三株具生防功能芽孢杆菌的分离鉴定及其生物活性研究

doi:10.13560/j.cnki.biotech.bull.1985.2022-0722

摘要/Abstract

摘要：

基于健康烟草根际土壤，分离筛选具功能多样化、活性高的菌株，为作物病害生物防治提供高效、多样化的生防资源。采用平板对峙法筛选活性高、抑菌谱广的菌株，测定菌株对番茄枯萎病病菌的抑制率、对菌丝生长及和孢子萌发率的影响；应用PCR技术，检测菌株抗生素合成基因，结合室内盆栽实验检测功能菌株对番茄枯萎病的防治效果，并测定其体外产酶、解磷、解钾、固氮及产铁载体能力；结合形态学、生理生化和16S rDNA通用引物对功能菌株进行鉴定；于健康烟草根际土壤分离得127个菌株，24株对番茄枯萎病病菌等指示病原菌具有抑制作用，活性较高的3个菌株SH-1471、SH-1464、SH-1439对番茄枯萎病菌抑制率分别为82.0%、74.0%、75.0%；可使番茄枯萎病菌菌丝扭曲变形，形成泡囊结构，对番茄枯萎病菌孢子萌发抑制率分别为62.7%、50.0%、37.0%；经测定，3个功能菌株具有产srfA、fenB、ituA、ituD、bymA等抗生素合成基因；盆栽实验结果表明SH-1471对番茄枯萎病的防效为83.7%，SH-1464对番茄枯萎病的防效为60.7%，SH-1439对番茄枯萎病的防效为59.0%；此外，3个菌株均具有产蛋白酶、纤维素酶、解磷、固氮、分泌铁载体的能力。经鉴定，SH-1471为贝莱斯芽孢杆菌（Bacillus velezensis）（Gen-bank ON417363）、SH-1464为拟蕈状芽孢杆菌（Bacillus paramycoides）（Gen-bank ON417365）、SH-1439为暹罗芽胞杆菌（Bacillus siamensis）（Gen-bank ON417364）；菌株SH-1471、SH-1464、SH-1439具有高效、广谱的抑菌能力，且功能多样化，可作为多种病害的高效生防资源，具有极大的开发潜力。

关键词: 烟草根际土壤, 生防菌株, 番茄枯萎病, 防效研究

Abstract:

Based on healthy tobacco rhizosphere soil, bacterial strains with diverse functions and high activity were isolated and screened to provide efficient and diversified biocontrol resources for the biological control of crop diseases. The plate confrontation method was used to screen the strains with high activity and wide antibacterial spectrum, and the inhibition rate of the strains on Fusarium oxysporum, mycelia growth and spore germination rate were determined. PCR was adapted to detect the antibiotic synthesis genes of the strains, and the control effects of the functional strains on tomato fusarium wilt were detected by indoor pot experiments. The enzyme production, phosphorus and potassium solubilization, nitrogen fixation and siderophorer production abilities of the functional strains were measured in vitro. The functional strains were identified by combining morphological, physiological and biochemical and 16S rDNA universal primers. A total of 127 strains were isolated from the rhizosphere soil of tobacco, and 24 strains presented the inhibitory effects on Fusarium oxysporum sp. of tomato. The inhibitory rates of 3 top-activity strain SH-1471, SH-1464 and SH-1439 against F. oxysporum sp. of tomato were 82.0%, 74.0% and 75.0%, respectively. It caused the mycelia distortion of F. oxysporum sp. of tomato and vesicle structure was formed, and thus inhibited the spore germination of F. oxysporum sp. of tomato by 62.7%, 50.0% and 37.0%, respectively. The three functional strains had genes for antibiotic synthesis, including srfA, fenB, ituA, ituD and bymA. The results of pot experiment showed that SH-1471, SH-1464 and SH-1439 had 83.7%, 60.7% and 59.0% control effect against tomato fusarium wilt. In addition, the three strains all had the ability of producing protease and cellulase, solubilizing phosphorus, fixing nitrogen, and secreting siderophores. The SH-1471 was identified as Bacillus amyloliquefaciens（Gen-bank ON417363）, and SH-1464 as Bacillus paramycoides（Gen-bank ON417365）and SH-1439 as Bacillus siamensis（Gen-bank ON417364）. Strains SH-1471, SH-1464 and SH-1439 have high-efficiency and broad-spectrum antibacterial ability and diversified functions, and they may be used as efficient biocontrol resources for a variety of diseases and had great development potential.

Key words: tobacco rhizosphere soil, biocontrol strain, tomato fusarium wilt, study on control effect

申云鑫, 施竹凤, 周旭东, 李铭刚, 张庆, 冯路遥, 陈齐斌, 杨佩文. 三株具生防功能芽孢杆菌的分离鉴定及其生物活性研究[J]. 生物技术通报, 2023, 39(3): 267-277.

SHEN Yun-xin, SHI Zhu-feng, ZHOU Xu-dong, LI Ming-gang, ZHANG Qing, FENG Lu-yao, CHEN Qi-bin, YANG Pei-wen. Isolation, Identification and Bio-activity of Three Bacillus Strains with Biocontrol Function[J]. Biotechnology Bulletin, 2023, 39(3): 267-277.

图/表 12

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物质种类Category	靶基因 Target gene	引物序列Primer sequence	扩增长度Amplification size/bp
Surfactins	Sfp srfA	5'-ATGAAGATTTACGGAATTTA-3' 5'-TTATAAAAGCTCTTCGTACG-3' 5'-ACACAGATATCAGGCAAGC-3' 5'-GTCCCATCGTTCCTTCACA-3'	675 1 300
Fengycins	fenA fenB	5'-GCTGTCCGTTCTGCTTTTTC-3' 5'-GTCGGTGCATGAAATGTACG-3' 5'-CTATAGTTTGTTGACGGCTC-3' 5'-CAGCACTGGTTCTTGTCGCA-3'	1 000 1 600
Iturins	ItuA ituC ituD	5'-ATGTATACCAGTCAATTCC-3' 5'-GATCCGAAGCTGACAATAG-3' 5'-TTCACTTTTGATCTGGCGAT-3' 5'-CGTCCGGTACATTTTCAC-3' 5'-GATGCGATCTCCTTGGATGT-3' 5'-ATCGTCATGTGCTGCTTGAG-3'	1 047 575 647
Bacillomycin D	bymA bymB bymC	5'-AAAGCGGCTCAAGAAGCGAAACCC-3' 5'-CGATTCAGCTCATCGACCAGGTAGGC-3 5'-AATAGAAGAACTGCTGGCGT-3' 5'-GCCTTCCCGACACGACACT-3' 5'-GAAGGACACGGCAGAGAGGTC-3' 5'-CACTGATGACTGTTCCTGCT-3'	1 200 983 875

物质种类Category	靶基因 Target gene	引物序列Primer sequence	扩增长度Amplification size/bp
Surfactins	Sfp srfA	5'-ATGAAGATTTACGGAATTTA-3' 5'-TTATAAAAGCTCTTCGTACG-3' 5'-ACACAGATATCAGGCAAGC-3' 5'-GTCCCATCGTTCCTTCACA-3'	675 1 300
Fengycins	fenA fenB	5'-GCTGTCCGTTCTGCTTTTTC-3' 5'-GTCGGTGCATGAAATGTACG-3' 5'-CTATAGTTTGTTGACGGCTC-3' 5'-CAGCACTGGTTCTTGTCGCA-3'	1 000 1 600
Iturins	ItuA ituC ituD	5'-ATGTATACCAGTCAATTCC-3' 5'-GATCCGAAGCTGACAATAG-3' 5'-TTCACTTTTGATCTGGCGAT-3' 5'-CGTCCGGTACATTTTCAC-3' 5'-GATGCGATCTCCTTGGATGT-3' 5'-ATCGTCATGTGCTGCTTGAG-3'	1 047 575 647
Bacillomycin D	bymA bymB bymC	5'-AAAGCGGCTCAAGAAGCGAAACCC-3' 5'-CGATTCAGCTCATCGACCAGGTAGGC-3 5'-AATAGAAGAACTGCTGGCGT-3' 5'-GCCTTCCCGACACGACACT-3' 5'-GAAGGACACGGCAGAGAGGTC-3' 5'-CACTGATGACTGTTCCTGCT-3'	1 200 983 875

编号 No.	番茄枯萎病 Tomato wilt	香蕉枯萎病 Banana fusarium wilt	烟草枯萎病 Tobacco fusarium wilt	烟草赤星病 Tobacco brown spot	葡萄溃疡病 Botryosphaeria dieback	玉米大斑病 Northern leaf blight of corn
SH-1471	82.0±0.01^a	74.0±0.04^a	74.0±0.02^b	79.0±0.02^b	75.0±0.01^b	78.0±0.00^b
SH-1464	74.0±0.01^b	73.9±0.03^a	70.0±0.02^b	67.0±0.00^c	68.0±0.02^c	77.0±0.02^b
SH-1439	75.0±0.01^c	74.0±0.01^a	80.0±0.04^a	81.0±0.02^a	81.0±0.02^a	81.0±0.03^a

编号 No.	番茄枯萎病 Tomato wilt	香蕉枯萎病 Banana fusarium wilt	烟草枯萎病 Tobacco fusarium wilt	烟草赤星病 Tobacco brown spot	葡萄溃疡病 Botryosphaeria dieback	玉米大斑病 Northern leaf blight of corn
SH-1471	82.0±0.01^a	74.0±0.04^a	74.0±0.02^b	79.0±0.02^b	75.0±0.01^b	78.0±0.00^b
SH-1464	74.0±0.01^b	73.9±0.03^a	70.0±0.02^b	67.0±0.00^c	68.0±0.02^c	77.0±0.02^b
SH-1439	75.0±0.01^c	74.0±0.01^a	80.0±0.04^a	81.0±0.02^a	81.0±0.02^a	81.0±0.03^a

指标 Item	CK1	CK2	处理组 Treatment group
指标 Item	CK1	CK2	SH-1471	SH-1464	SH-1439
病情指数Disease index	89.0±0.032c	-	15.3±0.014a	31.7±0.027b	32.0±0.020b
防效Control effect/%	-	-	83.7±0.024c	60.7±0.009b	59.0±0.023a