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浙江大学学报（农业与生命科学版）
浙江大学学报（农业与生命科学版）  2020, Vol. 46 Issue (2): 177-188    DOI: 10.3785/j.issn.1008-9209.2019.05.301
植物保护     
1株生防菌的鉴定及其发酵条件优化
陈海念1(),冯蓉1,2,杨胜竹1,曹本福1,3,文明江1,刘丽1,2(),陆引罡1,2,3
1.贵州大学农学院，贵阳 550025
2.贵州大学新型肥料资源与技术研究所，贵阳 550025
3.贵州省烟草品质研究重点实验室，贵阳 550025
Identification of a biocontrol strain and optimization of its fermentation conditions
Hainian CHEN1(),Rong FENG1,2,Shengzhu YANG1,Benfu CAO1,3,Mingjiang WEN1,Li LIU1,2(),Yingang LU1,2,3
1.Agricultural College, Guizhou University, Guiyang 550025, China
2.Institute of New Fertilizer Resources and Technology, Guizhou University, Guiyang 550025, China
3.Guizhou Key Laboratory for Tobacco Quality Study, Guiyang 550025, China
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摘要：

对草莓灰霉病菌具有较强拮抗活性的生防菌株F11进行分类学鉴定和抑菌效果评价，并采用正交试验和单因素试验对该菌株发酵配方和发酵条件进行优化，以期提高该菌株的发酵效率。结果表明，F11菌株对水稻稻瘟病菌、核盘菌等12种常见病原菌抑制率均达79.88%以上，表现出高效、广谱的抑菌特性，可作为植物病害生物防治的潜在资源。经菌落形态观察、生理生化测定、16S rRNA及gyrB基因测序分析，确定菌株F11为解淀粉芽孢杆菌（Bacillus amyloliquefaciens）。通过L16(43×26)正交试验优化，确定该菌株适宜的发酵培养基配方为：3.00 g/L牛肉膏、10.00 g/L蛋白胨、5.00 g/L NaCl、3.00 g/L葡萄糖、2.00 g/L MgCl2。单因素试验结果进一步表明，基于优化配方，菌株F11培养的适宜初始pH为7.0、温度为30 ℃、振荡速率为150 r/min；平板计数显示，上述培养条件下活菌数为4.67×109 CFU/mL。
关键词： 生防菌;  鉴定;  抑菌效果;  正交试验;  培养基优化    
Abstract:

The taxonomic identification and antimicrobial effect of a strain called F11 which has inhibitory effect on strawberry gray mold was studied. In order to improve the fermentation efficiency of the strain, the fermentation formula and conditions were optimized by orthogonal experiment and single factor test. The results showed that the strain F11 could inhibit the growth and reproduction of 12 common pathogens with high inhibition rate reaching 79.88% or more, which showed that this strain possessed broad-spectrum and high-efficiency antimicrobial effect, and that it would be a hidden promising resource for biological control agent to resist plant diseases. The strain F11 was identified as Bacillus amyloliquefaciens by observation of colony morphology, physiological and biochemical tests, 16S rRNA and gyrB gene sequencing. The fermentation medium of strain F11 was optimized from L16 (43×26) orthogonal test: 3.00 g/L beef extract, 10.00 g/L peptone, 5.00 g/L NaCl, 3.00 g/L glucose, 2.00 g/L MgCl2. Furthermore, the single factor test further showed that based on the optimized medium, the suitable initial pH of strain F11 culture was 7.0, and the befitting temperature was 30 ℃, and the proper oscillation rate was 150 r/min, which the number of viable cells were 4.67×109 CFU/mL.
Key words: biocontrol bacterium    identification    inhibitory effect    orthogonal experiment    medium optimization
收稿日期: 2019-05-30 出版日期: 2020-05-22
CLC:  S 476.1  
基金资助: 国家自然科学基金(31760133);贵州省科技计划（〔2017〕0106）;贵州省科技计划项目“黔科合平台人才”（〔2017〕5788号）;贵州大学人才引进项目（〔2015〕10号）;贵州省生物学一流学科建设项目（GNYL〔2017〕009）
通讯作者: 刘丽     E-mail: 571661476@qq.com;liuliz706@sina.com
作者简介: 陈海念（https://orcid.org/0000-0003-3541-7116），E-mail：571661476@qq.com
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引用本文:

陈海念,冯蓉,杨胜竹,曹本福,文明江,刘丽,陆引罡. 1株生防菌的鉴定及其发酵条件优化[J]. 浙江大学学报（农业与生命科学版）, 2020, 46(2): 177-188.

Hainian CHEN,Rong FENG,Shengzhu YANG,Benfu CAO,Mingjiang WEN,Li LIU,Yingang LU. Identification of a biocontrol strain and optimization of its fermentation conditions. Journal of Zhejiang University (Agriculture and Life Sciences), 2020, 46(2): 177-188.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2019.05.301    或    http://www.zjujournals.com/agr/CN/Y2020/V46/I2/177

处理

Treatment

牛肉膏

Beef extract

蛋白胨

Peptone

NaCl

葡萄糖

Glucose

MnMoBFeMg
MnSO4?2H2ONa2MoO4H3BO3FeSO4?7H2OMgCl2
11(1.00)1(2.00)1(2.00)1(0.00)1(0.00)1(0.00)1(0.00)1(0.00)1(0.50)
21(1.00)2(5.00)2(5.00)1(0.00)1(0.00)2(0.05)2(0.05)2(0.10)2(2.00)
31(1.00)3(7.00)3(7.00)2(3.00)2(0.50)1(0.00)1(0.00)1(0.00)2(2.00)
41(1.00)4(10.00)4(10.00)2(3.00)2(0.50)2(0.05)2(0.05)2(0.10)1(0.50)
52(2.00)1(2.00)2(5.00)2(3.00)2(0.50)1(0.00)2(0.05)2(0.10)1(0.50)
62(2.00)2(5.00)1(2.00)2(3.00)2(0.50)2(0.05)1(0.00)1(0.00)2(2.00)
72(2.00)3(7.00)4(10.00)1(0.00)1(0.00)1(0.00)2(0.05)2(0.10)2(2.00)
82(2.00)4(10.00)3(7.00)1(0.00)1(0.00)2(0.05)1(0.00)1(0.00)1(0.50)
93(3.00)1(2.00)3(7.00)1(0.00)2(0.50)2(0.05)2(0.05)2(0.10)2(2.00)
103(3.00)2(5.00)4(10.00)1(0.00)2(0.50)1(0.00)1(0.00)1(0.00)1(0.50)
113(3.00)3(7.00)1(2.00)2(3.00)1(0.00)2(0.05)2(0.05)2(0.10)1(0.50)
123(3.00)4(10.00)2(5.00)2(3.00)1(0.00)1(0.00)1(0.00)1(0.00)2(2.00)
134(5.00)1(2.00)4(10.00)2(3.00)1(0.00)2(0.05)1(0.00)1(0.00)2(2.00)
144(5.00)2(5.00)3(7.00)2(3.00)1(0.00)1(0.00)2(0.05)2(0.10)1(0.50)
154(5.00)3(7.00)2(5.00)1(0.00)2(0.50)2(0.05)1(0.00)1(0.00)1(0.50)
164(5.00)4(10.00)1(2.00)1(0.00)2(0.50)1(0.00)2(0.05)2(0.10)2(2.00)
表1  L16(43×26)正交试验设计方案

病原菌

Pathogen

相对抑制率

Relative inhibition

rate/%

病原菌直径

Pathogen diameter

对照组

Control group/cm

F11拮抗组

Antagonism group of F11/cm

t检验

t test

P值

P value

烟草黑胫病菌

Phytophthora parasitica var.

80.535.63±1.021.70±0.266.460.002 9

烟草炭疽病菌

Colletotrichum nicotianae Av.

90.695.05±0.351.15±0.0715.300.002 1

樟树胶孢炭疽病菌

Colletotrichum gloeosp orioides

86.679.00±0.001.85±0.0712.350.000 1

高粱斑点病菌

Pseudomonas syringae pv. syringae Van Hall

80.978.63±0.642.25±0.4911.810.001 3

玉兰炭疽病菌

Colletotrchum magnoliae camara

83.518.63±0.632.05±0.2113.540.008 7

核盘菌

Sclerotinia sclerotiorum

100.009.00±0.000.75±0.0111.820.000 1

禾谷炭疽菌

Colletotrichum graminicola Cesati Wilson

82.156.63±0.491.80±0.2015.730.000 1

玉米大斑病菌

Setosphaeria turcica

94.747.40±0.961.10±0.2011.080.000 2

烟草赤星病菌

Alternaria alternata (Freis) Keissler

81.427.30±0.561.97±0.2115.540.000 1

辣椒炭疽菌

Colletotrichum capsici

86.069.00±0.001.90±0.2012.310.001 4

水稻稻瘟病菌

Pyricularia oryzae

79.886.30±0.361.87±0.2118.440.000 1

猕猴桃葡萄座腔菌

Botryosphaeria dothidea

91.529.00±0.001.45±0.2114.130.000 2
表2  菌株F11对12种病原菌的抑制作用
图1  菌株F11的形态A.肉眼观察；B.光学显微镜下观察。
指标 IndexF11LX1[25]WTD[26]H-2[27]H-7[28]
β-半乳糖苷酶 β-galactosidase+
接触酶 Catalase++
氧化酶 Oxidase+－+
脲酶 Urease++－－
精氨酸双水解酶 Arginine dihydrolase++
淀粉水解 Starch hydrolysis+++
明胶水解 Gelatin hydrolysis+++++
酪素水解 Casein hydrolysis++
鸟氨酸脱羧酶 Ornithine decarboxylase－－
赖氨酸脱羧酶 Lysine decarboxylase－
硝酸盐还原 Nitrate reduction++
柠檬酸生长 Citric acid growth+－++
50 ℃生长 Growth at 50 ℃+
VP反应 Voges-Proskauer reaction+++++
甲基红反应 Methyl red reaction－－
硫化氢产生 Hydrogen sulfide production－－+－－
甘露醇 Mannitol++－－
山梨醇 Sorbitol++－－
鼠李糖 L-(+)-rhamnose－－－－
蔗糖 D(+)-sucrose++－++
蜜二糖 Melibiose－++
革兰氏染色 Gram stain+++
表3  菌株F11与其他解淀粉芽孢杆菌生理生化鉴定结果对比
图 2  16S rRNA的系统发育树
图3  gyrB基因的系统发育树
图4  L16（43×26）正交试验各处理及NB培养基处理的F11菌悬液D600 nm值比较短栅上不同小写字母表示在P＜0.05水平差异有统计学意义; n=5。
图5  F11菌悬液D600 nm值在各正交试验因子水平间的比较数据点上不同小写字母表示同一因子水平间在P＜0.05水平差异有统计学意义。

因子

Factor

极差

Range

P值

P value

优选量

Optimal dosage/(g/L)

牛肉膏 Beef extract0.259 22.96×10－203.00
蛋白胨 Peptone0.390 49.54×10－2510.00
NaCl0.343 31.65×10－225.00
葡萄糖 Glucose0.320 15.64×10－263.00
Mn0.517 01.64×10－320
Mo0.039 14.13×10－40
B0.012 80.200
Fe0.012 80.210
Mg0.159 76.82×10－172.00
表4  各正交试验因子的极差、P值与优选量
图6  不同初始pH对菌株F11生长的影响短栅上不同小写字母表示同一配方下不同处理间在P＜0.05水平差异有统计学意义；n=5。
图7  不同培养温度对菌株F11的影响短栅上不同小写字母表示同一配方下不同处理间在P＜0.05水平差异有统计学意义；n=5。
图8  不同初始振荡速率对菌株F11生长的影响短栅上不同小写字母表示同一配方下不同处理间在P＜0.05水平差异有统计学意义；n=5。
图9  优化配方和NB液体培养基中F11活菌数比较短栅上不同大写字母表示在P＜0.01水平差异有高度统计学意义； n=5。
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