马铃薯是世界第四大粮食作物，对保障粮食安全具有重要作用。马铃薯青枯病是由青枯雷尔氏菌（Ralstonia solanacearum E. F. Smith，原名Pseudomonas solanacearum）引起的一种严重土传病害，其危害程度仅次于马铃薯晚疫病，主要分布在热带、亚热带和温带地区，在我国南方各省市均有严重发生[1⇓-3]。青枯菌的寄主范围非常广泛，除危害马铃薯外，也可侵染包括烟草、番茄和花生在内的50多个科的数百种植物，其中以茄科植物受害最为严重[4]。部分高发青枯病地区田块的发病率达到100%，每年造成10%~15%的产量损失，发病严重的地块减产可达80%，甚至绝收，严重威胁着各地马铃薯产业的健康发展[5-6]。
植物根际是一个独特的土壤区域，包含众多种类的微生物及腐殖质等物质，农作物、根际微环境和根际微生物之间相互依赖，相互影响，共同维持农业生态系统平衡[10]。根际微生物是提供这些生态系统服务的关键因素，在植物抗病过程中起着不可或缺的作用，根际土壤中的有益微生物对植物土传病害的生物防治具有重要意义[11⇓⇓-14]。目前，与植物病害抑制相关的微生物大多为链霉菌属（Streptomyces）[15-16]、假单胞菌属（Pseudomonas）[17⇓-19]、芽孢杆菌属（Bacillus）[20⇓-22]和噬菌体等[23]。目前，国内外报道的防治青枯病的研究主要针对番茄、辣椒和烟草，拮抗菌主要为芽孢杆菌和假单胞菌，针对马铃薯青枯病生物防治的研究相对较少。邱清华等[24]筛选出的假单胞杆菌对马铃薯和烟草青枯病菌都具有较强的拮抗力。Kheirandish等[25]从马铃薯根际土壤中分离出的恶臭假单胞菌Pp17对马铃薯青枯菌表现出了较强的拮抗活性，温室盆栽防治效果可达51.50%。青枯菌生理小种具有复杂的遗传多样性，不同来源青枯菌对不同植物的致病性有较大差异，筛选广谱高效的拮抗菌对于作物青枯病的防治具有重要意义。本研究从多地采集健康马铃薯、番茄、茄子及辣椒根际土壤，分离筛选拮抗马铃薯、番茄和烟草青枯菌的菌株，并用温室盆栽防病试验检测拮抗菌防治马铃薯青枯病的效果，为马铃薯青枯病生物防控提供新的有效拮抗菌株，为防治马铃薯青枯病提供菌种资源及理论依据。
1 材料与方法
1.1 供试材料
供试青枯菌菌种：青枯菌[P.solanacearum （Smith）] M1.12分离自马铃薯；青枯菌（R. solanacearum）TY2和FJAT-91分离自烟草和番茄，菌株均购于宁波明舟生物科技有限公司。
供试马铃薯品种“陇薯7号”和“大西洋”购自甘肃省定西市“钰妹农场”。
供试土壤样品采自海南、宁夏、四川的健康马铃薯、番茄、茄子及辣椒根际土壤。
1.2 拮抗菌的分离与筛选
1.2.1 初筛
将青枯菌接种于液体NA培养基（10 g/L葡萄糖、3 g/L牛肉膏、0.5 g/L酵母粉、5 g/L蛋白胨，pH 7.0）中，30 ℃、200转/min振荡培养48 h，调整菌液OD600nm=5.0，将青枯菌按1:50加入到约50 °C未凝固的NA固体培养基中混合均匀，制成含菌平板，备用。
称取1 g供试土壤倒入装有20 mL无菌水和玻璃珠的三角瓶中，剧烈振荡10 min，静置30 min。取上清液以10倍梯度稀释，取10-3~10-5稀释度的上清液各150 μL涂布至含青枯菌的NA平板上，每个梯度3次重复。30 °C培养48 h，菌落周围出现透明抑菌圈的为拮抗菌株。挑选有抑菌圈的菌落划线纯化培养得到初筛菌株，4 °C保存备用。
1.2.2 复筛
采用1.2.1中相同方法制备含病原菌平板，用牛津杯打孔法（Φ=6 mm）进行平板抑菌试验。每孔分别注入50 μL不同初筛菌株的培养液（OD600nm=5.0），每个处理重复3次，30 °C培养48 h后根据拮抗圈大小得到复筛菌株。
1.3 拮抗菌16S rRNA基因及基因间隔区序列鉴定
采用试剂盒提取拮抗菌基因组DNA，以16S rRNA通用引物27F和1492R，16S rRNA基因间隔区（ITS序列）引物1405f（5′-TGCACACACCGCC CGT-3′）和456r（5′-CCTTTCCCTCACGGTACTG- 3′）进行PCR扩增。扩增程序为95 °C 3 min；95 °C 15 s，55 °C 15 s，72 °C 30 s，30次循环；72 °C5 min。PCR产物经电泳检测后直接测序，或电泳后回收扩增产物连接到pMD19-T载体，转化大肠杆菌，筛选阳性转化子测序，根据测序结果在NCBI核酸数据库上采用Blast软件进行比对确定拮抗菌种属。
1.4 拮抗菌抑菌广谱性
参照1.2.1和1.2.2的方法分别制备番茄青枯菌和烟草青枯菌平板，病原菌平板每孔分别注入不同拮抗菌株的培养液50 μL（OD600nm=5.0），每个处理重复3次，30 °C培养48 h，观察记录抑菌圈，采用十字交叉法测量其大小，每个试验重复3次。
1.5 拮抗菌产相关抑菌物质分析
1.5.1 产蛋白酶能力检测
用直径5 mm的无菌打孔器在含1%牛奶的LB固体平板上打孔，每孔注入20 μL拮抗菌菌液（OD600nm=5.0），30 °C培养48 h，观察菌落周围有无透明圈产生来判断菌株产胞外蛋白酶能力。
1.5.2 产铁载体能力检测
采用熊汉琴[26]报道的方法进行产铁载体检测，将20 μL拮抗菌菌液（OD600nm=5.0）与等体积铬天青显色液混合均匀后室温显色5 min，若颜色由青蓝色变为紫红色，则说明有铁载体产生，同时设置空白试验对照。
1.5.3 生物膜形成能力检测
利用结晶紫染色法测定生物膜，采用Zhou等[27]的方法并简单修改。将待测菌株活化后按10%转种量转接于2 mL液体NA培养基，30 °C或37 °C静置培养48 h，弃菌液后用无菌生理盐水轻洗一遍试管，加入1 mL 1%的结晶紫染色15 min，用无菌水将多余染液洗净，若在管内壁的空气与培养基的接触面上形成一道清楚的紫色环痕，即说明该菌株产生物膜。
1.6 盆栽防效试验
1.6.1 盆栽试验设计
表1 盆栽防治效果试验设计
Table 1
| 处理Treatment | 添加物（盆）Supplement (pot) |
|---|---|
| R | 20 mL无菌水+10 mL青枯菌菌液 |
| FR | 20 mL拮抗菌菌液+10 mL青枯菌菌液 |
| F | 20 mL拮抗菌菌液 |
| CK | 30 mL无菌水 |
每盆2~5株马铃薯苗，每个处理设9个（陇薯7号）或5个重复（大西洋）。
1.6.2 防治效果试验判定标准
病情指数（DI）=[∑（每个病级的植株数×级别数）/（调查总植株数×最高级别数）]×100；
防治效果（BE，%）=[（R的病情指数-试验处理的病情指数）/R的病情指数]×100。
1.7 数据处理
采用Microsoft Excel 2010软件进行数据统计处理，釆用SPSS 18.0软件进行数据分析，用Duncan法进行差异显著性多重比较（P<0.05）。
2 结果与分析
2.1 拮抗菌的分离筛选及其抑菌广谱性
图1
图1
拮抗菌对3株青枯菌的抑制作用
(a)~(c) 培养基里依次包埋马铃薯、番茄和烟草青枯菌，样品孔里为不同拮抗菌的菌液50 μL（OD600nm=5.0）。
Fig.1
Inhibitory effect of antagonistic bacteria against three strains of R.solanacearum
Agar medium (a)-(c) were embedded with R. solanacearum strains from potato, tomato and tobacco, respectively. The loading volume of antagonistic bacteria was 50 μL (OD600nm=5.0) per well.
表2 6株拮抗菌对3种青枯菌的抑菌圈直径
Table 2
| 菌株名称 Strain name | 青枯菌来源Source of R. solanacearum | ||
|---|---|---|---|
| 马铃薯Potato | 番茄Tomato | 烟草Tobacco | |
| XC-1 | 4.40±0.74c | 3.91±0.30d | 4.38±0.74d |
| XC-2 | 7.38±0.98b | 9.41±0.45b | 9.03±0.59b |
| FC-11 | 6.61±0.34b | 5.54±0.82c | 6.64±0.34c |
| FC-17 | 6.90±0.79b | 8.26±0.62b | 8.41±0.34b |
| HN-9 | 7.11±0.91b | 8.83±0.80b | 8.96±0.81b |
| NX-18 | 10.14±0.74a | 11.98±0.80a | 10.70±0.53a |
不同小写字母表示差异显著（P < 0.05）。
Different lowercase letters indicate significant differences (P < 0.05).
2.2 拮抗菌的分子鉴定及形态特征
对分离到的6株拮抗菌提取基因组DNA，分别扩增16S rRNA基因序列和基因间隔区（ITS）序列，电泳（图2）显示目的基因片段分别在1450和1000 bp左右。根据16S rRNA基因序列及ITS序列比对结果，判断菌株XC-1为Pseudomonas palleroniana，菌株XC-2和HN-9为Lysinibacillus fusiformis；菌株FC-11为Kosakonia cowanii，菌株FC-17为Pseudomonas frederiksbergensis；菌株NX-18为Lysinibacillus sphaericus，6株菌与各参比菌株的16S rRNA相似性均高于99.5%，ITS序列相似性均高于98.5%。菌株XC-2、HN-9和NX-18为革兰氏阳性菌，菌株XC-1、FC-11和FC-17为革兰氏阴性菌。6株菌在NA平板上的菌落形态如图3所示，菌株XC-2和HN-9菌落具有较强的粘附性；菌株FC-17菌落呈浅黄色；菌株NX-18菌落呈微黄色。
图2
图2
拮抗菌16S rRNA基因及基因间隔区PCR扩增产物电泳图
(a)为16S rRNA基因扩增产物电泳图，M为λ-Eco I T-14 digest；(b)为16S至23S基因间隔区扩增产物电泳图，M为1kb plus DNA Ladder。1~6分别为菌株XC-1、XC-2、FC-11、FC-17、HN-9和NX-18。
Fig.2
Electrophoresis of PCR products amplified from antagonistic bacteria 16S rRNA and internal transcribed spacer
(a): Amplification of 16S rRNA, M represents λ-Eco I T-14 digest marker; (b): Amplification of internal transcribed spacer between 16S and 23S rRNA, M represents 1 kb plus DNA Ladder; 1-6 represent XC-1, XC-2, FC-11, FC-17, HN-9 and NX-18, respectively.
图3
图3
拮抗菌菌落形态
菌株划线后在30 ℃静置培养48 h。
Fig.3
Colony morphology of antagonistic bacteria
Strains were incubated at 30 ℃ for 48 h after streaking.
2.3 拮抗菌产相关抑菌物质分析
拮抗菌可通过分泌次生代谢产物、抗菌肽、铁载体、防御酶、蛋白酶等物质直接作用于病原菌产生拮抗作用。本试验对拮抗菌进行了蛋白酶、铁载体及生物膜形成能力检测，以初步分析不同拮抗菌的可能作用机制。蛋白酶测定（图4a）显示，菌株XC-1、XC-2和HN-9周围出现较大透明水解圈，表明3株菌均产蛋白酶，其中XC-1菌株产蛋白酶能力最强。铁载体试验中，XC-1、FC-11和FC-17菌株均使铬天青染液由蓝色变为紫红色，表明能产生铁载体，其中FC-17菌株产铁载体能力最强（图4b）；产生物膜试验中，菌株XC-1和FC-11的试管壁上出现紫色环痕，表明其具有形成生物膜的能力（图4c）。6株拮抗菌中，菌株XC-1同时具有产蛋白酶、铁载体和生物膜的3种能力；菌株FC-11具有产铁载体及生物膜的2种能力；菌株XC-2和HN-9只产蛋白酶；菌株FC-17只具有产铁载体的能力。菌株NX-18不具有试验所检测的3种拮抗物质，可能通过其他机制发挥拮抗作用。
图4
图4
拮抗菌抑菌物质的测定
(a) 蛋白酶；(b) 铁载体；(c) 生物膜；“+”：短小芽孢杆菌SCU11，“-”：大肠杆菌MG1655，CK：空白对照。
Fig.4
Determination of antibacterial substances of antagonistic bacteria
(a) protease; (b) siderophore; (c) biofilm.“+”: Bacillus pumilus SCU11,“-”: Escherichia coli MG1655, CK: blank control.
2.4 温室盆栽防效试验
马铃薯青枯菌拮抗试验结果显示，拮抗活性较好的菌株为XC-2、FC-17、HN-9和NX-18；其中XC-2和HN-9菌株具有产蛋白酶能力，FC-17具有较好的产铁载体能力，NX-18菌株的抑菌物质暂不明确。综合考虑拮抗效果及拮抗机制，选用XC-2和FC-17进行盆栽防效预试验，预试验中FC-17表现出更好的生防效果，因此进行盆栽试验。
图5
图5
拮抗菌FC-17对马铃薯青枯病的防治作用
(a)和(b)分别为“陇薯7号”和“大西洋”马铃薯，均拍摄于播种后45 d，最右侧为患病叶特写。
Fig.5
Effects of FC-17 on the suppression of potato bacterial wilt
(a) and (b) were Longshu 7 and Atlantic, respectively, pictures were taken at 45 days after seeding, and the enlargement of infected leaf is shown on the right of the picture.
表3 盆栽试验FC-17对马铃薯青枯病的防治效果统计
Table 3
| 马铃薯品种 Potato cultivar | 处理 Treatment | 病情指数 Disease index | 防治效果 Control effect (%) |
|---|---|---|---|
| 陇薯7号 Longshu 7 | R | 45.37±18.49 | － |
| FR | 9.52±16.49* | 79.46 | |
| F | － | － | |
| CK | － | － | |
| 大西洋 Atlantic | R | 52.68±16.04 | － |
| FR | 15.18±15.64* | 70.83 | |
| F | － | － | |
| CK | － | － |
“*”表示同一品种FR和R处理的发病率在P < 0.05水平有显著差异。
“*” indicates significant differences between FR and R treatments at P < 0.05 level.
3 讨论
青枯病是世界范围内的毁灭性细菌病害，其预防和控制一直是研究的热点和难点，生物防治菌株的应用为其防治提供了一条重要的解决途径。各国学者一直在从事针对马铃薯青枯病生防菌的研究，虽取得了一定的进展，但马铃薯青枯病生物防治菌种资源仍相对较少。
假单胞菌是一类根际有益菌，在植物根围繁殖能力强，具有杀虫、改善植物营养、降解有毒物质、产生抗生素和植物生长调节物质、改善植物微环境和诱导系统抗性等生物防治作用。相关研究[30⇓⇓-33]发现，P. frederiksbergensis还具有解磷作用，该菌能够将土壤中的不溶性磷源溶解成为可溶性磷，并显著促进植物生长。Weller[34]认为，假单胞菌能够作为生防微生物，主要有以下几个优点：（1）生长速度快，适合大量发酵生产；（2）能迅速利用种子和根系分泌物；（3）不管是土壤习居还是植物内生假单胞菌，都有很强的定殖和繁殖能力；（4）多数假单胞菌能合成有益次生代谢物，如抗生素、铁载体及其他促生物质；（5）适应环境压力能力强，假单胞菌可优先利用根分泌物和根际土壤中的养料，获得竞争优势，占据根际生态位，减少病原菌生长所必需的营养物质，从而抑制病原菌生长[35]。国内外报道的用于防治青枯病的假单胞菌较多，Anuratha等[36]利用荧光假单胞菌进行烟草盆栽试验取得了良好的防治效果，推测假单胞菌产生的抗生素和噬铁素对烟草青枯病有较好的抑制作用。Ramesh等[37]从稻谷、茄子和秋葵等多种作物的根际土壤中分离筛选出18株对茄子青枯菌有较好拮抗活性的假单胞菌。魏春妹等[38]报道在温室条件下，假单胞菌B4-2-2对番茄青枯病的生物防治效果可达70%以上。除假单胞菌可用于马铃薯青枯病的防治外，芽孢杆菌对青枯病也具有良好的防治效果。徐进等[39]用筛选的2株枯草芽孢杆菌制成生物防治菌剂不仅可以控制马铃薯青枯病的发生，而且对马铃薯有促生增产作用。丁传雨[40]从健康植株根际土壤中分离出的解淀粉芽孢杆菌LH23和枯草芽孢杆菌LH36对马铃薯青枯菌也有较好拮抗作用，温室盆栽防治效果分别可达53.9%和50.0%。郑新艳[41]将分离得到的芽孢杆菌T3制备生物有机肥，温室防控效果高达79.44%。
假单胞菌是一种重要的生防菌，本试验中拮抗菌FC-17是从土壤中分离筛选出的假单胞菌，温室防效可达70.83%和79.46%，有效地控制了青枯病的发生。本试验仅在温室条件下对拮抗菌进行了防治效果验证，拮抗菌的田间应用会受降水、气温和土壤酸碱度等诸多因素影响，因此与室内防治效果可能存在差异。后续要应用于田间试验，需要进一步优化菌株发酵条件，确定其营养载体、配施药剂、田间控病效果、最佳施用次数和时期等。假单胞菌FC-17在马铃薯土传病害防控中的应用潜力还需要通过研发微生物制剂并开展大田防治效果试验加以验证。
4 结论
本研究共筛选得到6株对马铃薯青枯菌有抑制效果的拮抗菌，它们对分离自番茄和烟草的青枯菌也具有较好的抑制活性。采用拮抗菌假单胞菌FC-17进行马铃薯青枯病的生物防治效果试验，结果表明其对“陇薯7号”和“大西洋”的青枯病生防效率分别达到79.46%和70.83%。
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