三株无量山森林土壤芽孢杆菌鉴定及其生物活性挖掘

doi:10.13560/j.cnki.biotech.bull.1985.2023-0918

摘要/Abstract

摘要：

【目的】挖掘无量山国家自然保护区潜在的高活性微生物菌株，旨在为农业绿色投入品研发提供高效的菌种资源。【方法】以番茄枯萎病菌（Fusarium oxysporum）为靶标筛选高拮抗活性菌株，观察对菌丝生长的影响，测定对孢子萌发的抑制作用；检测脂肽类化合物合成基因以及菌株体外产酶、解磷、解钾、固氮及产铁载体能力；结合形态学、生理生化特征和16S rRNA、gyrA、rpoB基因进行菌株鉴定，室内盆栽试验验证防病和促生效果。【结果】分离筛选获得153株可培养细菌，其中，菌株SH-53、N4471和N9456抑菌效果分别为92.35%、87.29%和88.47%，能抑制病原菌菌丝生长和分生孢子萌发，且对多种病原菌表现出良好拮抗活性。3株功能菌均具有溶锌、产淀粉酶和蛋白酶的能力，菌株SH-53和N4471还具有解磷、固氮、分泌纤维素酶以及产铁载体能力。3个功能菌基因组中均含有srfA、fenA、ituA、ituC、ituD、bymC等脂肽类化合物合成基因。经鉴定菌株SH-53为解淀粉芽孢杆菌（Bacillus amyloliquefaciens），N4471为卡式芽孢杆菌（B. cabrialesii），N9456为暹罗芽孢杆菌（B. siamensis）。盆栽试验结果表明，3株功能菌防效分别为84.66%、54.96%和59.74%，对番茄幼苗的株高、茎粗、根长等农艺性状均具有较好促进作用。【结论】3个菌株具有高效、广谱的抑菌活性，具有多样化的生物活性，作为高效的微生物资源具有广阔的应用前景。

关键词: 解淀粉芽孢杆菌, 卡式芽孢杆菌, 暹罗芽孢杆菌, 鉴定, 生物活性, 番茄枯萎病, 促生

Abstract:

【Objective】 In order to provide efficient microbial resource for the development of green agricultural inputs, we identified potential high-activity microbial strains from the National Wilderness Preservation System of Mount Wuliangshan.【Method】 We used Fusarium oxysporum as a target to screen highly antagonistic strains, and observed the effect on hyphae growth and spore germination. We also tested the lipopeptide synthesis genes and the ability of the strain to produce enzymes, hydrolyze phosphorus, hydrolyze potassium, fix nitrogen and produce siderophore. In addition, the strains were identified according to their morphological, physiological and biochemical characteristics and 16S rRNA, gyrA and rpoB genes, and the effects of disease control and growth promotion were verified by pot experiment.【Result】 A total of 153 strains of culturable bacteria were isolated and screened. Among them, the inhibition rates of SH-53, N4471 and N9456 were 92.35%, 87.29% and 88.47%, respectively, it showed good antagonistic activity against many pathogens, and also inhibited the hypha growth and conidia germination of the pathogen. The three functional strains all had the abilities of dissolving zinc, producing amylase and protease, and the strains Sh-53 and N4471 also had the abilities of dissolving phosphorus, fixing nitrogen, secreting cellulase and producing siderophore. In addition, we found that srfA, fenA, ituA, ituC, ituD and bymC genes were found in the genomes of the three functional bacteria. Strain SH-53 was identified as Bacillus amyloliquefaciens, N4471 as B. cabrialesii, and N9456 as B. siamensis. The results of pot experiment showed that the control effects of the three functional bacteria were 84.66%, 54.96% and 59.74%, respectively. They promoted the agronomic characters of tomato seedlings, such as plant height, stem diameter and root length.【Conclusion】 As high-efficiency microbial resources, the three strains have broad-spectrum antibacterial activity, diverse biological activity, and have broad application prospects in the development of green agricultural inputs.

Key words: Bacillus amyloliquefaciens, Bacillus cabrialesii, Bacillus siamensis, identification, biological activity, tomato wilt disease, growth-promoting

王楠, 廖永琴, 施竹凤, 申云鑫, 杨童雨, 冯路遥, 矣小鹏, 唐加菜, 陈齐斌, 杨佩文. 三株无量山森林土壤芽孢杆菌鉴定及其生物活性挖掘[J]. 生物技术通报, 2024, 40(2): 277-288.

WANG Nan, LIAO Yong-qin, SHI Zhu-feng, SHEN Yun-xin, YANG Tong-yu, FENG Lu-yao, YI Xiao-peng, TANG Jia-cai, CHEN Qi-bin, YANG Pei-wen. Identification of Three Strains of Bacillus from the Forest Soil of Wuliang Mountain and Mining of Their Bioactivities[J]. Biotechnology Bulletin, 2024, 40(2): 277-288.

图/表 14

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培养基种类 Medium category	配方Formulation/L
马铃薯葡萄糖琼脂培养基 Potato dextrose agar medium（PDA）	去皮马铃薯200.0 g，葡萄糖20.0 g，琼脂20.0 g，pH自然
营养琼脂培养基 Nutrient agar medium（NA）	牛肉膏5.0 g，蛋白胨10.0 g，氯化钠5.0 g，琼脂20.0 g，pH 7.0
LB固体培养基 Luria-Bertani solid medium	胰蛋白胨10.0 g，酵母提取物5.0 g，氯化钠10.0 g，琼脂20.0 g，pH 7.4
营养肉汤培养基 Nutrient broth medium（NB）	牛肉膏3.0 g，蛋白胨10.0 g，氯化钠5.0 g，pH 7.4
水琼脂培养基 Water agar medium	琼脂20.0 g，pH 7.0
解磷选择培养基 Phosphate-solubilizing selection medium	葡萄糖10.0 g，硫酸铵0.5 g，氯化钠0.3 g，七水合硫酸镁0.3 g，七水合硫酸亚铁0.03 g，四水合硫酸锰0.03 g，氯化钾0.3 g，磷酸钙2.0 g，琼脂17.0 g，pH 7.2-7.4
解钾选择培养基 Potassium solution selection medium	蔗糖5.0 g，葡萄糖5.0 g，硫酸铵0.5 g，酵母膏0.5 g，七水合硫酸镁0.3 g，磷酸氢二钠2.0 g，七水合硫酸亚铁0.03 g，硫酸锰0.03 g，钾长石粉2.0 g，琼脂18.0 g，pH 7.0
固氮选择培养基 Nitrogen-fixing selection medium	磷酸二氢钾0.2 g，硫酸镁0.2 g，氯化钠0.2 g，碳酸钙5.0 g，甘露醇10.0 g，硫酸钙0.1 g，琼脂18.0 g，pH 6.8-7.0
酪蛋白选择培养基	葡萄糖10.0 g，酵母膏4.0 g，酪蛋白10.0 g，磷酸氢二钾1.0 g，七水合硫酸镁0.2 g，琼脂20.0 g，pH 7.0
纤维素培养基 Cellulose medium	磷酸氢二钾1.0 g，硫酸铵2.0 g，七水合硫酸镁0.5 g，氯化钠0.5 g，微晶纤维素2.0 g，琼脂20.0 g，pH 7.0
淀粉选择培养基 Starch selection medium	可溶性淀粉10.0 g，磷酸氢二钾1.0 g，硫酸镁1.0 g，氯化钠1.0 g，硫酸铵2.0 g，碳酸钙2.0 g，硫酸亚铁0.001 g，氯化锰0.001 g，硫酸锌0.001 g，琼脂20.0 g，pH 7.0-7.4
溶锌选择培养基^[17] Zinc-solubilized selection medium	氧化锌1.0 g，葡萄糖10.0 g，氯化钾0.2 g、硫酸铵1.0 g，硫酸镁0.2 g，磷酸氢二钾0.1 g，琼脂20.0 g，pH 7.0
CAS双层显色培养基^[18] CAS bilayer chromogenic medium	底层为5 mL水琼脂培养基（加有2 mL CAS显色液），上层为5 mL的PDA培养基

培养基种类 Medium category	配方Formulation/L
马铃薯葡萄糖琼脂培养基 Potato dextrose agar medium（PDA）	去皮马铃薯200.0 g，葡萄糖20.0 g，琼脂20.0 g，pH自然
营养琼脂培养基 Nutrient agar medium（NA）	牛肉膏5.0 g，蛋白胨10.0 g，氯化钠5.0 g，琼脂20.0 g，pH 7.0
LB固体培养基 Luria-Bertani solid medium	胰蛋白胨10.0 g，酵母提取物5.0 g，氯化钠10.0 g，琼脂20.0 g，pH 7.4
营养肉汤培养基 Nutrient broth medium（NB）	牛肉膏3.0 g，蛋白胨10.0 g，氯化钠5.0 g，pH 7.4
水琼脂培养基 Water agar medium	琼脂20.0 g，pH 7.0
解磷选择培养基 Phosphate-solubilizing selection medium	葡萄糖10.0 g，硫酸铵0.5 g，氯化钠0.3 g，七水合硫酸镁0.3 g，七水合硫酸亚铁0.03 g，四水合硫酸锰0.03 g，氯化钾0.3 g，磷酸钙2.0 g，琼脂17.0 g，pH 7.2-7.4
解钾选择培养基 Potassium solution selection medium	蔗糖5.0 g，葡萄糖5.0 g，硫酸铵0.5 g，酵母膏0.5 g，七水合硫酸镁0.3 g，磷酸氢二钠2.0 g，七水合硫酸亚铁0.03 g，硫酸锰0.03 g，钾长石粉2.0 g，琼脂18.0 g，pH 7.0
固氮选择培养基 Nitrogen-fixing selection medium	磷酸二氢钾0.2 g，硫酸镁0.2 g，氯化钠0.2 g，碳酸钙5.0 g，甘露醇10.0 g，硫酸钙0.1 g，琼脂18.0 g，pH 6.8-7.0
酪蛋白选择培养基	葡萄糖10.0 g，酵母膏4.0 g，酪蛋白10.0 g，磷酸氢二钾1.0 g，七水合硫酸镁0.2 g，琼脂20.0 g，pH 7.0
纤维素培养基 Cellulose medium	磷酸氢二钾1.0 g，硫酸铵2.0 g，七水合硫酸镁0.5 g，氯化钠0.5 g，微晶纤维素2.0 g，琼脂20.0 g，pH 7.0
淀粉选择培养基 Starch selection medium	可溶性淀粉10.0 g，磷酸氢二钾1.0 g，硫酸镁1.0 g，氯化钠1.0 g，硫酸铵2.0 g，碳酸钙2.0 g，硫酸亚铁0.001 g，氯化锰0.001 g，硫酸锌0.001 g，琼脂20.0 g，pH 7.0-7.4
溶锌选择培养基^[17] Zinc-solubilized selection medium	氧化锌1.0 g，葡萄糖10.0 g，氯化钾0.2 g、硫酸铵1.0 g，硫酸镁0.2 g，磷酸氢二钾0.1 g，琼脂20.0 g，pH 7.0
CAS双层显色培养基^[18] CAS bilayer chromogenic medium	底层为5 mL水琼脂培养基（加有2 mL CAS显色液），上层为5 mL的PDA培养基

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

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

菌株 Strain	初筛抑菌率Inhibition rate of initial screening/%	复筛抑菌率Inhibition rate of re-screening/%
SH-53	92.35±0.99a	89.59±1.18a
N4471	87.29±0.67b	88.06±0.90a
N9456	88.47±0.83b	85.35±0.67a
N1468	80.00±1.71cd	75.41±1.23b
N1475	82.35±1.03c	80.59±0.68b
N1477	74.47±1.52e	64.71±1.33c
82-a2	79.41±1.21d	79.61±0.68b
82-a3	77.06±1.33e	74.70±1.56b
SH-1	76.71±1.02e	73.13±1.23b
SH-6	75.29±1.55e	58.04±2.45d