贾慧慧1, 谢心悦1, 潘园园2, 任争光1, 刘钢2,3,4
, 魏艳敏1 1. 北京农学院农业应用新技术北京市重点实验室, 植物生产国家级实验教学示范中心, 北京 102206;
2. 中国科学院微生物研究所真菌学国家重点实验室, 北京 100101;
3. 中国科学院大学, 北京 100049;
4. 中国科学院种子创新研究院, 北京 100864
收稿日期：2019-09-03；修回日期：2019-11-17；网络出版日期：2020-11-25
基金项目：北京市教委（科技创新）服务能力建设-科研计划（KM201710020007）
*通信作者：刘钢, Tel/Fax:+86-10-64806017, E-mail:liug@im.ac.cn;
魏艳敏, Tel/Fax:+86-10-80794280, E-mail:yanminwei@139.com.
摘要：[目的] 为了探究从杏树根际土壤分离的芽胞杆菌BJ-6的分类地位及其防病促生作用。[方法] 本研究测定了芽胞杆菌BJ-6的形态和生理生化特征，通过PCR扩增了该菌的16S rRNA、gyrA和gyrB基因并进行了序列测定，通过多基因聚类分析确定其分类地位，平板对峙法测定抗菌谱，盆栽幼苗实验验证其对甜瓜细菌性果斑病的防治效果和对甜瓜的促生作用。[结果] 结合形态特征、生理生化特性及多基因序列分析建立的系统进化树，确定菌株BJ-6为解淀粉芽胞杆菌（B.amyloliquefaciens），抑菌实验发现该菌株对15种植物病原菌均有不同程度的抑菌活性，盆栽实验结果发现该菌株发酵液对甜瓜细菌性果斑病有很好的防治效果，并对甜瓜苗有很好的促生作用。[结论] BJ-6属于解淀粉芽胞杆菌，抑菌谱广，且具有防病促生作用，具有进一步开发为生防制剂的前景。
关键词：芽胞杆菌鉴定甜瓜细菌性果斑病生物防治促生
Identification of Bacillus sp. BJ-6 for biocontrol of bacterial fruit blotch of melon
Huihui Jia1, Xinyue Xie1, Yuanyuan Pan2, Zhengguang Ren1, Gang Liu2,3,4
, Yanmin Wei1 1. Beijing Key Laboratory of New Technology in Agricultural Application, National Demonstration Center for Experimental Plant Production Education, Beijing University of Agriculture, Beijing 102206, China;
2. State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China;
3. University of Chinese Academy of Sciences, Beijing 100049, China;
4. The Innovative Academy of Seed Design, Chinese Academy of Sciences, Beijing 100864, China
Received: 3 September 2019; Revised: 17 November 2019; Published online: 25 November 2020
*Corresponding author: Gang Liu, Tel/Fax: +86-10-64806017, E-mail: liug@im.ac.cn;
Yanmin Wei, Tel/Fax: +86-10-80794280, E-mail: yanminwei@139.com.
Foundation item: Supported by the Research Project of Beijing Municipal Education Commission (KM201710020007)
Abstract: [Objective] To identify strain BJ-6 and explore its biological control function. [Methods] We identified the strain BJ-6 by morphological characteristics, physiological and biochemical tests and 16S rRNA, gyrA and gyrB gene sequence analysis. We measured the antimicrobial spectrum of BJ-6 by antagonistic experiment and studied the biocontrol effects and growth promotion on melon seedlings by pot experiment. [Results] We identified strain BJ-6 as B. amyloliquefaciens through the phylogenetic analysis of 16S rRNA, gyrA and gyrB sequences, and the characteristics of morphology, physiology and biochemistry. We found that BJ-6 strain had the bioactivity against 15 plant pathogens by the antagonistic experiment. The fermentation broth of BJ-6 had very good control efficiency to bacterial fruit blotch of melon seedlings in the pot experiment. Pot tests also showed that BJ-6 strain could promote the melon seedlings growth significantly. [Conclusion] We identified BJ-6 strain as B. amyloliquefaciens. Due to its broad spectrum of inhibiting plant pathogens and plant growth promoting ability, this stain had a good development value as biocontrol agents in plant protection.
Keywords: Bacillusidentificationbacterial fruit blotchbiocontrolgrowth promotion
芽胞杆菌(Bacillus sp.)是一类革兰氏阳性细菌，广泛分布在江河、海水、土壤等自然环境中，也可以作为内生菌生长于植物体内。芽胞杆菌在生长过程中能够产生大量的次级代谢产物，这些物质能够有效抑制植物病原真菌或细菌的生长，达到防病和促生的效果[1-4]。此外，多数芽胞杆菌具有对植物无致病性、对环境无污染、对人畜无害、自身抗逆性强、抑菌谱广等优点，因而成为了植物病害生物防治的良好材料[5-9]。甜瓜细菌性果斑病(bacterial fruit blotch，BFB)是由西瓜食酸菌(Acidovorax citrulli)引起的西瓜和甜瓜上的毁灭性病害，在我国各地瓜类产区均有不同程度的发生，造成了巨大经济损失[10-12]。目前甜瓜细菌性果斑病防治主要以化学防治为主，但是长期大量使用化学农药已经带来一些负面影响，如农药在果蔬上的残留问题、农药对生态环境的污染问题以及病原菌的耐药性和病害的再次发生等问题[13]。因此，开发和利用拮抗微生物防治甜瓜细菌性果斑病具有重要的意义。本文以北京市门头沟区杏树根际土壤分离到的一株芽胞杆菌BJ-6为对象，明确其分类地位、抑菌活性和防病促生效果。
1 材料和方法 1.1 供试材料 供试拮抗菌株：芽胞杆菌BJ-6分离自杏树根际土壤，北京农学院植物保护实验室保存。
供试植物病原菌：供试的15种常见植物病原菌由北京农学院植物病理实验室提供并保存。
供试甜瓜：“绿翠”薄皮甜瓜，由大绿种苗科技有限公司生产。
供试农药：72%农用硫酸链霉素可湿性粉剂，由华北制药股份有限公司生产。90%新植霉素可溶性粉剂，由石家庄三农化工有限公司生产。
1.2 供试培养基 PDA培养基：马铃薯200 g/L，葡萄糖20 g/L，琼脂20 g/L；用于植物病原真菌培养。
发酵培养基：麦芽糖20 g/L，蛋白胨10 g/L，麸皮10 g/L，CaCO3 10 g/L，Na2HPO4 2 g/L，KH2PO4 1 g/L；用于拮抗菌BJ-6的发酵培养。
LB培养基：酵母膏5 g/L，胰蛋白胨10 g/L，氯化钠5 g/L；用于植物病原细菌的培养。
1.3 细菌形态与生理生化特征鉴定 细菌形态观察和生理生化鉴定参考《常见细菌系统鉴定手册》[14]进行。
1.4 16S rDNA、gyrB和gyrA基因序列分析 菌株BJ-6的单个菌落在LB液体培养基中于28 ℃、180 r/min培养36 h，离心收集菌体，用细菌DNA提取试剂盒提取细菌DNA。以提取的总DNA为模板，分别用细菌16S rDNA扩增通用引物27F和1492R[3, 15]、gyrB基因扩增引物gyrb-up1f和gyrb-up2r[16]、gyrA基因扩增序列引物gyrA-F和gyrA-R[9]进行PCR扩增。PCR反应体系为25 μL：2×Taq PCR Master Mix 12.5 μL，总DNA模板1 μL，上下游引物各1 μL，ddH2O补充到25 μL。PCR反应：95 ℃ 5 min；95 ℃ 30 s，54 ℃ 30 s，72 ℃ 1 min，共34个循环；72 ℃ 10 min延伸。将扩增产物纯化后送到生工生物工程(上海)股份有限公司进行测序。将测序结果提交到NCBI (https://www.ncbi.nlm.nih.gov/)数据库中，进行BLAST比对分析。从NCBI中下载其他芽胞杆菌的相关基因序列，利用Clustral W软件和MEGA 5.0软件构建系统进化树。
1.5 菌株BJ-6的抑菌活性测定 采用平板对峙法[15]测定拮抗菌株BJ-6对植物病原真菌的抑菌活性。采用抑菌圈法[17]测定菌株BJ-6对植物病原细菌的抑菌活性。
1.6 芽胞杆菌BJ-6对甜瓜细菌性果斑病的防病效果 采用盆栽幼苗喷雾法实验[16]测定菌株的防病效果。
拮抗菌发酵液的制备：将活化好的BJ-6菌液按照1%的接菌量加到装有100 mL的发酵培养基中，28 ℃、180 r/min振荡培养3 d，得到菌株BJ-6的发酵液，菌液浓度约7×109 CFU/mL。
待测植物病原菌液的制备：将活化好的甜瓜细菌性果斑病菌MH21的菌液按照终浓度1%的接菌量加到含有100 mL的LB液体培养基中，28 ℃、180 r/min振荡培养3 d，调整菌液浓度至1×109 CFU/mL，喷雾接种时加入1%的吐温80。
甜瓜幼苗处理和接种：首先，采用喷雾法将清水、72%农用链霉素可湿性粉剂1000倍液、90%新植霉素可溶性粉剂1000倍液、稀释5倍的BJ-6带菌发酵液(菌液浓度1.4×109 CFU/mL)、稀释5倍的BJ-6灭菌发酵液以及稀释5倍的培养基6个处理均匀喷洒在幼苗上，每个处理重复3盆，每盆5棵苗。然后，用喷雾法接种甜瓜细菌性果斑病菌，最后，保湿光照培养。
检查结果：分别在第5天和7天观察甜瓜幼苗的发病情况，计算病叶率(公式1)、病情指数(公式2)和防治效果(公式3)。细菌性果斑病分级标准见表 1。
表 1. 甜瓜细菌性果斑病分级标准 Table 1. The grading standard of bacterial fruit blotch of melon
| Grade | The severity of symptom |
| 0 | No lesions on leaves |
| 1 | 0–5% lesion area in total leaves |
| 3 | 6%–10% lesion area in total leaves |
| 5 | 11%–25% lesion area in total leaves |
| 7 | 25%–50% lesion area in total leaves |
| 9 | More than 50% lesion area in total leaves |
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 | 公式(1) |
 | 公式(2) |
 | 公式(3) |
2 结果和分析 2.1 芽胞杆菌BJ-6的形态特征观察结果 菌株BJ-6在LB培养基上生长24–48 h后，菌落颜色呈浅黄色，菌落边缘略显乳白色，边缘不规则且不透明，菌落表面不光滑，有褶皱隆起(图 1-A)。单菌落中心也会有褶皱隆起，革兰氏染色呈阳性。在扫描电镜下观察菌体成杆状，芽孢表面不光滑，侧面有脊状突起，芽孢顶部有局部突起(图 1-B)。
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| 图 1 菌株BJ-6的菌落(A)和芽孢(B) Figure 1 The colony of stain BJ-6 (A) and its spore structure under scanning microscope (B) |
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2.2 芽胞杆菌BJ-6的生理生化特征 菌株BJ-6生理生化实验发现：接触酶试验、硝酸盐试验、明胶液化试验、VP试验均呈阳性；硫化氢试验、亚硝酸盐还原试验、纤维素试验、甲基红试验均呈阴性。菌株BJ-6在以硝酸铵、氯化铵、硫酸铵、或硝酸钠为唯一氮源的培养基中均能生长。BJ-6利用葡萄糖、肌醇、麦芽糖、蔗糖、乳糖、半乳糖和山梨醇的效果比木糖和甘露醇好。参考《常见细菌系统鉴定手册》，菌株BJ-6与解淀粉芽胞杆菌的特征基本相似。
2.3 基于16S rDNA、gyrA和gyrB的序列分析结果 将PCR扩增菌株BJ-6的16S rDNA、gyrA和gyrB基因序列提交到GenBank中(16S rRNA基因登录号MN272328，gyrA登录号MN295596，gyrB登录号MN295597)。通过BLAST软件比对分析发现，菌株BJ-6的16S rRNA基因扩增序列与NCBI中的芽胞杆菌属序列相似度最高，菌株BJ-6与该属下的几个模式种的序列相似度均在100%。gyrB和gyrA基因序列的比对结果显示，BJ-6菌株与解淀粉芽胞杆菌高度相似。通过串联16S rRNA、gyrB和gyrA基因序列，利用MEGA 5.0邻接法构建系统进化发育树，结果发现(图 2)，菌株BJ-6与解淀粉芽胞杆菌DSM7(FN597644.1)聚类在一支上。根据菌落形态特征及生理生化特征，对照《常见细菌系统鉴定手册》，并结合系统发育分析，初步确定菌株BJ-6为解淀粉芽胞杆菌(B. amyloliquefaciens)。
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| 图 2 基于16S rRNA、gyrA和gyrB多基因序列的聚类分析 Figure 2 Phylogenetic tree based on the sequences of 16S rRNA, gyrA and gyrB. GenBank accession numbers of the aligned sequences are shown in the brackets. The bootstrap values are shown at the node. Bar 0.05 means the nucleotide substitution rate of 0.05. T represents the sequence from the type strain |
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2.4 芽胞杆菌BJ-6的抑菌活性测定 芽胞杆菌BJ-6对供试的15种植物病原菌的抑菌活性测定结果见图 3和表 2。菌株BJ-6对15种植物病原菌均表现出不同程度的抑制活性，其中对葡萄炭疽病菌、苹果树皮腐烂病菌、桃树细菌性穿孔病菌、葡萄白腐病菌、甜瓜细菌性果斑病菌抑菌活性最强，抑菌带宽度分别达到1.27、0.90、0.75、0.59、0.50 cm；对葡萄溃疡病菌、草莓炭疽病菌、玉米大斑病菌、南瓜炭疽病菌、富克葡萄孢盘菌、辣椒疫病菌、果生炭疽病菌、黄瓜枯萎病菌抑菌活性稍差，抑菌带宽度分别为0.47、0.43、0.43、0.35、0.30、0.22、0.22、0.13 cm；对草莓枯萎病菌、草莓立枯病菌仅有微弱的抑菌活性。
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| 图 3 菌株BJ-6的抗菌活性 Figure 3 Antimicrobial activity of BJ-6 strain. A: Colletotrichum fragariae; B: Rhizoctonia solani; C: Colletotrichum fragariae; D: Botryosphaeria dothidea; E: Pilidiella diplodiella; F: Phytophthora capsici; G: Valsa mali; H:Fusarium oxysporum; I: Exserohilum turcicum; J: Botryotinia fuckelliana; K: Fusarium oxysporum; L: Colletotrchum fructicola; M: Colletotrichum orbiculare; N: Acidovorax citrull; O: Xanthomonas campestris |
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表 2. 菌株BJ-6对15种植物病原菌的抑制作用 Table 2. Inhibition activity of BJ-6 against 15 plant pathogens
| Diseases | Pathogen | Inhibition belt/cm |
| Grape anthracnose | Colletotrichum fragariae | 1.27±0.08 |
| Apple canker | Valsa mali | 0.90±0.25 |
| Peach perforation | Xanthomonas campestris | 0.75±0.05 |
| Grape white rot | Pilidiella diplodiella | 0.59±0.19 |
| Bacterial fruit blotch of melon | Acidovorax citrulli | 0.50±0.00 |
| Grape canker | Botryosphaeria dothidea | 0.47±0.06 |
| Strawberry anthracnose | Colletotrichum fragariae | 0.43±0.07 |
| Northern leaf blight of corn | Exserohilum turcicum | 0.43±0.07 |
| Pumpkin anthracnose | Colletotrichum orbiculare | 0.35±0.05 |
| Grape canker | Botryotinia fuckelliana | 0.30±0.10 |
| Phytophthora blight of pepper | Phytophthora capsici | 0.22±0.03 |
| Strawberry anthracnose | Colletotrchum fructicola | 0.22±0.11 |
| Cucumber fusarium wilt | Fusarium oxysporum | 0.13±0.03 |
| Strawberry fusarium wilt | Fusarium oxysporum | 0±0 |
| Strawberry seedling disease | Rhizoctonia solani | 0±0 |
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2.5 芽胞杆菌BJ-6对甜瓜细菌性果斑病的防病效果 采用盆栽喷雾接种的方法测定了菌株BJ-6对甜瓜细菌性果斑病的防治效果(图 4和表 3)。结果显示，甜瓜幼苗在接种病原细菌5 d后，处理组带菌发酵液、农用链霉素、新植霉素和灭菌发酵液的病叶率分别为10%、10%、15.63%、43.33%，病情指数分别为1.85、7.78、10.07、18.52，防治效果分别为95.84%、82.49%、77.34%、58.33%、水和培养基的对照组处理发病较重，病叶率分别为80%和70%，病情指数分别为44.44和47.04，由此可见BJ-6的带菌发酵液防效最好，达到了95%以上，高于农用链霉素和新植霉素。接种甜瓜细菌性果斑病菌7 d后，处理组带菌发酵液、农用链霉素、新植霉素和灭菌发酵液病情指数分别为9.62、7.78、24.55、26.30，防治效果分别为79.55%、83.46%、47.81%、44.09%。
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| 图 4 BJ-6菌株发酵液对盆栽甜瓜细菌性果斑病的防治效果 Figure 4 The biocontrol effect of BJ-6 fermentation broth on bacterial fruit blotch of melon seedlings. A: water; B: culture medium; C: fermentation liquid with bacteria; D: sterile fermentation liquid; E: 72% agricultural streptomycin (72% streptomycin sulfate); F: 90% Xinzhimycin (35% streptomycin sulfate and 55% oxytetracycline hydrochloride) |
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表 3. 菌株BJ-6发酵液对盆栽甜瓜细菌性果斑病的防治效果 Table 3. The biocontrol effect of BJ-6 fermentation broth on bacterial fruit blotch of sweet melon
| Treatments | 5 d | 7 d | |||||
| Incidence of diseased leaves/% | Disease index | Control effect/% | Incidence of diseased leaves/% | Disease index | Control effect/% | ||
| Water | 80.00 | 44.44 | 0.00 | 76.67 | 47.04 | 0.00 | |
| 5 times dilution of culture medium | 70.00 | 47.04 | –5.85 | 80.00 | 55.56 | –18.11 | |
| 5 times dilution of fermentation broth with bacteria | 10.00 | 1.85 | 95.84 | 23.33 | 9.62 | 79.55 | |
| 5 times dilution of sterile fermentation broth | 43.33 | 18.52 | 58.33 | 46.67 | 26.30 | 44.09 | |
| 1000 times dilution of 90% Xinzhimycin | 15.63 | 10.07 | 77.34 | 28.13 | 24.55 | 47.81 | |
| 1000 times dilution of 72% agricultural streptomycin | 10.00 | 7.78 | 82.49 | 10.00 | 7.78 | 83.46 | |
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2.6 室内盆栽甜瓜促生作用测定结果 室内盆栽测定结果表明，用BJ-6菌株发酵液灌根处理甜瓜对甜瓜生长具有显著的促生作用(表 4)。用1 mL的BJ-6发酵液灌根处理后，甜瓜植株的株高、茎粗、鲜重、叶片个数分别为20.64 cm、2.90 mm、1.73 g、4.80个，比对照分别增加了32.14%、7.41%、12.34%、14.29%。用5 mL的BJ-6发酵液灌根后，甜瓜植株的株高、茎粗、鲜重、叶片个数分别为25.16 cm、2.80 mm、1.75 g、5.25个，比对照分别增加61.08%、3.70%、13.64%、25.00%。
表 4. 菌株BJ-6发酵液对甜瓜苗生长的影响 Table 4. The effect of BJ-6 fermentation broth on the growth of melon seedlings
| Treatment | Stem length/cm | Stem dameter/mm | Fresh weight/g | Number of leaves |
| Water | 15.62 | 2.70 | 1.54 | 4.20 |
| 1 mL of fermentation broth with bacteria | 20.64 | 2.90 | 1.73 | 4.80 |
| 5 mL of fermentation broth with bacteria | 25.16 | 2.80 | 1.75 | 5.25 |
表选项
3 讨论 通过形态特征、生理生化指标以及基于16S rRNA、gyrA和gyrB序列的多基因聚类分析，初步将菌株BJ-6鉴定为解淀粉芽胞杆菌(B. amyloliquefaciens)。16S rRNA基因由于高度保守，因此仅能区别属水平上的细菌，无法区分亲缘关系较近的种间关系。gyrA和gyrB基因存在于所有细菌中，是编码DNA促旋酶A和B亚单位蛋白的基因，分子进化率要比16S rRNA基因大很多，且芽胞杆菌属不同种的gyrA和gyrB基因存在很大差异。通过gyrA和gyrB基因构建系统发育树，从而判断不同种之间的亲缘关系，是芽胞杆菌(Bacillus)的常用分类鉴定方法[18-19]。因此，本实验采用多基因聚类对16S rRNA、gyrA和gyrB三个基因进行了分析。抑菌活性测定发现，菌株BJ-6对供试的绝大部分植物病原菌都有不同程度的抑菌活性，其中对葡萄炭疽病菌、苹果树皮腐烂病菌、桃树细菌性穿孔病菌、葡萄白腐病菌、甜瓜细菌性果斑病菌抑菌活性最强。菌株BJ-6对甜瓜细菌性果斑病的盆栽防病效果发现，BJ-6带菌发酵液防病效果较好，接种5 d后防病效果高于农用链霉素、新植霉素和灭菌发酵液。而灭菌发酵液对甜瓜细菌性果斑病仍有一定防病作用，推测BJ-6发酵液中有某些抗菌物质起到了抑制病原菌的作用。带菌发酵液防效优于灭菌发酵液，说明可能还有其他生防机理存在，比如生防菌占据生态位点，与病原菌竞争营养和空间位点，使得病原菌不能侵染植物等；或者生防菌在植物表面存活并且产生次级代谢产物，破坏病原菌的细胞结构和改变生活环境，使得病原菌侵染效果下降；还有可能是生防菌诱导植物产生抗病性和激活植物防卫反应，从而抑制有害微生物的生长[20-23]。无菌发酵液对甜瓜细菌性果斑病有很好的防治作用，证实了BJ-6能够产生一些抗菌物质，这些抗菌物质能够外泌到培养基中[24]，这类物质应该包括脂肽类物质[25-26]、聚酮类物质、胞外酶等物质，为此下一步将研究BJ-6的最佳发酵条件，更好地利用BJ-6产生的次级代谢产物。总之，菌株BJ-6抑菌谱广，抑菌活性强，具有良好的防病促生效果，具有一定的开发利用潜能。
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