大豆根际促生菌的分离、鉴定及其耐盐促生作用

doi:10.3864/j.issn.0578-1752.2024.21.007

摘要/Abstract

摘要：

【目的】植物根际促生菌（plant growth-promoting rhizobacteria，PGPR）长期与植物互惠共生，可有效促进植物生长。从大豆根际土壤中筛选鉴定根际促生放线菌并研究其促生机制，以期为微生物菌剂的开发利用打下基础。【方法】采用土壤稀释涂布法、平板划线接种法收集放线菌分离株；利用溶磷、解钾、铁载体检测培养基分析分离株的促生特性；通过显微观察、生理生化试验和多基因序列分析，对目标促生菌进行种类鉴定；设置不同盐浓度（0—10%）对目标分离株进行耐盐性测定；通过盆栽试验检测其对大豆幼苗的促生能力并评估在盐胁迫条件下喷洒该分离株培养液是否可提升大豆植株的抗逆性。【结果】经筛选获得13株PGPR，其中Sg-7分离株不仅具有较强的溶磷、解钾、铁载体和吲哚乙酸（IAA）产生能力，还可有效拮抗丁香假单胞菌大豆致病变种（Pseudomonas syringae pv. glycinea，Psg）。根据形态特征、生理生化、多基因分析将大豆根际促生菌Sg-7鉴定为灰肉色链霉菌（Streptomyces griseocarneus）。灰肉色链霉菌Sg-7最高可耐受7% NaCl，盆栽试验表明该菌株培养液可有效促进大豆种子的萌发以及植株根长、茎粗、鲜重、叶面积等生长指标的提升。在150 mmol·L^-1盐胁迫下，浇灌Sg-7培养液50×稀释液可有效提升大豆叶片SOD活性，为盐胁迫处理组的1.84倍；浇灌培养液100×稀释液可提升CAT活性，为盐胁迫处理组的4.33倍；而浇灌培养液10×稀释液POD活性为盐胁迫处理组的1.10倍。Sg-7菌株培养液还可提高大豆植株根系活力，促进叶片中类胡萝卜素含量的增加以抵御逆境胁迫，分别为盐胁迫处理组的3.05和1.12倍。【结论】灰肉色链霉菌Sg-7是一株潜在的植物根际促生菌，具有广阔的开发潜力与应用价值。

关键词: 灰肉色链霉菌, 促生评价, 盐胁迫, 抗氧化酶活性, 大豆, 根系活力

Abstract:

【Objective】Plant growth-promoting rhizobacteria (PGPR) can effectively promote plant growth through long-term mutualistic symbiosis with plants. The objective of this study is to screen and identify rhizosphere growth-promoting actinomycetes from soybean rhizosphere soil and clarify their growth-promoting mechanisms, and to lay a foundation for the development and utilization of microbial agents. 【Method】Different actinomycetes were collected using soil dilution coating method and plate streak inoculation method. The growth-promoting characteristics of isolates were analyzed using phosphorus solubilization, potassium solubilization, and siderophore assay media. Through microscopic observation, physiological and biochemical experiments, and multi-gene sequence analysis, the target growth-promoting actinomycetes were identified. Different salt concentrations (0-10%) were set to determine the salt tolerance of the target isolate. The growth-promoting ability of the isolate on soybean seedlings was detected by pot experiments and whether spraying the culture medium of the isolate under salt stress conditions could improve the stress resistance of soybean plants was evaluated. 【Result】Thirteen PGPR isolates were screened and obtained. Among then, the Sg-7 isolate not only had strong abilities to dissolve phosphorus, potassium, siderophore, and IAA production, but also effectively antagonized Pseudomonas syringae pv. glycinea (Psg). Based on morphological characteristics, biochemical, and multi-gene analyses, the soybean rhizosphere growth-promoting bacterium Sg-7 was identified as Streptomyces griseocarneus. The Sg-7 strain had certain salt tolerance characteristics, with a maximum tolerance to 7% NaCl salt concentration. Pot experiments showed that the diluted culture fluid after irrigation could effectively promote the germination of soybean seeds and improve growth indicators such as root length, stem diameter, fresh weight, and leaf area. Under 150 mmol·L^-1 salt stress, irrigation with a 50× dilution of the Sg-7 culture fluid could effectively enhance SOD enzyme activity of soybean leaf, which was 1.84 times that of the salt stress treatment group. The CAT enzyme activity after irrigation with 100× dilution of culture fluid was 4.33 times higher than that of the salt stress treatment group. The POD enzyme activity after irrigation with 10× dilution of culture fluid was 1.10 times that of the salt stress treatment group. The culture fluid of Sg-7 strain could also enhance the root activity of soybean plants and promote the increase of carotenoid content in leaves to resist stress, which was 3.05 and 1.12 times that of salt stress treatment group, respectively. 【Conclusion】S. griseocarneus Sg-7 strain is a potential plant rhizosphere growth-promoting bacterium with broad development potential and practical application value.

Key words: Streptomyces griseocarneus, growth-promoting evaluation, salt stress, antioxidant enzyme activity, soybean, root activity

邵嘉朱, 吕雯, 廖鑫琳, 袁歆瑜, 宋振, 蒋冬花. 大豆根际促生菌的分离、鉴定及其耐盐促生作用[J]. 中国农业科学, 2024, 57(21): 4248-4263.

SHAO JiaZhu, LÜ Wen, LIAO XinLin, YUAN XinYu, SONG Zhen, JIANG DongHua. Isolation and Identification of Soybean Rhizosphere Growth-Promoting Bacteria and Their Salt Tolerance and Growth-Promoting Effects[J]. Scientia Agricultura Sinica, 2024, 57(21): 4248-4263.

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基因 Gene	引物序列 Primer sequence (5′-3′)
16S rRNA	27F AGAGTTTGATCCTGGCTCAG 1492R CTACGGCTACCTTGTTACGA
recA	recA F CCGCRCTCGCACAGATTGAACGSCAATTC recA R GCSAGGTCGGGGTTGTCCTTSAGGAAGTTGCG
rpoB	rpoB F GAGCGCATGACCACCCAGGACGTCGAGGC rpoB R CCTCGTAGTTGTGACCCTCCCACGGCATGA
gyrB	gyrB F GAGGTCGTCATGACCCAGCA gyrB R GTCTTGGTCTGGCCCTCGAACTG

分离株编号 Isolate number	固氮Nitrogen fixation	溶磷Phosphorus solubilization	解钾Potassium solubilization	产铁载体Siderophore production	产吲哚乙酸 IAA production
Sg-7	-	++	+++	++	+++
Sc-8	++	+	-	+	-
Sl-9	-	+	+	-	-
Sv-9	++	-	-	-	-
Sn-1	+	+	-	-	+
Sa-4	++	-	+	+	-
Sb-17	-	-	+	++	-
Sl-10	++	+	-	+	-
Sc-7	-	+	+	+	-
Sm-11	+	+	-	-	+
Sc-2	-	+	+	+	-
St-4	+	+	-	-	+
Sw-6	++	-	+	++	-

特征性培养基 Characteristic medium	气生菌丝 Aerial mycelium	基内菌丝 Substrate mycelium	可溶性色素 Soluble pigment
无机盐淀粉琼脂ISP4	白色White	白色White	-
甘油天门冬素ISP5	黄褐色Yellow brown	黄色Yellow	-
马铃薯葡萄糖琼脂PDA	白色White	黑色Black	黄色Yellow
察氏Cha’s	黄色Yellow	黄色Yellow	-
葡萄糖天门冬素琼脂 Glucose asparagine agar	黄色Yellow	黄棕色Yellow brown	-
克氏1号Keshi No. 1	黄色Yellow	褐色Brown	-

项目Item	结果Result	项目Item	结果Result
淀粉酶Amylase	++	硫化氢Hydrogen sulfide	-
纤维素酶Cellulase	++	黑色素Melanin	+
明胶液化Gelatin liquefaction	++	蛋白酶Protease	+++
甲基红Methyl red (MR)	-	脲酶Urease	+++
V-P	-	脂肪酶Lipase	+++

碳源种类 Carbon source	Sg-7分离株 Sg-7 isolate	标准菌株 <BOLD>S</BOLD>tandard strain	氮源种类 Nitrogen source	Sg-7分离株 Sg-7 isolate	标准菌株 <BOLD>S</BOLD>tandard strain
葡萄糖Glucose	+	+	蛋白胨Peptone	+	+
α-乳糖α-lactose	-	+	KNO₃	+	+
L-鼠李糖L-rhamnose	+	-	(NH₄)₂SO₄	+	+
D-木糖D-xylose	-	-	甲硫氨酸Methionine	+	+
蔗糖Sucrose	-	-	赖氨酸Lysine	+	-
D-麦芽糖D-maltose	+	+	亮氨酸Leucine	+	-
肌醇Inositol	+	+	组氨酸Histidine	+	+