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