三株马铃薯炭疽病生防菌的分离鉴定及其高抑菌活性培养物发酵参数优化

doi:10.13560/j.cnki.biotech.bull.1985.2025-0209

摘要/Abstract

摘要：

目的马铃薯炭疽病由球炭疽菌（Colletotrichum coccodes）引起，严重影响产量品质，而芽胞杆菌（Bacillus spp.）是一类常见的生防菌，寻找高效低毒生防菌防治马铃薯炭疽病意义重大。本研究以3株高抑菌活性芽胞杆菌及其复合菌群为对象，旨在通过优化复合芽胞杆菌发酵参数，以提高其对该病菌抑菌活性，为研制复合生防菌剂提供参考。方法通过组织分离法和划线法从马铃薯块茎中分离内生菌，通过药敏纸片法筛选对马铃薯炭疽病具有较强抑制作用的菌株，结合形态学和分子生物学鉴定确定活性菌株的分类地位，通过药敏纸片法确定3株菌的最佳配比比例，以单因素试验和响应面设计对复合菌群HQS6513的发酵培养基和发酵条件进行优化。结果从马铃薯块茎中筛选出3株对马铃薯球炭疽菌（Colletotrichum coccodes）具有明显抑菌作用的菌株QS10-6、QS2-5和QS2-13，QS10-6和QS2-5鉴定为萎缩芽胞杆菌（Bacillus atrophaeus），QS2-13为贝莱斯芽胞杆菌（Bacillus velezensis）；响应面法分析优化得到复合菌群HQS6513最佳发酵培养基为麸皮2.5%、酵母浸粉2.6%、NaCl 1.0%，最佳发酵条件为培养温度32 ℃、pH 5.0、接种量2.0%、摇床转速190 r/min，优化后的复合菌群HQS6513的抑菌率达到69.66%。结论分离得到3株芽胞杆菌，由这3株芽胞杆菌组成的复合菌群HQS6513对马铃薯球炭疽菌有高抑菌活性，在优化发酵配方和培养条件下，复合菌群HQS6513产胞量和抑菌活性增加。

关键词: 马铃薯球炭疽菌, 复合菌群, 萎缩芽胞杆菌, 贝莱斯芽胞杆菌, 响应面法, 发酵参数

Abstract:

Objective Potato anthracnose, caused by Colletotrichum coccodes, severely impacts both yield and quality. Bacillus spp. are commonly used as biocontrol agents, making the search for efficient and low-toxicity strains to manage this disease highly significant. This study focused on three Bacillus strains with strong antibacterial activity and their composite microbial community. By optimizing the fermentation parameters of the composite Bacillus, the aim is to enhance its inhibitory effect against the pathogen, providing a reference for the development of composite biocontrol agents. Method Endophytic bacteria were isolated from potato tubers by tissue isolation and scribing methods, strains with strong inhibitory effect on potato anthrax were screened by drug-sensitive paper slide method, the taxonomic status of the active strains was determined by combining morphological and molecular biological identification, and the optimal ratio of the three strains was determined by drug-sensitive paper slide method, and the composite group was optimized by one-factor test and response surface design. The fermentation medium and fermentation conditions of HQS6513 were optimized by one-way test and response surface design. Result Three strains QS10-6, QS2-5 and QS2-13 with obvious inhibitory effect on potato coccodes (Colletotrichum coccodes) were screened out from potato tubers and identified as Bacillus atrophaeus, Bacillus atrophaeus and Bacillus velezensis, response surface method analysis was optimized to obtain the optimal fermentation medium for the composite colony HQS6513 as bran 2.5%, yeast extract powder 2.6%, NaCl 1.0%, and the optimal fermentation conditions as incubation temperature of 32 ℃, pH 5, inoculum amount of 2.0%, and shaking table speed of 190 r/min, and the optimized bacterial inhibition rate of the composite colony HQS6513 reached 69.66%. Conclusion Three strains of Bacillus spp. were isolated, and the composite colony HQS6513 composed of these three strains of Bacillus spp. had high inhibitory activity against C. coccodes, and the optimization of fermentation formula and culture conditions for it resulted in the increase of cell production and inhibitory activity of the composite flora HQS6513.

Key words: Colletotrichun coccodes, complex flora, Bacillus atrophicus, Bacilhas velezensis, response surface methodology, fermentation parameters

罗明凯, 张豪杰, 石晖琴, 李亚楠, 封瑞超, 沈硕. 三株马铃薯炭疽病生防菌的分离鉴定及其高抑菌活性培养物发酵参数优化[J]. 生物技术通报, 2025, 41(12): 313-327.

LUO Ming-kai, ZHANG Hao-jie, SHI Hui-qin, LI Ya-nan, FENG Rui-chao, SHEN Shuo. Isolation and Identification of Biocontrol Bacteria for Potato Anthracnose in Three Strains and Optimization of Fermentation Parameters for Cultures with High Antibacterial Activity[J]. Biotechnology Bulletin, 2025, 41(12): 313-327.

图/表 16

表1 芽胞杆菌QS10-6、QS2-5、QS2-13分子鉴定的引物信息

Table 1 Primers for molecular identification of Bacillus QS10-6, QS2-5 and QS2-13

菌株

Strain

基因

Gene

GenBank 登录号

GenBank No.

引物序列

Primer sequence （5′-3′）

产物长度

Product size （bp）

QS10-6

16S rDNA

PQ856826

F：TGCAGTCGAGCGGACAGATGGGA

R：CACCCGAAGTCGGTGAGGTAACCT

F：GAGCGGACAGATGGGAGCTTGCT

R：CCGAAGTCGGTGAGGTAACCTTTA

F：GTCGAGCGGACAGATGGGAGCTTG

R：TAACACCCGAAGTCGGTGAGGTAA

1 394

表2 3种菌株含量的比例设置

Table 2 The set ratio of the contents of three strains

处理 Treatment	QS10-6∶QS2-5∶QS2-13
T1	1∶1∶1
T2	1∶2∶1
T3	1∶3∶3
T4	2∶1∶2
T5	2∶2∶3
T6	2∶3∶1
T7	3∶1∶3
T8	3∶2∶1
T9	3∶3∶2

图1 不同细菌对马铃薯炭球炭疽菌的抑菌活性A：CK；B：QS10-1；C：QS10-2；D：QS10-3；E：QS10-4；F：QS10-5；G：QS10-6；H：QS2-4；I：QS2-5；J：QS2-7；K：QS2-11；L：QS2-12；M：QS2-13；N：QS10-15；O：QS10-16；P：QS2-15；Q：QS2-17；R：QS2-19；S：QS10-18；T：QS10-19；U：QS10-20

Fig. 1 Antibacterial activities of different bacteria against Colletotrichum coccodes on potatoes

表3 细菌抑制马铃薯炭疽病病原菌的活性

Table 3 Activities of bacteria inhibiting pathogens of potato anthracnose

菌株 Strain	菌落直径 Colony diameter （mm）	抑菌率 Inhibition rate （%）
QS10-5	29.52±0.27d	50.55±0.46c
QS10-6	34.48±0.14b	42.25±0.24e
QS2-4	31.49±1.54c	47.25±2.58d
QS2-5	25.82±0.16e	56.75±0.27a
QS2-7	26.43±0.45e	55.74±0.77b
QS2-11	29.91±0.37d	49.91±0.06c
QS2-13	23.90±0.33f	59.97±0.55a
QS10-15	30.25±0.12cd	49.35±0.20cd
QS10-16	27.02±0.21e	54.75±0.34b
QS2-15	26.67±0.13e	55.34±0.21b
QS2-17	26.37±0.18e	55.83±0.29b
QS2-19	26.55±0.41e	55.53±0.07b
QS10-18	29.66±0.12d	50.33±0.21c
QS10-19	29.48±0.27d	50.07±0.22c
QS10-20	34.59±0.12b	42.07±0.19e
CK	59.71±0.50a

图2 菌株的菌落形态及系统发育树A：LB培养基上QS10-6的菌落形态，下同；B：QS2-5；C：QS2-13；A1：QS10-6菌株革兰氏染色形态；B1：QS2-5；C1：QS2-13；A2：基于16S rDNA、gyrA和rpoD构建的QS10-6菌株及相关基因的多基因系统发育树，下同；B2：QS2-5；C2：QS2-13

Fig. 2 Colony morphology and phylogenetic tree of strainsA: Colony morphology of QS10-6 on LB medium,the same below; B: QS2-5; C: QS2-13; A1:Gram stain morphology of strain QS10-6, the same below; B1: QS2-5; C1: QS2-13. A2: Polygenic phylogenetic tree of QS10-6 strain and related genes based on 16S rDNA, gyrA and rpoD, the same below; B2: QS2-5; C2: QS2-13

图3 溶血安全性评价A：金黄色葡萄球菌；B：QS10-6-萎缩芽胞杆菌；C：QS2-5-萎缩芽胞杆菌；D：QS2-13-贝莱斯芽胞杆菌

Fig. 3 Evaluation of hemolytic safetyA: Staphylococcus aureus; B: QS10-6-Bacillus atrophaeus; C: QS2-5-Bacillus atrophaeus; D: QS2-13- Bacillus velezensis

图4 3株活性菌相容性测定A: QS10-6 to QS2-5; B: QS10-6 to QS2-13; C: QS2-5 to QS2-13; D: QS2-5 to QS10-6; E: QS2-13 to QS10-6; F: QS2-13 to QS2-5

Fig. 4 Compatibility determination of 3 strains of active bacteria

表4 菌株QS10-6、QS2-5、QS2-13及组成的复合菌群的抑菌活性

Table 4 Antibacterial activities of strains QS10-6, QS2-5, and QS2-13 and the composed compound microbial community

试验 Test	序号 Number	处理 Treatment	菌圈直径 Colony diameter （mm）	抑菌率 Inhibition rate （%）
A：平板试验	1	QS10-6	35.97±0.34b	36.31±0.60e
	2	QS2-5	24.27±0.33e	57.04±0.58b
	3	QS2-13	23.54±0.19e	58.32±0.34ab
	4	HQS65	28.07±0.35c	50.29±0.63b
	5	HQS613	25.41±0.17d	54.99±0.29c
	6	HQS513	26.18±0.32d	53.64±0.57c
	7	HQS6513	23.20±0.23e	58.91±0.42a
	8	CK	56.48±0.71a
B：防治（发酵液）	1	QS10-6	21.41±1.28b	23.11±4.59c
	2	QS2-5	7.99±0.07d	71.32 ±0.26a
	3	QS2-13	7.87±0.04d	71.73±0.14a
	4	HQS65	10.09±0.03c	63.77±0.11b
	5	HQS613	7.87±0.03d	71.73±0.10a
	6	HQS513	8.06±0.03d	71.06±0.12a
	7	HQS6513	7.80±0.03d	71.99±0.09a
	8	百菌清	8.11±0.03d	70.88±0.12a
	9	多菌灵	8.17±0.03d	70.65±0.10a
	10	H₂O	7.91±0.02d
	11	CK	27.85±0.46a
C：防治（菌悬液）	1	QS10-6	21.10±0.26ef	26.61±0.91c
	2	QS2-5	20.77±0.15fj	27.77±0.54c
	3	QS2-13	22.05±0.14cd	23.31±0.49d
	4	HQS65	22.63±0.15c	21.30±0.51e
	5	HQS613	28.45±0.06a	1.04±0.20f
	6	HQS513	21.64±0.10de	24.74±0.36d
	7	HQS6513	20.22±0.29j	29.68±1.01b
	8	百菌清	8.11±0.03h	71.79±0.11a
	9	多菌灵	8.17±0.03h	71.58±0.09b
	10	H₂O	7.91±0.02h
	11	CK	27.85±0.46b
D：预防（发酵液）	1	QS10-6	8.29±0.09b	71.08±0.32f
	2	QS2-5	8.00±0.03b	72.11±0.10c
	3	QS2-13	8.00±0.01 b	72.09±0.04cd
	4	HQS65	8.01±0.01b	72.06±0.04cde
	5	HQS613	8.12±0.02b	71.68±0.06e
	6	HQS513	8.11±0.03b	71.70±0.09de
	7	HQS6513	7.95±0.03b	72.26±0.09bc
	8	百菌清	7.77±0.01b	72.89±0.04a
	9	多菌灵	7.85±0.02b	72.63±0.07ab
	10	H₂O	7.91±0.02b
	11	CK	28.67±0.61a
E：预防（菌悬液）	1	QS10-6	28.20±0.04a	1.65±0.13d
	2	QS2-5	8.31±0.38b	71.01±1.31c
	3	QS2-13	8.31±0.02b	71.00±0.07c
	4	HQS65	28.35±0.08a	1.10±0.26d
	5	HQS613	7.99±0.16bc	72.13±0.56bc
	6	HQS513	8.18±0.13bc	71.48±0.45c
	7	HQS6513	8.18±0.01bc	71.47±0.02c
	8	百菌清	7.64±0.08bc	73.34±0.29ab
	9	多菌灵	7.42±0.17c	74.13±0.58a
	10	H₂O	7.91±0.02bc
	11	CK	28.67±0.61a

表4 菌株QS10-6、QS2-5、QS2-13及组成的复合菌群的抑菌活性

Table 4 Antibacterial activities of strains QS10-6, QS2-5, and QS2-13 and the composed compound microbial community

试验 Test	序号 Number	处理 Treatment	菌圈直径 Colony diameter （mm）	抑菌率 Inhibition rate （%）
A：平板试验	1	QS10-6	35.97±0.34b	36.31±0.60e
	2	QS2-5	24.27±0.33e	57.04±0.58b
	3	QS2-13	23.54±0.19e	58.32±0.34ab
	4	HQS65	28.07±0.35c	50.29±0.63b
	5	HQS613	25.41±0.17d	54.99±0.29c
	6	HQS513	26.18±0.32d	53.64±0.57c
	7	HQS6513	23.20±0.23e	58.91±0.42a
	8	CK	56.48±0.71a
B：防治（发酵液）	1	QS10-6	21.41±1.28b	23.11±4.59c
	2	QS2-5	7.99±0.07d	71.32 ±0.26a
	3	QS2-13	7.87±0.04d	71.73±0.14a
	4	HQS65	10.09±0.03c	63.77±0.11b
	5	HQS613	7.87±0.03d	71.73±0.10a
	6	HQS513	8.06±0.03d	71.06±0.12a
	7	HQS6513	7.80±0.03d	71.99±0.09a
	8	百菌清	8.11±0.03d	70.88±0.12a
	9	多菌灵	8.17±0.03d	70.65±0.10a
	10	H₂O	7.91±0.02d
	11	CK	27.85±0.46a
C：防治（菌悬液）	1	QS10-6	21.10±0.26ef	26.61±0.91c
	2	QS2-5	20.77±0.15fj	27.77±0.54c
	3	QS2-13	22.05±0.14cd	23.31±0.49d
	4	HQS65	22.63±0.15c	21.30±0.51e
	5	HQS613	28.45±0.06a	1.04±0.20f
	6	HQS513	21.64±0.10de	24.74±0.36d
	7	HQS6513	20.22±0.29j	29.68±1.01b
	8	百菌清	8.11±0.03h	71.79±0.11a
	9	多菌灵	8.17±0.03h	71.58±0.09b
	10	H₂O	7.91±0.02h
	11	CK	27.85±0.46b
D：预防（发酵液）	1	QS10-6	8.29±0.09b	71.08±0.32f
	2	QS2-5	8.00±0.03b	72.11±0.10c
	3	QS2-13	8.00±0.01 b	72.09±0.04cd
	4	HQS65	8.01±0.01b	72.06±0.04cde
	5	HQS613	8.12±0.02b	71.68±0.06e
	6	HQS513	8.11±0.03b	71.70±0.09de
	7	HQS6513	7.95±0.03b	72.26±0.09bc
	8	百菌清	7.77±0.01b	72.89±0.04a
	9	多菌灵	7.85±0.02b	72.63±0.07ab
	10	H₂O	7.91±0.02b
	11	CK	28.67±0.61a
E：预防（菌悬液）	1	QS10-6	28.20±0.04a	1.65±0.13d
	2	QS2-5	8.31±0.38b	71.01±1.31c
	3	QS2-13	8.31±0.02b	71.00±0.07c
	4	HQS65	28.35±0.08a	1.10±0.26d
	5	HQS613	7.99±0.16bc	72.13±0.56bc
	6	HQS513	8.18±0.13bc	71.48±0.45c
	7	HQS6513	8.18±0.01bc	71.47±0.02c
	8	百菌清	7.64±0.08bc	73.34±0.29ab
	9	多菌灵	7.42±0.17c	74.13±0.58a
	10	H₂O	7.91±0.02bc
	11	CK	28.67±0.61a

图5 菌株QS10-6、QS2-5、QS2-13及组成的复合菌群的抑菌活性A：平板试验。a：CK；b：QS10-6；c：QS2-5；d：QS2-13；e：HQS65；f：HQS613；g：HQS513；h：HQS6513。B：菌株发酵液对马铃薯块茎（防治）；C：菌株菌悬液对马铃薯块茎（防治）；D：菌株发酵液对马铃薯块茎（预防）；E：菌株菌悬液对马铃薯块茎（预防）。a：CK；b：H2O；c：百菌清；d：多菌灵；e：HQS6513；f：QS10-6；g：QS2-5；h：QS2-13；i：HQS65；j：HQS613；k：HQS513

Fig. 5 Antibacterial activities of strains QS10-6, QS2-5, and QS2-13 and their composite floraA: Plate test. a: CK; b: QS10-6; c: QS2-5; d: QS2-13; e: HQS65; f: HQS613; g: HQS513; h: HQS6513. B: Strain fermentation solution on potato tubers(prevention and cure). C: Strain bacterial suspension on potato tubers (prevention and cure). D: Strain fermentation solution on potato tubers (guard against). E: Strain bacterial suspension on potato tubers (guard against). a: CK; b: H2O; c: Chlorothalonil; d: carbendazim; e: HQS6513; f: QS10-6; g: QS2-5; h: QS2-13; i: HQS65; j: HQS613; k: HQS513

图6 复合菌群HQS6513不同复配比例对马铃薯炭疽病的抑菌效果

Fig. 6 Bacteriostatic effects of different proportions of complex bacteria HQS6513 on potato anthracnose

图7 不同培养基组分及其浓度对复合菌群HQS6513生长和抑菌活性的影响柱状图：处理后复合菌群HQS6513发酵液的OD600；折线：处理后复合菌群HQS6513的抑菌率；图柱或折线上不同小写字母表示差异显著（P<0.05）。下同

Fig. 7 Effects of different medium components and their concentrations on the growth and antibacterial activity of complex flora HQS6513Column chart: OD600 of complex bacterial group HQS6513 fermentation solution after treatment. Broken line: Inhibition rate of complex bacteria HQS6513 after treatment. Different lowercase letters on the column or line indicate significant difference (P<0.05). The same below

表5 复合菌群HQS6513发酵培养基组分优化试验的因素和水平

Table 5 Factors and levels of optimized test for fermentation medium components of complex flora HQS6513

编号 Number	麸皮（A） Bran （A）	酵母浸粉（B） Yeast extract powder （B）	NaCl（C）	OD₆₀₀	抑菌率 Inhibition rate （%）
1	2.0	2.0	1.0	3.021	54.99
2	3.0	2.0	1.0	3.162	55.17
3	2.0	3.0	1.0	3.174	57.23
4	3.0	3.0	1.0	3.213	56.41
5	2.0	2.5	0.5	2.972	56.31
6	3.0	2.5	0.5	3.051	56.18
7	2.0	2.5	1.5	3.113	57.64
8	3.0	2.5	1.5	3.072	56.52
9	2.5	2.0	0.5	3.085	56.68
10	2.5	3.0	0.5	3.126	56.72
11	2.5	2.0	1.5	3.159	56.32
12	2.5	3.0	1.5	3.132	58.56
13	2.5	2.5	1.0	3.157	63.78
14	2.5	2.5	1.0	3.156	63.26
15	2.5	2.5	1.0	3.148	63.57
16	2.5	2.5	1.0	3.162	63.97
17	2.5	2.5	1.0	3.168	63.48

表6 复合菌群HQS6513培养基组分优化的回归模型显著性检验

Table 6 Significance test of regression model for component optimization of complex flora HQS6513 medium

方差来源 Variance source	平方和 Quadratic sum	自由度 Degree of freedom	均方 Mean square	F	P	显著性 Significance
模型 Model	185.59	9	20.62	272.09	<0.000 1	**
麸皮（A） Bran	0.446 5	1	0.446 5	5.89	0.0456	*
酵母浸粉（B） Yeast extract powder	4.15	1	4.15	54.72	0.0001	**
NaCl（C）	1.24	1	1.24	16.37	0.004 9	*
AB	0.250 0	1	0.25	3.3	0.112 2
AC	0.245 0	1	0.245	3.23	0.115 2
BC	1.21	1	1.21	15.97	0.005 2	*
A²	68.54	1	68.54	904.44	<0.000 1	**
B²	55.40	1	55.4	730.97	<0.000 1	**
C²	35.77	1	35.77	472.01	<0.000 1	**
残差 Residual	0.530 5	7	0.075 8
失拟项 Lack of fit	0.231 0	3	0.077	1.03	0.469 3
纯误差 Pure error	0.299 5	4	0.074 9
总误差 Cor total	186.12	16
R²=0.9971；R²_adj=0.9935；Adeq precision=39.7638

图8 不同培养基组分的交互作用对复合菌群HQS6513抑菌活性影响的等高线及响应面图A、D：麸皮与酵母浸粉交互作用；B、E：麸皮与NaCl交互作用；C、F：酵母浸粉与NaCl交互作用

Fig. 8 Effects of interaction of different medium components on antibacterial activity of complex flora HQS6513： Contour and response surfaceA, D: Interaction between bran and yeast extract powder. B, E: Interaction between bran and NaCl. C, F: Interaction between yeast extract powder and NaCl

图9 复合菌群HQS6513的最佳培养基组分

Fig. 9 Optimal medium component of complex flora HQS6513

图10 发酵条件对复合菌群HQS6513生长和抑菌活性的影响

Fig. 10 Effects of fermentation conditions on the growth and antibacterial activities of complex flora HQS6513

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