李生樟#, 陈颖#, 杨瑞环, 张翠萍, 刘昭, 李逸朗, 陈涛, 陈功友, 邹丽芳
上海交通大学农业与生物学院, 上海 200240
收稿日期：2018-11-14；修回日期：2019-01-24；网络出版日期：2019-03-14
基金项目：上海市科技兴农推广项目[沪农科推字（2016）第1-2-3号]；国家重点研发计划资助（2017YFD0200400）
*通信作者：邹丽芳, E-mail:zoulifang202018@sjtu.edu.cn.
#并列第一作者
摘要：[目的] 为了筛选防治水稻条斑病（bacterial leaf streak，BLS）的生防细菌。[方法] 以水稻条斑病菌（Xanthomonas oryzae pv.oryzicola，Xoc）的模式菌株RS105为靶标菌，采用平板稀释和抑菌圈法，从空心菜根际土壤中筛选到一株对RS105具有拮抗作用的细菌菌株504。通过形态学、生理生化特征以及16S rDNA和gyrA序列分析对菌株504进行了鉴定。利用牛津杯法测定504对植物病原黄单胞菌的拮抗活性及其无菌发酵液拮抗活性的稳定性。通过PCR扩增预测504编码合成脂肽类和聚酮类化合物的合成相关基因。采用苗期水稻注射接菌法来评价水稻组织中504对Xoc的拮抗活性。[结果] 菌株鉴定结果表明504为贝莱斯芽孢杆菌，命名为Bacillus velezensis 504。抑菌实验显示，B.velezensis 504对黄单胞菌属的细菌具有较好的抑菌活性，对水稻白叶枯病菌（X.oryzae pv.oryzae，Xoo）的拮抗效果最显著。基因预测结果显示，B.velezensis 504含有fenA、dhbA、sfrA、bmyA、beaS、dfnA及bacA等编码脂肽类和聚酮糖类抑菌化合物的基因簇。其无菌发酵液的活性物质耐高温和蛋白酶降解，但不耐强酸、强碱，在pH值为5.5-8.9时仍具有稳定的拮抗活性。在高感水稻品种原丰早上，B.velezensis 504对Xoc在水稻叶片中引起的水渍症状具有显著的抑制作用。[结论] B.velezensis 504能够特异性拮抗黄单胞菌，在黄单胞菌引起的细菌性病害的生物防治中将具有较大的应用潜力。
关键词：水稻条斑病菌贝莱斯芽孢杆菌生物防治拮抗活性
Isolation and identification of a Bacillus velezensis strain against plant pathogenic Xanthomonas spp.
Shengzhang Li#, Ying Chen#, Ruihuan Yang, Cuiping Zhang, Zhao Liu, Yilang Li, Tao Chen, Gongyou Chen, Lifang Zou
School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China
Received: 14 November 2018; Revised: 24 January 2019; Published online: 14 March 2019
*Corresponding author: Lifang Zou, E-mail:zoulifang202018@sjtu.edu.cn.
Foundation item: Supported by the Shanghai Agriculture Applied Technology Development Program, China (T2016010203) and by the National Key R&D Program of China (2017YFD0200400)
#Those authors contributed equally to this work
Abstract: [Objective] To obtain biocontrol bacteria to control bacterial leaf streak (BLS) of rice caused by Xanthomonas oryzae pv. oryzicola (Xoc). [Methods] A bacterial strain 504 with antibacterial activity was screened from water spinach rhizosphere soil by agar disk dilution and inhibition zone method using an Xoc model strain RS105 as target. Strain 504 was identified through morphological, physiological and biochemical characteristics in combination with 16S rDNA and gyrA sequence analysis. Antagonistic activity of 504 against some plant pathogenic Xanthomonas spp. was measured and stability of its sterile fermentation broth against Xoc was analyzed by Oxford Cup method. We predicted the genes encoding the synthesis of multiple lipopeptide and polyketide compounds by PCR amplification. Antibacterial activities of 504 against Xoc in the leaves of rice seedlings were evaluated by injection inoculation method. [Results] Strain 504 was identified as Bacillus velezensis, named B. velezensis 504. B. velezensis 504 showed specific inhibitory effects on some plant pathogenic Xanthomonas, especially on X. oryzae pv. oryzae. The secondary metabolites prediction showed that B. velezensis 504 contains genes such as fenA, dhbA, sfrA, bmyA, beaS, dfnA and bacA encoding the biosynthesis of various peptidoglycans and polyketose antibacterial compounds. The cell-free supernatant showed stable inhibitory effects against Xoc under high temperature and by protease treatment, however, there is no inhibitory activities when the pH value of supernatant is beyond 5.5 to 8.9. B. velezensis 504 exhibits significantly inhibitory effect on water-soaked lesions caused by Xoc in the leaves of the high susceptible rice cultivar Yuanfengzao. [Conclusion] B. velezensis 504 exhibits the broad-spectrum antagonistic activity against Xanthomonas spp., and has the potential as an effective biocontrol agent on diseases caused by plant pathogenic Xanthomonas spp..
Keywords: Xanthomonas oryzae pv. oryzicolaBacillus velezensisbiological controlantagonistic activity
稻黄单胞菌种下的稻生致病变种水稻条斑病菌(Xanthomonas oryzae pv. oryzicola，Xoc)侵染水稻引起细菌性条斑病(bacterial leaf streak，BLS)[1]。在我国南方水稻产区，条斑病已逐渐成为水稻上的第4大病害，每年造成水稻减产10%-30%，严重时达60%[2-3]。国内****发现，在已有的稻种资源中存在抗Xoc的水稻品种，未发现对Xoc具有完全免疫的水稻品种[4]。现阶段，国内的普栽水稻品种对于Xoc都表现为感病性，一些杂交水稻品种表现为高度感病性[5]；同时，也发现对水稻白叶枯病菌(X. oryzae pv. oryzae，Xoo)具有抗性的水稻品种对Xoc却不表现抗性。目前，水稻条斑病的防治主要依赖于化学药剂，如噻唑类杀菌剂[6]。化学药剂的大量使用不仅会破坏生态环境，并且会导致病原菌产生抗药性[7]。近年来，随着生物防治措施的应用和推广，筛选拮抗微生物来防治水稻条斑病也成为广泛关注的研究热点。
目前，应用最多的生防菌主要包括芽孢杆菌(Bacillus spp.)、假单胞杆菌(Pseudomonas spp.)、链霉菌(Streptomyces spp.)以及其他一些有益细菌[8]，其中芽孢杆菌的应用最广泛。芽孢杆菌广泛存在于空气、水和土壤等环境中，广泛应用于植物病害的生物防治，通过产生抑菌蛋白或抗菌肽等发挥其生防作用[9]。例如，枯草芽孢杆菌(Bacillus subtilis) F3能够产生一种抑制桃褐腐病菌(Monilinia fructicola)的抗菌蛋白[10]；蜡样芽孢杆菌(Bacillus cereus)能够对梨黑斑病菌(Altrnaria alternata)和柑橘绿霉菌(Penicillium digitatum)具有较强的抑菌活性[11]；张荣胜等利用解淀粉芽孢杆菌(Bacillus amyloliquefuciens)防治水稻条斑病，防效为60%以上[12]。
贝莱斯芽孢杆菌(Bacillus velezensis)作为一种新型的生防芽孢杆菌，由Ruiz-García等发现并命名[13]。已有的报道显示，贝莱斯芽孢杆菌能够对棉花黄萎病菌(Verticillium dahliae Kleb)[14]、白菜黑斑病菌(Alternaria brassicae)[15]、番茄灰霉病菌(Botrytis cinerea Pers)[16]、兰花枯萎病菌(Fusarium oxysporum f. sp. cattleyae)[17]、草莓枯萎病菌(Fusarium oxysporum f. sp. fragariae)[18]、莴苣根腐病菌(Pythium)[19]以及小麦全蚀病菌(Gaeumannomyces graminis var. tritic)[20]等多种植物病原菌具有拮抗作用。贝莱斯芽孢杆菌产生的次生代谢产物主要包括脂肽类和聚酮类化合物，脂肽类和聚酮类化合物的合成功能基因有srfAA、bmyB、ituC和fenD等[16]。目前，关于贝莱斯芽孢杆菌对Xoc具有拮抗作用的报道较少。
本研究从空心菜根际土壤中分离筛选到1株对Xoc具有显著拮抗作用的生防菌，鉴定为贝莱斯芽孢杆菌，命名B. velezensis 504；明确了该菌株对黄单胞菌具有特异的拮抗作用以及拮抗活性物质的稳定性；初步预测了该菌株的抗生素合成相关基因。这些研究为水稻条斑病的生物防治提供了新的微生物资源，为后续生防机理的探究奠定了理论基础。
1 材料和方法 1.1 菌株 贝莱斯芽孢杆菌B. velezensis 504从福建省三明市尤溪县西滨镇西洋村空心菜根际土壤中分离。供试的Xoo和Xoc菌株为本实验收集或者从各省水稻病样中分离；其他植物病原黄单胞菌为本实验室保存的菌株(表 1)，这些菌株置于NA和NB (NA broth)中培养，培养温度为28 ℃。
表 1. 实验菌株 Table 1. Strains used in this study
| Strains | Relevant characteristic | Source |
| B. velezensis 504 | Isolated from rhizosphere soil of spinach | This study |
| X. oryzae pv. oryzicola, Xoc | ||
| RS105 | Xoc wild type strain, Chinese race 2 | This lab |
| HNB07-3 | Xoc strain isolated from Hunan province | This lab |
| RS85 | Xoc strain isolated from Jiangsu province | This lab |
| HNB3-17 | Xoc strain isolated from Hunan province | This lab |
| HANB12-26 | Xoc strain isolated from Hainan province | This lab |
| ZJB01-25 | Xoc strain isolated from Zhejiang province | This lab |
| HANB1-19 | Xoc strain isolated from Hainan province | This lab |
| JSB1-39 | Xoc strain isolated from Jiangsu province | This lab |
| AHB3-7 | Xoc strain isolated from Anhui province | This lab |
| HNB8-47 | Xoc strain isolated from Hunan province | This lab |
| AHB1-58 | Xoc strain isolated from Anhui province | This lab |
| YNB01-3 | Xoc strain isolated from Yunnan province | This lab |
| X. oryzae pv. oryzae, Xoo | ||
| PXO99A | Xoo wild type strain, Philippine race 6 | [21] |
| YC2 | Xoo strain isolated from Yinchuan city | This lab |
| AH1 | Xoo strain isolated from Anhui province | This lab |
| YC6 | Xoo strain isolated from Yinchuan city | This lab |
| YC11 | Xoo strain isolated from Yinchuan city | This lab |
| YN04-1 | Xoo strain isolated from Yunnan province | This lab |
| LYG46 | Xoo strain isolated from Jiangsu province | This lab |
| JL3 | Xoo strain isolated from Jilin province | This lab |
| 8569 | Xoo strain isolated from Yinchuan city | This lab |
| YC18 | Xoo strain isolated from Yinchuan city | This lab |
| XZ35 | Xoo strain isolated from Tibet | This lab |
| YC7 | Xoo strain isolated from Yinchuan city | This lab |
| Xanthomonas campestris pv. juglandis | Causing black spot of walnut | This lab |
| Xanthomonas axonopodis pv. vignicola | Causing cowpea bacterial blight and pustule | This lab |
| Xanthomonas campestris pv. phaseoli | Causing bacterial blight of bean | This lab |
| Xanthomonas campestris pv. vesicatoria | Causing pepper spot disease | This lab |
| Xanthomonas campestris pv. malvacearum | Causing cotton bacterial angular leaf spot | This lab |
| Xanthomonas axonopodis pv. vasculorum | Causing sugarcane gummosis | This lab |
| Xanthomonas axonopodis pv. allii | Causing bacterial blight of onion | This lab |
| Xanthomonas campestris pv. musacearum | Causing banana bacterial wilt | This lab |
表选项
1.2 拮抗Xoc细菌的分离 利用五点采样法，从全国14个省或者自治区的蔬菜和大田作物根际收集了107份土样，从中筛选能够拮抗Xoc的细菌。每份土样称取10 g，置于三角锥形瓶中，加入90 mL灭菌水和少许钢珠，28 ℃、200 r/min振荡培养20 min，室温静置20 min，制成土壤菌悬液。吸取1 mL菌悬液，稀释成10-3、10-4和10-5 3个浓度梯度[22]。吸取100 μL菌液，均匀涂布在含有RS105菌液的NA培养基上，28 ℃培养24 h后，观察抑菌圈的形成，挑出对RS105具有抑菌活性的菌落，纯化为单菌落。单菌落接菌至NB培养液中，28 ℃、200 r/min振荡培养12-24 h，调整菌液浓度为OD600=2.0。利用牛津杯法，加入50 μL菌液，在含有RS105菌液的NA培养基上，进行抑菌活性的复筛实验，保存抑菌圈直径大于40 mm的菌落，进行后续菌株的鉴定。菌株504从编号为50号的土样中筛选获得，土样于2018年2月21日采集自福建省三明市尤溪县西滨镇西洋村空心菜根际土壤。
1.3 504菌株分类地位的鉴定
1.3.1 形态和生理生化特征的分析: 单菌落的504菌株送至中国典型培养物保藏中心(中国，武汉)进行形态学观察和生理生化特性的测定。根据《伯杰细菌鉴定手册》[23]和《常见细菌系统鉴定手册》[24]对菌株504产酸产气、柠檬酸利用、糖醇类发酵、色氨酸脱氨酶试验、吲哚试验等生理生化特性进行比较分析。
1.3.2 分子生物学的鉴定: 采用细菌基因组试剂盒(美基，中国广州)，提取菌株504的基因组DNA，以细菌16S rDNA基因的特异性引物27F (5′-AGAGTTTGATCCTGGCTCAG-3′)和1492R (5′-TACGGCTACCTTGTTACGACTT-3′)以及持家基因gyrA的引物GyrA-F (5′-CAGTCAGGA AATGCGTACGTCCTT-3′)和GyrA-R (5′-CAAGGT AATGCTCCAGGCATTGCT-3′)进行PCR扩增。PCR反应体系(20 μL)：1 μL模板DNA，2 μL primers (F/R)，2 μL dNTPs，10 μL buffer GC，0.2 μL Ex taq，2.8 μL ddH2O。PCR扩增条件为：95 ℃ 10 min，95 ℃ 30 s，56 ℃ 30 s，72 ℃ 90 s，35个循环；72 ℃ 10 min。PCR产物纯化后，送至铂尚生物技术(上海)有限公司进行测序分析。利用NCBI网站的BLAST功能对所测的16S rDNA和gyrA序列进行同源性分析，确定亲缘关系，最后使用MEGA 6.0软件的Neighbor-Joining法[25]构建系统发育树。
1.4 504菌株对黄单胞菌拮抗活性的分析 采用牛津杯法测定菌株504对12株水稻条斑病菌、12株水稻白叶枯病菌及8株其他黄单胞属病菌的拮抗效果。将各供试菌株单菌落分别接种于3 mL NB培养基中，28 ℃中培养24 h，调节菌液浓度为OD600=2.0。吸取200 μL的黄单胞菌液加入到冷却至45 ℃左右的NA培养基中，混合均匀。在平板的中央上放置无菌的牛津杯，向牛津杯中加入50 μL的504发酵液，每个处理设3个重复，以灭菌水为阴性对照，置于28 ℃培养24 h，测量和统计抑菌圈直径大小。数据用Excel 2010和SPSS 22.0软件进行统计分析。
1.5 水稻组织中菌株504对Xoc的抑制活性测定 高感Xoc的原丰早种植于上海交通大学水稻温室中，生长2周左右的水稻苗进行接种实验。供试菌株504和RS105单菌落接菌至5 mL的NB培养基中，28 ℃中培养24 h，菌株504的菌液浓度为OD600=1.0，RS105的菌液浓度为OD600=0.3。采用2种接种方式，观察和测定504对于RS105的抑制活性。治疗处理的接种法：利用无针头的注射器将RS105的菌液注射入水稻叶片中，2 h后再注射接种菌株504。预防处理的接种法：先利用无针头的注射器将504的菌液注射入水稻叶片中，2 h后再注射接种RS105菌液。每种方法接种大约30张叶片，连续7 d观察水渍状病斑的形成，统计病斑的扩展情况。原丰早由湖南农业科学院植物保护研究所肖友伦博士提供。7 d后测量条斑病斑的大小，按照公式(1)计算治疗处理和预防处理的抑菌效果。
 | 公式(1) |
1.7 抗生素相关基因PCR检测分析 根据McSpadden、Arguelles-Arias等[28-31]的方法设计了11对的抗生素相关基因的引物，15 μL PCR反应体系：1 μL模板DNA，1.5 μL primers (F/R)，1.5 μL dNTPs，7.5 μL buffer GC，0.15 μL Ex Taq，3.35 μL ddH2O。PCR扩增条件：95 ℃ 5 min；95 ℃ 45 s，58 ℃ 1 min，72 ℃ 2 min，35个循环；72 ℃ 10 min。取2 μL的PCR产物进行电泳，回收电泳条带，送至铂尚生物技术(上海)有限公司进行测序，测序结果在NCBI数据库中进行比对分析。
2 结果和分析 2.1 Xoc拮抗细菌的分离和鉴定 为了筛选能够抑制Xoc的细菌，从全国14个省或者自治区收集了107份土样，以Xoc的模式菌株RS105为指示菌，通过平板稀释涂布法和介质含菌法，从50份土样中分离得到66株具有明显拮抗效果的细菌菌株。进一步复筛发现，菌株504对RS105的拮抗作用非常明显，抑菌圈的平均直径可达40.66 mm (图 1-A)。在NA培养基上，菌株504形成乳白色菌落，表面粗糙，边缘不规则、不透明(图 1-B)。进行革兰氏染色，结果表明为阳性菌，短杆状，能够产生芽孢，芽孢为圆形或椭圆形(图 1-C)。酶活、碳源同化和碳源产酸等生理生化特性分析显示，菌株504可以产生H2S、乙酰甲基甲醇，能分泌明胶酶，不能产生吲哚(表 2)；可以发酵/氧化蔗糖，不能氧化葡萄糖、甘露醇、肌醇、山梨醇、鼠李糖、密二糖和苦杏仁苷(表 3)；能够利用甘油、葡萄糖、果糖、甘露糖、甘露醇、山梨醇等17种碳源(表 3)。根据《伯杰细菌鉴定手册》[23]和《常见细菌系统鉴定手册》[24]进行分析，这些生理生化特性与革兰氏阳性菌芽孢杆菌具有较大的相似性。
 |
| 图 1 菌株504的特征 Figure 1 Characteristics of the isolate 504. A: Antagonistic ability of the isolate 504 against the Xoc strain RS105; B: the colony form of 504 on NA medium; C: the morphology of 504 under microscope (1000×). |
| 图选项 |
表 2. 拮抗菌株504生理生化特性(酶活、碳源同化) Table 2. Physiological and biochemical characteristics of the isolate 504 (enzyme activity, carbon source) assimilation
| Substrate | Enzyme | Results |
| O-nitrobenzene-galactoside | β-galactosidase | - |
| Arginine | Arginine double hydrolase | - |
| Lysine | Lysine decarboxylase | - |
| Ornithine | Ornithine decarboxylase | - |
| Sodium citrate | Citric acid utilazation | - |
| Sodium thiosulfate | H2S production | + |
| Ure | Urease | - |
| Tryptophan | Tryptophan deaminase | - |
| Tryptophan | Indole production | - |
| Pyruvate | 3-hydroxybutanone produces acetylmethylmethanol | + |
| Kohn gelatin | Gelatinase | + |
| Glucose | Fermentation/Oxidation (4) | - |
| Mannitol | Fermentation/Oxidation (4) | - |
| Mannitol | Fermentation/Oxidation (4) | - |
| Sorbitol | Fermentation/Oxidation (4) | - |
| Rhamnose | Fermentation/Oxidation (4) | - |
| Sucrose | Fermentation/Oxidation (4) | + |
| Melibiose | Fermentation/Oxidation (4) | - |
| Amygdalin | Fermentation/Oxidation (4) | - |
| Arabinose | Fermentation/Oxidation (4) | - |
| +: positive reaction; -: negative reaction; W: weakly positive reaction. | ||
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表 3. 拮抗菌株504生理生化特性(利用碳源产酸) Table 3. Physiological and biochemical characteristics of the isolate 504 (acid production using carbon source)
| Subtrates | Results |
| CK | - |
| Glycerin | + |
| Erythritol | - |
| D-arabinose | - |
| L-arabinose | W |
| Ribose | W |
| D-xylose | - |
| L-xylose | - |
| Adonol | - |
| β-methyl-D-xylose | - |
| Galactose | - |
| Glucose | + |
| Fructose | + |
| Mannose | + |
| Sorbinose | - |
| Rhamnose | - |
| Dulcitol | - |
| Inositol | + |
| Mannitol | + |
| Sorbitol | + |
| α-methyl-D-mannose | - |
| α-methyl-D-glucoside | W |
| N-acetyl-glucosamine | - |
| Amygdalin | - |
| Arbutin | W |
| Esculin | + |
| Salicyl alcohol | + |
| Cellobiose | + |
| Maltose | + |
| Lactose | + |
| Melibiose | - |
| Sucrose | W |
| Trehalose | W |
| Inulin | - |
| Melezitose | - |
| Raffinose | - |
| Starch | - |
| Glycogen | - |
| Xylitol | - |
| Geraniol | - |
| D-turanose | - |
| D-lyxose | - |
| D-tagatose | - |
| D-fucose | - |
| L-fucose | - |
| D-arabitol | - |
| L-arabitol | - |
| Gluconate | - |
| 2-keto-gluconate | - |
| 5-keto-gluconate | - |
| +: positive reaction; -: negative reaction; W: weakly positive reaction. | |
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为了明确菌株504的分类地位，将504的16S rDNA序列在NCBI中进行BLAST分析，发现其与Bacillus velezensis CR-502 (AY603658)、Bacillus siamensis KCTC 13613 (AJVF01000043)、Bacillus nakamurai NRRLB-41091 (LSAZ01000028)、Bacillus amyloliquefaciens DSM7 (FN597644)、Bacillus subtilis subsp. subtilis NCIB 3610 (ABQL01000001)等菌株的16S rDNA同源性达到99.24%。系统发育树结果显示，504与Bacillus velezensis、Bacillus siamensis和Bacillus amyloliquefaciens处于同一个分支(图 2-A)。进一步利用gyrA基因序列在NCBI中进行BLAST分析，结果显示其与Bacillus velezensis的gyrA基因序列最为相似，同源性达98.91%。系统发育树结果也表明504与Bacillus velezensis处于同一个分支(图 2-B)。综合上述结果最终确定菌株504为Bacillus velezensis，命名为贝莱斯芽孢杆菌504 (B. velezensis 504)。
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| 图 2 菌株504系统发育树 Figure 2 Neighbour-joining phylogenetic trees based on 16S rRNA (A) and gyrA (B) gene sequences of strains 504. Numbers in parentheses represent the sequences accession number in GenBank. The number at each branch point is the percentage supported by bootstrap. Bar 0.5% sequence divergence. |
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2.2 B. velezensis 504拮抗植物病原黄单胞菌活性的测定 为了明确B. velezensis 504是否能够特异性拮抗水稻黄单胞菌，选取12株Xoc菌株和12株Xoo菌株进行拮抗活性的分析，发现B. velezensis 504对这些菌株都具有拮抗作用(图 3-A，3-B)，对Xoo菌株的拮抗作用明显强于Xoc菌株，特别是对来自银川市的8569和YC2菌株，抑菌直径分别为62.25 mm (图 3-B-1)和49.83 mm (图 3-B-2)。
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| 图 3 B. velezensis 504对黄单胞菌的拮抗谱 Figure 3 The antagonistic spectrum of B. velezensis 504 against Xanthomonas. Inhibition zones against different strains were shown. A: 1-12 are Xoc strains. 1: HNB07-3, 2: RS105, 3: RS85, 4: HNB3-17, 5: HANB12-26, 6: ZJB01-25, 7: HANB1-19, 8: JSB1-39, 9: AHB3-7, 10: HNB8-47, 11: AHB1-58, 12: YNB01-3. B: 1-12 are Xoo strains. 1: 8569, 2: YC2, 3: AH1, 4: YC6, 5: YC11, 6: YN041, 7: LYG46, 8: JL3, 9: PXO99A, 10: YC18, 11: XZ35, 12: YC7. C: 1-8 are other phytopathogenic Xanthomonas. 1: X. campestris pv. musacearum, 2: X. axonopodis pv. vignicola, 3: X. campestris pv. juglandis, 4: X. axonopodis pv. allii, 5: X. campestris pv. vesicatoria, 6: X. campestris pv. malvacearum, 7: X. campestris pv. phaseoli, 8: X. axonopodis pv. vasculorum. Black numbers denote the inhibition zone and SD (mm). |
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进一步选取8株黄单胞菌的细菌进行拮抗活性的分析，发现B. velezensis 504对香蕉细菌性青枯病菌(X. campestris pv. musacearum)、核桃细菌性黑斑病菌(X. campestris pv. juglandis)、豇豆细菌性疫病菌(X. axonopodis pv. vignicola)、洋葱细菌性叶枯病菌(X. axonopodis pv. allii)、辣椒斑点病菌(X. campestris pv. vesicatoria)和菜豆细菌性疫病菌(X. campestris pv. phaseoli)具有明显的拮抗作用；对棉花细菌性角斑病菌(X. campestris pv. malvacearum)和甘蔗流胶病菌(X. axonopodis pv. vasculorum)仅存在微弱的拮抗作用(图 3-C)。这些结果暗示，B. velezensis 504对大多数黄单胞菌属的细菌有明显的拮抗作用，对于Xoo的拮抗效果最显著。
2.3 水稻组织中B. velezensis 504对Xoc抑制效果的分析 为了测定B. velezensis 504对Xoc的抑制效果，利用高感Xoc的原丰早水稻品种，采用苗期注射接种法，在水稻叶片中同时接种B. velezensis 504和RS105菌株。接种2 d后，野生型菌株RS105在原丰早叶片上引起明显的水渍状病斑(图 4-A)，经过B. velezensis 504预防处理(504/RS105)和治疗处理(RS105/504)的原丰早叶片在第3天才展现微弱的水渍状病斑(图 4-A)。连续5-7 d的观察结果显示，RS105在注射了B. velezensis 504的原丰早叶片上引起的病斑明显变短(图 4-A和图 4-B)。预防处理的抑菌效果为75.95%，治疗处理的抑菌效果为72.65%。这表明B. velezensis 504中含有的抑菌活性物质在水稻组织中能够有效抑制Xoc的生长，限制水渍状病斑的扩展。
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| 图 4 盆栽试验检测B. velezensis 504对水稻条斑病菌的抑制活性 Figure 4 Effectors of B. velezensis 504 for suppression of rice bacterial leaf streak in pot under glasshouse conditions. The lesions length were observed at 2 d, 3 d, 5 d and 7 d after infiltration (A). Representative lesions on rice leaves (A) and lesions length at 7 d after infiltration (B) are shown. |
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2.4 B. velezensis 504抗生素合成相关基因的检测 利用11种芽孢杆菌中常有的抗生素合成相关基因的引物对B. velezensis 504的gDNA进行PCR扩增，结果显示(图 5)能够扩增到Fengycin合成相关基因fenA、Bacillibactin合成相关基因dhbA、Surfactin合成相关基因sfrA、BacillomycinD合成相关基因bmyA、Bacillaene合成相关基因beaS、Difficidin合成相关基因dfnA及Bacillysin合成相关基因bacA，但是没有检测到吩嗪、2, 4-二乙酰藤黄酚及藤黄绿菌素的合成基因phzFA、phlD、pltC。这表明B.velezensis 504的基因组含有Fengycin、Fengycin、Surfactin、BacillomycinD、Bacillaene、Difficidin及Bacillysin 7种脂肽类和聚酮类化合物。
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| 图 5 B. velezensis 504抗生素合成基因PCR凝胶电泳检测结果 Figure 5 Detection of antibiotics biosynthesis genes in B. velezensis 504 by PCR. M: DNA marker; 1: fenA; 2: dhbA; 3: ituC; 4: sfrA; 5: bmyA; 6: beaS; 7: dfnA; 8: phzFA; 9: bacA; 10: phlD; 11: pltC; 12: non-template control. |
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2.5 B. velezensis 504抑菌活性物质的特性分析 B. velezensis 504的上清发酵液经过细菌滤器后仍具有抑制Xoc的活性。将无菌发酵液经过40-100 ℃的7个不同温度处理，发现与常温(25 ℃)相比，不同高温处理对B. velezensis 504拮抗Xoc的活性无显著影响(图 6-A)。将无菌发酵液经过胃蛋白酶、胰蛋白酶和蛋白酶K的处理，也发现其拮抗Xoc的活性无明显影响(图 6-B)。无菌发酵液的pH值约为6.3，对其进行加酸、加碱的调节，当pH值为2.29、3.37和4.56以及pH值为10.15、11.19时，无菌发酵液丧失了拮抗Xoc的活性。上述结果表明，发酵液中的活性物质能够耐高温，具有热稳定性，可能不属于蛋白；发酵液的pH值偏酸或者偏碱都会影响活性物质的稳定性，理想发酵液的pH值应维持5.5-8.9 (图 6-C)。
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| 图 6 不同条件对B. velezensis 504无菌发酵液抑菌活性的影响 Figure 6 The inhibitory stability of the cell-free supernatant of B. velezensis 504 against Xoc RS105. The inhibitory effect of B. velezensis 504 on Xoc was detected under different temperature (A), proteases (B) and pH values (C). Columns marked with asterisks indicate significant differences (Student's t test, P < 0.01). |
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3 讨论 本研究从空心菜根际土壤中筛选到一株贝莱斯芽孢杆菌B. velezensis 504，该菌株对Xoo 和Xoc具有明显的拮抗活性，对其他一些植物病原黄单胞菌也具有较好的抑制效果，其发酵液能够耐高温和蛋白酶降解，为黄单胞菌引起的细菌病害的生物防治提供了新的微生物资源。
芽孢杆菌抗逆性较强，是植物病害的生物防治中应用最广泛的细菌[32]。本研究根据形态和生理生化特征将菌株504的分类地位鉴定为芽孢杆菌属。利用16S rDNA序列进行分析，发现其与贝莱斯芽孢杆菌(B. velezensis)、解淀粉芽孢杆菌(B. amyloliquefaciens)以及西姆芽孢杆菌(B. siamensis)的同源性达到99.24%，无法有效地确定菌株504的分类地位。进一步选取保守的gyrA序列进行分析，发现504与B. velezensis的相似性为98.91%；再结合能够产H2S的特性，将其分类地位确定为B. velezensis。B. velezensis是芽孢杆菌属的一个新种，最初被认为是枯草芽孢杆菌(B. subtilis)的一个亚种，与B. amyloliquefaciens的相似性也较高[33]。在利用分子技术进行鉴定时，除了考虑16S rDNA序列之外，同时也需要结合一些持家基因的同源性进行分析。也有研究显示，解淀粉芽孢杆菌(B. amyloliquefaciens)和甲基营养型芽孢杆菌(B. methylotrophicus)与B. velezensis具有相似的分子特性，可能是B. velezensis的同物异名菌[34]。
已有研究显示，B. velezensis能够促进植物生长、抗病虫和诱导植物的系统抗病性，特别对于病原真菌如大丽轮枝菌(Verticillium dahlia Kleb)、芸薹链格孢菌(Alternaria brassicae)具有明显的抑制作用，已成功应用于生产实践[35]。但是，关于B. velezensis对黄单胞菌特别是水稻黄单胞菌Xoo和Xoc具有拮抗活性的报道较少。本研究筛选到的B. velezensis 504可以抑制多种植物病原黄单胞菌，对于Xoo的抑制效果最明显(图 3)，将B. velezensis的抑制谱扩展到了植物病原黄单胞菌。张荣胜等曾利用解淀粉芽孢杆菌LX-11菌株来防治水稻条斑病，防治效果可达60.2%[12]。刘冰等也利用生防菌GN233来防治水稻条斑病，防效接近于化学农药[36]。本研究发现B. velezensis 504在水稻叶片中能够抑制Xoc的生长，限制条斑症状的扩展(图 4)。这暗示，B. velezensis 504可能也具有防治水稻条斑病的潜力，但是需要进一步田间生防实验的论证。
芽孢杆菌产生的抗菌活性物质主要包括脂肽类、聚酮类及抗菌蛋白等[35]。已有研究显示，B. velezensis含有的抗菌蛋白对棉花黄萎病菌具有较好的拮抗活性[14]；基因组中含有srfAA、srfAB、ituC、bmyB、bioA及fen等6种脂肽类抗生素相关基因[16]。与B. velezensis亲缘关系较近的B. amyloliquefaciens也对Xoo和Xoc具有拮抗活性，拮抗活性物质为Difficidin和Bacilysin [37]。本研究利用11种抗生素合成基因的引物对B. velezensis 504的基因组DNA进行PCR扩增，能检测到Fengycin、Bacillibactin、Surfactin、Bacillomycin D、Bacillaene等6种抗生素合成相关基因(图 5)。B. velezensis 504的无菌发酵液能够耐高温、蛋白酶降解，在pH值5.5-8.9都能够维持稳定的抑菌活性(图 6)。这暗示，B. velezensis 504拮抗黄单胞菌的活性物质可能为脂肽类物质，是否为Difficidin或Bacilysin，有待于进一步分析。
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