2023, Vol. 63
表1(Table 1)
柞蚕空胴病病原拮抗菌的分离、鉴定及防治效果研究
http://dx.doi.org/10.13343/j.cnki.wsxb.20220422
中国科学院微生物研究所，中国微生物学会
中国科学院微生物研究所，中国微生物学会
文章信息
- 贾姝, 张俊涛, 李喜升, 赫英姿, 于庭洪, 赵翀, 宋策. 2023
- JIA Shu, ZHANG Juntao, LI Xisheng, HE Yingzi, YU Tinghong, ZHAO Chong, SONG Ce.
- 柞蚕空胴病病原拮抗菌的分离、鉴定及防治效果研究
- Screening, identification, and biocontrol effect of bacteria against empty-gut disease of Antheraea pernyi
- 微生物学报, 63(2): 670-682
- Acta Microbiologica Sinica, 63(2): 670-682
-
文章历史
- 收稿日期：2022-06-05
- 网络出版日期：2022-08-04
引用本文 |
贾姝, 张俊涛, 李喜升, 赫英姿, 于庭洪, 赵翀, 宋策. 柞蚕空胴病病原拮抗菌的分离、鉴定及防治效果研究[J]. 微生物学报, 2023, 63(2): 670-682.
Jia Shu, Zhang Juntao, Li Xisheng, He Yingzi, Yu Tinghong, Zhao Chong, Song Ce. Screening, identification, and biocontrol effect of bacteria against empty-gut disease of Antheraea pernyi[J]. Acta Microbiologica Sinica, 2023, 63(2): 670-682.
柞蚕空胴病病原拮抗菌的分离、鉴定及防治效果研究
辽宁省蚕业科学研究所, 辽宁 凤城 118100
摘要：[目的] 本研究从辽宁省蚕业科学研究所柞园土壤中分离筛选对柞蚕空胴病病原菌有显著拮抗作用的细菌，为该病的生物防治奠定研究基础。[方法] 采用稀释涂布平板法分离柞园土壤中细菌，利用抑菌圈法筛选拮抗效果显著的菌株；根据形态学、生理生化及分子生物学对拮抗菌进行鉴定；利用自然转化法对拮抗菌进行荧光蛋白标记，测定其在柞树叶片和柞蚕肠道内的定殖规律，并对其室内和野外防效进行测定。[结果] 从柞园土壤中分离获得87株细菌，其中BF-49对柞蚕肠球菌的拮抗效果显著(P < 0.001)；鉴定结果显示该BF-49与贝莱斯芽孢杆菌(Bacillus velezensis)的亲缘关系最近，故将该菌株鉴定为Bacillus velezensis，命名为B. velezensis BF-49。荧光蛋白标记菌株BF-49-GFP在柞蚕肠道中能定殖5 d，在柞树叶片上接种20 d后浓度仍达1.25×104 CFU/g。BF-49发酵液的10倍稀释液对柞蚕空胴病的室内防效为78.25%，野外防效为74.42%，均显著高于对照药剂。[结论] 筛选获得的B. velezensis BF-49对柞蚕空胴病防效显著，可作为开发柞蚕空胴病生防制剂的候选菌株。
关键词：柞蚕肠球菌 拮抗菌 鉴定 生防效果
Screening, identification, and biocontrol effect of bacteria against empty-gut disease of Antheraea pernyi
Sericultural Research Institute of Liaoning Province, Fengcheng 118100, Liaoning, China
Abstract: [Objective] To isolate and screen bacteria against Enterococcus pernyi (the pathogen of Antheraea pernyi empty-gut disease) from the oak garden soil of Sericultural Research Institute of Liaoning Province and thus to lay a foundation for the biocontrol of the disease. [Methods] Bacteria were isolated from soil by spread plate technique and the antagonistic strain was screened out with the inhibition zone method. The strain was identified according to morphological features, physiological and biochemical characteristics, and molecular biology. The antagonistic strain was labeled with green fluorescent protein with natural transformation method, and the colonization of the antagonistic strain in oak leaves and gut of A. pernyi was determined. The control of empty-gut disease of A. pernyi was identified by indoor and field experiments. [Results] A total of 87 bacterial strains were isolated from soil. Among them, BF-49 was found to have significant antagonistic activity against E. pernyi. The identification results revealed that BF-49 had the closest genetic relationship with Bacillus velezensis. Therefore, it was identified as B. velezensis and named B. velezensis BF-49. The fluorescence-labeled strain BF-49-GFP could colonize in the gut of A. pernyi for 5 days, and the count of BF-49-GFP reached 1.25×104 CFU/g 20 days after inoculation on the oak leaves. The indoor and field control of empty-gut disease by the 10-fold diluted BF-49 fermentation broth was 78.25% and 74.42%, respectively, which were significantly higher than those of the control agents. [Conclusion] The B. velezensis BF-49 has significant control effect on empty-gut disease of A. pernyi, being a candidate strain for developing the biological control agent against empty-gut disease.
Keywords:
Enterococcus pernyi antagonistic bacterium identification control effect
柞蚕(Antheraea pernyi)是一种重要的经济昆虫，我国柞蚕茧产量达8.5万t/年，占全球总产量的90%[1]。柞蚕茧及其副产品可有效带动服装、纺织、医药及食品等产业的发展，另一方面也为人们提供了更为丰富的蛋白摄入食用资源[2]。柞蚕肠球菌(Enterococcus pernyi)引起的空胴病[3]是柞蚕生产上的主要病害之一，分布范围广，为害时间长。一般年份造成减产20%–30%，严重年份可达80%以上，给柞蚕生产造成巨大损失[4]。柞蚕1–5龄幼虫都能受到肠球菌侵染[5]，柞园内病蚕粪便或尸体中的柞蚕肠球菌可存活17.5–35个月[6]，并且该菌可在柞蚕与其他昆虫间交叉感染[7]，最终造成柞蚕空胴病扩大传染。柞蚕肠球菌对不同环境条件和化学药剂(如乙醇、1%氢氧化钠、1%高锰酸钾等)有一定的耐受性，需要高温干热、高温蒸汽和高压蒸汽等才能达到灭菌的目的[8]。目前，生产上主要采用放养前消毒的方法，对该病起到消灭垂直传播的作用，在种源上控制病害的发生。虽然相继研发出一些防治药剂如蚕得乐、保蚕宁、蚕康宁等[9]，但受到发病原因不明确、侵染方式复杂、寄主易产生抗药性等因素制约，化学药剂始终无法发挥有力作用[10]。同时，由于柞蚕自身对于药剂的耐受性较差[11]，化学农药的不正确使用常会引起柞蚕中毒[12]。因此，柞蚕生产上亟需一种安全、环保、高效的生物农药的研发与应用。
柞蚕病害生物防治相关报道较少，1982年，曾繁启等从病蚕中分离出了8株柞蚕软化病链球菌噬菌体，用其对病卵进行卵面消毒，取得明显效果[13-14]，但并未见其后续研究。赫英姿等[15]分离出8种植物提取液对柞蚕肠球菌具有抑菌活性，但并未对其防治效果进行研究。Qu等[16]利用木霉菌株培养滤液合成银纳米颗粒，能有效控制柞蚕空胴病，提高柞蚕存活率，但仅限于感染14 d内的室内防效。在动物生物防治方面，一些芽孢杆菌能够产生对胃酸和胆盐具有抗性的孢子，在肠道环境的生存能力比其他益生菌具有优势，因此芽孢杆菌被认为是动物益生菌补充剂的候选菌株，并在包括家禽、猪、反刍动物和水产养殖的益生菌研究中展现出良好的抗菌活性、促生作用和生物安全性[17]。在柞蚕病害防治中尚未见芽孢杆菌属用于防治柞蚕相关病害的研究，本研究从柞园土壤中分离、筛选对柞蚕肠球菌有拮抗作用的细菌，对其进行鉴定和定殖规律测定，并确定其对柞蚕空胴病的防效，为该病生防菌剂的开发提供候选菌株。
1 材料与方法 1.1 供试材料和培养基菌株和质粒：柞蚕肠球菌为模式菌株78501，保存于中国科学院微生物研究所(菌号：AS1.1010)；pHT0-P43GFPmut3a为携带氯霉素和绿色荧光蛋白基因的质粒，由辽宁省农业科学院植物保护研究所水稻病害研究室提供。
培养基：柞蚕肠球菌培养基(酵母膏3 g，蛋白胨20 g，葡萄糖20 g，NaCl 5 g，琼脂20 g，pH 9–10，水1 000 mL)用于柞蚕肠球菌培养，LB用于细菌培养。
供试柞蚕品种为抗大，孵化后的柞蚕幼虫在室内饲养(24 ℃、RH 75%)至4龄第2天用于室内防效测定，饲料为麻栎(Quercus acutissima Carruth.)；孵化后的柞蚕幼虫在麻栎柞园饲养至4龄第2天用于野外防效测定。
1.2 柞蚕肠球菌拮抗菌的分离和筛选土样细菌的分离：采用5点取样法[18]从辽宁省蚕业科学研究所柞园(N123°99′，E40°48′)土壤表层5–10 cm柞树根际周围取土壤样品5份，各200–300 g。混合均匀，称取各份土样20 g放入装有50 mL灭菌水的三角瓶中，28 ℃、180 r/min振荡培养1 h后，静置沉淀，吸取上清液，采用稀释涂布平板法，取10 μL稀释103、104、105、106和107倍的土壤悬浮液均匀涂布于LB平板上，28 ℃培养24 h，挑取单菌落进行纯化。
拮抗细菌的筛选：采用抑菌圈法[19]。将柞蚕肠球菌配制成浓度为1×107 CFU/mL的菌悬液，以5%的接种量接种到温度为45 ℃左右的肠球菌培养基中，混匀后倒入培养皿。冷却后在培养基中央接种细菌发酵液，28 ℃培养2 d后调查抑菌效果。
1.3 拮抗菌株的分类鉴定 1.3.1 生理生化鉴定参照文献[20]的方法对拮抗菌株进行生理生化测定，包括革兰氏染色、甲基红、淀粉水解、蛋白胨水解、明胶液化试验、VP试验、过氧化氢酶试验以及耐盐试验。
1.3.2 分子鉴定使用细菌快速提取试剂盒提取细菌基因组DNA。采用表 1中[21]引物对拮抗菌株16S rRNA和gyr B基因进行扩增，所得PCR产物送至生物工程技术有限公司测序。测序结果与GenBank中核酸数据进行Blast比对，下载同源性较高的序列，使用邻接法(neighbour-joining)构建系统发育树[22]。
1.4 拮抗菌株对柞蚕幼虫安全性测定
柞蚕幼虫室内饲养参照方法1.1，饥饿胁迫1 d后用于安全性测定。设置4个处理，每个处理10头幼虫，设置3次重复，分别添食浓度为1.0×108、1.0×107、1.0×106和1.0×105 CFU/mL的拮抗菌株发酵液，剂量为10 μL/头。连续添食7 d，每隔24 h观察幼虫粪便、活动力和死亡情况。
1.5 拮抗菌的荧光蛋白标记 1.5.1 拮抗菌株自然转化拮抗菌株的自然转化参照唐萍等[23]的方法，拮抗菌种培养于GMI和GMII培养基中，制备感受态细胞，将pHT01-P43GFPmut3a质粒导入到感受态细胞中，摇瓶培养2–4 h后涂布于LB平板上(氯霉素浓度10 μg/mL)，过夜培养，选取荧光较强的转化子进行后续试验。
1.5.2 标记菌株的稳定性、生长速率和生物膜测定将荧光标记菌株连续转接于不含抗生素的液体LB培养基中10次，每次转接前进行计数，测得荧光菌占比，检测荧光菌在无胁迫压力下继代培养稳定性。将标记菌株与原始菌株配置成浓度为108 CFU/mL菌悬液，按1%接种量接种到100 mL培养液中，每4 h用细菌计数板计算浓度，试验处理设置3次重复。分别以测定时间、细菌浓度为横、纵坐标轴，绘制标记菌株与原始菌株的生长曲线[23]。分别挑取标记菌株与原始菌株单菌落[23]，接种于LB培养液中，35 ℃、180 r/min摇瓶培养1 d，使用无菌液体LB培养基稀释菌液并调整OD600=0.1，将菌液以1% (体积分数)的比例加入装有无菌液体LB培养基的玻璃管中，混匀后于25 ℃静置培养1 d，记录生物膜的形成情况，测定生物膜OD590的光吸收值，每个处理设置3次重复。
1.6 标记菌株定殖测定 1.6.1 标记菌株在柞树叶片上的定殖将标记菌株发酵液(浓度为1×107 CFU/mL)以雾状均匀喷施在长势基本一致的盆栽麻栎叶片表面，设置3次重复。分别收集未接种叶片和接种第1、3、5、10、15、20 d叶片1片，用无菌水洗涤，并在无菌滤纸上干燥。称取3 g置于装有20 mL无菌水的50 mL离心管中，150 r/min振荡1.5 h，10倍系列稀释涂布抗性平板(氯霉素浓度10 μg/mL)[24]，培养48 h后计菌落数，根据每皿菌落数计算每克鲜重组织中的细菌数(CFU/g)。
1.6.2 标记菌株在柞蚕肠道内的定殖柞蚕幼虫室内饲养参照方法1.1，用无菌微量进样器取10 μL标记菌株(浓度为1×107 CFU/mL)经口添食，设置3次重复，分别取未添食和添食第1、3、5、10、15天柞蚕幼虫1头，解剖取中肠组织2 g置于灭菌研磨内，加入4 mL水研磨，取上清做10倍系列稀释涂布抗性平板(氯霉素浓度10 μg/mL)[24]，培养48 h后计菌落数，根据每皿菌落数计算每克中肠组织中的细菌数(CFU/g)。
1.6.3 荧光菌落观察取标记菌株处理3 d的柞树叶片和柞蚕幼虫，用无菌刀片将叶片和柞蚕肠道切成0.5 cm左右的切片，铺展在预先滴有无菌水的载玻片上，盖上盖玻片后用共聚焦荧光显微镜(Nikon Eclipsev 80i)观察，激发波长为488 nm。
1.7 拮抗菌株对柞蚕空胴病的防治效果 1.7.1 拮抗菌发酵液制备取1 mL浓度为1.0×108 CFU/mL的BF-49菌悬液添加到装有100 mL LB培养基的三角瓶中，置于35 ℃、180 r/min摇床中培养24 h，得到BF-49发酵液，BF-49发酵液经0.22 μm滤膜过滤，即得无菌发酵液。
1.7.2 室内防治效果柞蚕幼虫室内饲养参照方法1.1，设5个处理，每个处理10头蚕，设置3次重复：(1) 空白对照；(2) 发病对照；(3) 蚕得乐30倍稀释液对照；(4) BF-49无菌发酵液10倍稀释液处理；(5) BF-49发酵液10倍稀释液处理，处理方法为经口添食，剂量为10 μL/头，每隔5 d处理1次，第2次添食1 d后接种肠球菌(浓度为1×107 CFU/mL)。发病后调查，发病率=发病幼虫数量/调查幼虫数量×100%；校正防治效率(以下简称防效)=(对照组发病率−处理组发病率)/对照组发病率×100%；结茧率=调查茧数量/10×100%[25]。
1.7.3 野外防治效果柞蚕幼虫野外饲养参照方法1.1，设5个处理，每个处理100头蚕，设置3次重复：(1) 空白对照；(2) 发病对照；(3) 蚕得乐30倍稀释液处理；(4) BF-49发酵液10倍稀释液处理；(5) BF-49发酵液100倍稀释液处理，处理方法为叶面喷施，以雾状均匀喷施在麻栎叶片表面至叶面湿润而不滴水，每隔5 d喷施1次，第2次喷施1 d后接种肠球菌(叶面喷施，浓度为1×107 CFU/mL)。发病后调查，发病率和防效计算方法同1.7.1；结茧率=调查茧数量/100×100%。
2 结果与分析 2.1 生防细菌的分离与筛选将采集的土壤样品进行涂布分离，共获得87株细菌。其中，13株对柞蚕肠球菌具有拮抗作用(表 2)，选择拮抗效果显著(P < 0.001)且稳定的BF-49菌株(抑菌圈直径为41.04 mm)开展后续研究(图 1)。
表 2. 不同细菌对柞蚕肠球菌的拮抗作用
Table 2. Antagonistic effect of different bacteria on Enterococcus pernyi
| Strain No. | Inhibition zone Diameters (mm) | Strain No. | Inhibition zone Diameters (mm) | |
| BF-03 | 21.15±0.25f | BF-53 | 8.03±0.11a | |
| BF-12 | 34.06±0.38i | BF-56 | 14.20±0.29d | |
| BF-13 | 12.64±0.33c | BF-58 | 17.82±0.15e | |
| BF-21 | 31.42±0.38h | BF-69 | 37.54±0.46j | |
| BF-27 | 17.99±0.222e | BF-78 | 38.04±0.25j | |
| BF-38 | 10.92±0.22b | BF-86 | 23.95±0.19g | |
| BF-49 | 41.10±0.07k | |||
| Data are presented as x±s (n=3). Values by different lowercase letters mean significant difference (P < 0.05), and with the same letter mean no significant difference (P > 0.05). A one-way ANOVA was conducted to evaluate differences. | ||||
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| 图 1 BF-49菌株带菌发酵液的抑菌效果 Figure 1 Inhibitory effect of fermentation broth from strain BF-49 against growth of Enterococcus pernyi. A: CK. B: Fermentation broth. |
2.2 生防菌株的分类鉴定 2.2.1 形态学与生理生化鉴定
菌株BF-49菌落在LB培养基上呈近圆形，表面干燥褶皱，乳白色不透明，边缘不整齐(图 2A)；光学显微镜观察显示BF-49菌株呈杆状，大小为0.5 μm×(1.5–3.5) μm。菌株BF-49生理生化鉴定结果如表 3所示。结合形态学及生理生化检测结果，初步将菌株BF-49归类为芽胞杆菌属(Bacillus)。
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| 图 2 BF-49菌株形态学特征和系统发育分析 Figure 2 The morphological features and phylogenetic analysis of strain BF-49. A: The colonial morphology of BF-49 strain. B: PCR products of 16S rRNA and gyr B gene amplified from strain BF-49. C: The phylogenetic tree of strain BF-49 based on 16S rRNA gene sequence. D: The phylogenetic tree of strain BF-49 based on gyr B gene sequence. Numbers at the nodes represented the bootstrap values based on neighbor-joining analyses of 1 000 resampled datasets; Numbers in parentheses represented the sequences' accession number in GenBank; The scale bar indicates 0.5% sequence divergence; Target strain was labeled in bold. |
表 3. BF-49菌株生理生化特征
Table 3. Physiological and biochemical characteristics of BF-49 strain
| Test item | Results |
| Gram staining | + |
| Starch hydrolysis | + |
| Gelatine liquefication test | + |
| Oxidase | + |
| VP test | + |
| Catalase | + |
| Nitrate reduction | + |
| Methyl red | − |
| Peptone hydrolysis | − |
| 3% NaCl | + |
| 5% NaCl | + |
| 7% NaCl | + |
| 9% NaCl | + |
| 11% NaCl | + |
| +: Positive (growth or reaction); –: Negative (no growth or no reaction). | |
2.2.2 分子生物学鉴定
对BF-49菌株进行16S rRNA和gyr B基因序列测定，分别得到1 452 bp和1 145 bp的序列(图 2B)，基因序列提交至NCBI，登录号分别为OK442649和ON929823。经Blast比对，BF-49菌株16S rRNA和gyr B基因序列与贝莱斯芽孢杆菌(Bacillus velezensis)的相似率分别为99.45%和99.91%，系统发育分析结果显示BF-49与贝莱斯芽胞杆菌(MT119763.1)聚合为一支(图 2C、2D)。确定菌株BF-49为B. velezensis，命名为B. velezensis BF-49。
2.3 BF-49菌株对柞蚕幼虫安全性测定以浓度为1.0×108、1.0×107、1.0×106和1.0× 105 CFU/mL的BF-49发酵液给柞蚕连续添食7 d，添食过程中柞蚕排便正常，活动力良好，无中毒和死亡现象。说明BF-49发酵液对柞蚕安全。
2.4 BF-49菌株的荧光标记 2.4.1 BF-49菌株的自然转化获得的标记菌株在共聚焦荧光显微镜下可呈现绿色荧光(图 3)，将该菌株命名为BF-49-GFP。
|
| 图 3 共聚焦荧光显微镜中BF-49-GFP的菌落形态及颜色 Figure 3 Colony morphology and color of strain BF-49-GFP under the Confocal fluorescence microscope. |
2.4.2 BF-49-GFP菌株的稳定性、生长曲线和生物膜形成能力测定
在无选择压力条件下连续转接培养50 h后，BF-49-GFP菌落占比仍可达91.04% (图 4)，稳定性较好；BF-49-GFP的生长趋势与BF-49基本一致(图 5)，表明质粒的转入未对BF-49的生长造成不良影响；图 6可见，BF-49和BF-49-GFP的生物膜形成能力无显著差异(P > 0.05)，故可推断BF-49-GFP的定殖能力未发生明显改变，BF-49-GFP可以用于BF-49的定殖研究。
|
| 图 4 BF-49-GFP菌株的稳定性 Figure 4 Stability of BF-49-GFP strain. Means represent the value of three replicates; Lines above the bars represent standard error (SE) of the mean within the treatments. |
|
| 图 5 BF-49与BF-49-GFP菌株的生长曲线 Figure 5 Growth curves of BF-49 and BF-49-GFP strains. |
|
| 图 6 BF-49与BF-49-GFP菌株的生物膜形成 Figure 6 Biofilm formation of BF-49 and BF-49-GFP strain. A: Biofilm formation. B: OD590 of biological film. Means represent the value of three replicates; Lines above the bars represent standard error (SE) of the mean within the treatments; The different lowercase letters mean significant difference (P < 0.05), and the same lowercase letter mean no significant difference (P > 0.05). |
2.5 BF-49-GFP在柞树叶片和柞蚕肠道内的定殖及观察
定殖试验结果显示(表 4)，未喷施BF-49-GFP菌悬液时，标记菌株浓度为0，说明柞树叶片和柞蚕肠道内原没有BF-49-GFP，现有的BF-49-GFP均来源于添食。喷施1 d后，叶片上BF-49-GFP菌株的浓度为5.99×105 CFU/g，随后逐渐减少，3 d后，BF-49-GFP菌株的浓度为3.25×105 CFU/g，20 d后仍在柞树叶片上检测到BF-49-GFP菌株，浓度为1.25×104 CFU/g。添食1 d后，柞蚕肠道内BF-49-GFP菌株浓度为3.6×104 CFU/g，3 d后急剧下降，5 d后浓度为1.33×103 CFU/g，10 d后未检测到BF-49-GFP。BF-49-GFP菌株处理3 d的定殖情况见图 7。
表 4. BF-49-GFP在柞树叶片和柞蚕肠道内的定殖情况
Table 4. Colonization of BF-49-GFP strain in oak leaf and Antheraea pernyi intestinal tract
| Group | Time after inoculation (d) | ||||||
| 0 | 1 | 3 | 5 | 10 | 15 | 20 | |
| Oak leaves (×104 CFU/g) | 0 | 57.67±3.26a | 32.13±2.01b | 29.26±1.73bc | 22.93±3.17c | 3.55±0.33d | 1.25±0.17d |
| Larvae guts (×103 CFU/g) | 0 | 36±3.33a | 2.13±0.53b | 1.33±0.31b | 0c | 0c | Undetected |
| Data are presented as x±s (n=3). Data in the same line with the different letters mean significant difference (P < 0.05), and with the same letter mean no significant difference (P > 0.05). A one-way ANOVA was conducted to evaluate differences. | |||||||
|
| 图 7 BF-49-GFP的定殖观察 Figure 7 Colonization of BF-49-GFP strain. A: In oak leaf. B: A. pernyi intestinal tract. |
2.6 室内防效
室内防效试验结果(表 5)表明，BF-49发酵液10倍稀释液对柞蚕空胴病的防效显著，达到78.25%，显著高于蚕得乐30倍液(防效65.21%)和BF-49无菌发酵液10倍稀释液(防效39.13%) (P < 0.05)。其结茧率与空白对照无明显差异(P > 0.05)。
表 5. BF-49菌株室内防治效果
Table 5. The control effect of BF-49 strain in indoor experiment
| Treatment | Incidence rate | Control effect | Cocoon rate |
| 1 | 23.33±8.82ab | 69.57±10.35c | 60.00±5.77bc |
| 2 | 76.67±3.33c | 0a | 0a |
| 3 | 26.67±8.82ab | 65.21±11.50c | 53.33±13.33bc |
| 4 | 46.67±8.82b | 39.13±11.50b | 36.67±6.67b |
| 5 | 16.67±3.33a | 78.25±7.53d | 63.33±6.67c |
| Data are presented as x±s (n=3). Data in the same column with the different letters mean significant difference (P < 0.05), and with the same letter mean no significant difference (P > 0.05); A one-way ANOVA was conducted to evaluate differences. 1: Blank control; 2: Disease control; 3: Pathogen inoculation after treatment with 30-fold dilution of Silkworm Dele; 4: Pathogen inoculation after treatment of 10-fold dilution of BF-49 aseptic fermentation broth; 5: Pathogen inoculation after treatment of 10-fold dilution of BF-49 carrier fermentation broth. | |||
2.7 野外防效
野外防效试验结果(表 6、图 8)表明，BF-49发酵液10倍稀释液的防效为74.42%，显著高于蚕得乐30倍液(防效31.00%)和BF-49带菌发酵液100倍稀释液(防效39.53%) (P < 0.05)，其结茧率与空白对照无明显差异(P > 0.05)。
表 6. BF-49菌株野外防治效果
Table 6. The control effect of BF-49 strain in field assay
| Treatment | Incidence rate | Control effect | Cocoon rate |
| 1 | 17.67±1.45ab | 58.91±5.45d | 52.33±2.60c |
| 2 | 43.00±4.58d | 0a | 30.33±2.96a |
| 3 | 29.67±4.67c | 31.00±3.85b | 38.00±1.53ab |
| 4 | 11.00±1.52a | 74.42±3.55e | 45.00±5.03bc |
| 5 | 26.00±4.04bc | 39.53±6.40c | 35.33±5.04ab |
| Data are presented as x±s (n=3). Data in the same column with the different letters mean significant difference (P < 0.05), and with the same letter mean no significant difference (P > 0.05). A one-way ANOVA was conducted to evaluate differences. 1: Blank control; 2: Disease control; 3: Pathogen inoculation after treatment with 30-fold dilution of Silkworm Dele; 4: Pathogen inoculation after treatment of 10-fold dilution of BF-49 aseptic fermentation broth; 5: Pathogen inoculation after 100× dilution treatment of BF-49 carrier fermentation broth. | |||
|
| 图 8 柞蚕幼虫野外发病情况 Figure 8 Field incidence of larvae. A: Diseased larvae. B: Healthy larvae. |
3 讨论与结论
在动物生产上，拮抗益生菌能够在动物肠道内定殖并形成种群优势，将有效减少益生菌的使用剂量和使用频率。目前益生菌研究是通过添加到饲料中使其在动物肠道中保持足够菌量，使其稳定地发挥益生和控制病害的作用[26]。已有研究显示鳞翅目昆虫肠道呈碱性[27]，对于非本土肠道菌而言，在高碱性肠道条件下无法长期定殖。绿色荧光蛋白标记的大肠杆菌(Escherichia coli)引入斜纹夜蛾(Spodoptera littoralis)肠道中4 d就消失了，引入弹尾目动物的肠道后1 d就消失了[28]。在本研究中，BF-49标记菌株在柞蚕肠道内能保持5 d，但野外放养中常使用喷雾法施用生防制剂，BF-49-GFP在柞树叶片上定殖时间较长，且在接种20 d后仍能达到4个数量级，若能在第5d强化处理一次，使其在叶片上保持种群优势，将对柞蚕空胴病发挥更明显的防治效果。
益生菌是家畜常用的饲料添加剂，它们能够作为抗生素的替代品，提高生长性能，降低发病率[29-31]。在我国，可以直接应用于动物饲养的常见菌种包含双歧杆菌属(Bifidobacterium)、芽孢杆菌属(Bacillus)、乳杆菌属(Lactobacillus)、酵母菌属(Saccharomyces)等[32]。它们大多可以产生抗菌物质，如抗菌肽、乳酸和细菌素等[33]，改善肠道微生物群的生态条件，降低病原菌定殖的风险，或通过竞争性排斥抑制病原菌生长[34]。虽然B. velezensis属内分类地位的确定比其他已报道的生防芽胞杆菌相对较晚[35]，相关研究较少，但已经展现出良好的应用潜力。Bampidis等[36]报道了B. velezensis符合添加剂授权条件，是动物生产上极具潜力的益生菌物种。B. velezensis具有广谱抑菌活性，能够分泌抗菌蛋白、脂肽类抗生素、多肽类物质和聚酮类等抗菌物质[37]，喂食B. velezensis能够提高宿主的抗病能力。例如Li等[38]的研究表明，B. velezensis K2对多种水生物致病菌具有良出好的拮抗作用，明确了该菌可增强石斑鱼对哈维氏弧菌的抗病力；在实验室条件下，饲喂B. velezensis LF01能够显著增强罗非鱼对无乳链球菌的抗病力[39]；B. velezensis DY-6菌株对多种病原菌均有良好的抑制作用，通过浸浴和投喂均可降低刺参的发病率[40]。在本研究中，B. velezensis BF-49对柞蚕肠球菌具有显著抑制作用，对柞蚕空胴病的室内防效为78.25%，野外防效为74.42%，室内防效显著高于陈卓等[9]报道的蚕康宁(74.44%)、保蚕宁3号(41.11%)和蚕得乐的室内防效(33.33%) (P < 0.05)，但陈卓的研究中未设置清水对照和发病对照，也并未对野外防效进行测定。另外毒性测试显示蚕康宁浓度为2.0 mg/mL时会使个别柞蚕行动略迟缓，对蚕有一定影响，而B. velezensis BF-49在安全性测试中柞蚕幼虫未表现出不良影响，对柞蚕幼虫具有良好的安全性。这与王金燕等[40]和高艳侠等[41]的研究结果一致，B. velezensis DY-6与B. velezensis LF01也表现出良好的生物安全性。
以往的研究证实了B. velezensis具有产生纤维素酶、半纤维素酶、淀粉酶、过氧化氢酶和氧化酶的能力[42]，可有效促进宿主消化食物、帮助宿主吸收营养物质。这提示B. velezensis BF-49可能还具有帮助柞蚕幼虫消化吸收营养物质、促进生长发育的作用，后续研究将继续探讨该生防细菌对柞蚕幼虫的促进生长发育的作用。高艳侠等[41]从罗非鱼肠道中分离的B. velezensis LF01具有耐热、耐酸碱、耐蛋白酶消化以及耐低温储藏等特性。从B. velezensis DH82发酵液提取的抗菌蛋白具有较好的热稳定性且对常见蛋白酶不敏感[43]，说明B. velezensis可能也具有一定抗逆性，这确保B. velezensis BF-49在柞蚕肠道内对肠球菌具有稳定的拮抗作用。
芽孢杆菌的抗菌活性通常与产生的抗菌物质有关，后续研究将在本研究基础上探讨该拮抗菌的抑菌机制和发酵条件等，为在生产中利用该菌株进行柞蚕空胴病的生物防治奠定研究基础。
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