CN117603864A - 用于烟草青枯病防治的嗜水气单胞菌ztyl3及其应用 - Google Patents
用于烟草青枯病防治的嗜水气单胞菌ztyl3及其应用 Download PDFInfo
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- CN117603864A CN117603864A CN202311574287.4A CN202311574287A CN117603864A CN 117603864 A CN117603864 A CN 117603864A CN 202311574287 A CN202311574287 A CN 202311574287A CN 117603864 A CN117603864 A CN 117603864A
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- Prior art keywords
- ztyl3
- acid
- strain
- aeromonas hydrophila
- ralstonia
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Abstract
本申请公开了一种用于烟草青枯病防治的嗜水气单胞菌ZTYL3及其应用,嗜水气单胞菌(Aeromonas hydrophila)菌株ZTYL3保藏证号:CCTCC No:M 2019922。所得嗜水气单胞菌(Aeromonas hydrophila)ZTYL3的防效评价,不以拮抗效果为评价指标,直接以控病能力为指标,经平板拮抗能力测定发现菌株ZTYL3对罗尔斯通氏菌的无抑菌能力,说明菌株ZTYL3的控病机理与传统的拮抗菌不同,说明菌株ZTYL3发挥控病作用不以产生抗生素为主,可能通过生态位竞争、诱导植株抗病、调节根际微生物群落等机制防病。嗜水气单胞菌(Aeromonas hydrophila)ZTYL3为作物青枯病的防治提供新的生防资源。
Description
技术领域
本申请涉及微生物技术领域,特别是一种用于烟草青枯病防治的嗜水气单胞菌ZTYL3及其应用。
背景技术
青枯病由罗尔斯通氏菌(Ralstonia spp.)侵染引起,是作物重要的土传病害。罗尔斯通氏菌寄主范围广泛,能侵染400多种作物,在香蕉、番茄、马铃薯、烟草上为害较重。在我国,烟草青枯病主要由烟草罗尔斯通氏菌(R.nicotianae)侵染引起,该病在长江流域及其以南各烟区普遍发生(Liu Jun-Ying,Zhang Jian-Feng,Wu Han-Lian,et al.Proposalto classify Ralstonia solanacearum phylotype I strains as Ralstonianicotianae sp.nov.,and a genomic comparison between members of the genusRalstonia.Frontiers in Microbiology,2023,14:1135872.doi:10.3389/fmicb.2023.1135872.)。卢灿华,李军营,米梦鸽等于2021年发表的“蒲桃雷尔氏菌(Ralstonia syzygii)LLRS-1的基因组学分析”([C]//中国植物病理学会.植物病理科技创新与绿色防控——中国植物病理学会2021年学术年会论文集.中国农业科学技术出版社,2021:1.DOI:10.26914/c.cnkihy.2021.063949.)一文已探明,该病在云南省文山、保山、临沧、红河、昆明、玉溪、曲靖、昭通、大理、丽江、楚雄和德宏等12个州市的43个区(县)有发生,其中文山、临沧、红河、普洱烟区发病较重。虽然烟草青枯病为害较重,但因抗病资源匮乏,加之无化学防治的特效药,该病一直是制约烟草产量和品质提升的重要因素。
生物防治因其对环境友好而受到广泛关注。已有大量生防资源作为土传病害生物防治手段得到深入研究,例如已报道的青枯病生防菌主要包括:假单胞菌(Pseudomonasspp.)、芽孢菌(Bacillus spp.)、链霉菌(Streptomyces spp.)、不动杆菌(Acinetobacterspp.)、伯克霍尔德氏菌(Burkholderia spp.)和类芽孢菌(Paenibacillus spp.)。
我国已针对烟草青枯病开发注册11种生防菌,主要为荧光假单胞菌、解淀粉芽孢杆菌、多粘类芽孢杆菌、枯草芽孢杆菌等杀菌剂,多数菌剂具有拮抗罗尔斯通氏菌的能力。国内已报道的烟草青枯病生防菌的相关专利主要包括:烟草肠杆菌(Enterbacter tabaci)65B7(CN115678806A)、指示器寡养单胞菌(Stenotrophomonas indicatrix)菌株107E3(CN115612651A)、罗尔斯通氏菌(Ralstonia sp.)56D2(CN113502250A)、副黄假单胞菌(P.parafulva)DW15(CN114934001A)、韩国假单胞菌(P.koreensis)CLP-23(CN111705016A)、解淀粉芽孢杆菌(B.amyloliquefaciens)TBA03(CN111117936A)、多粘类芽孢杆菌NX1-4-4(CN107365729A)、枯草芽孢杆菌(B.subtilis)生防菌株Trb3(CN102747013A);复合菌剂主要有3株假单胞菌(P.lurida)FGD5-2、(P.koreensis)HCH2-3和(P.rhodesiae)MTD4-1组合(CN112920965A),细黄链霉菌(Streptomyces microflavus)CGMCC 12841、微白黄链霉菌(S.albidoflavus)CGMCC 12842、链霉菌(S.pratensis)CGMCC12843和1株铜绿假单胞菌(P.aeruginosa)组合(CN106754563A),浅多色链霉菌、累西菲链霉菌、沙福芽孢杆菌、中孢短芽孢杆菌、褐红木霉菌组合(CN106465734A),里氏木霉(Trichoderma ressei)ACCC 30150和黄赭色链霉菌(S.silaceus)ACCC 40021组合(CN103315005A),多粘菌与枯草菌组合(PD20200380)等。
目前无气单胞菌属细菌在作物病害防治中的应用,更未见可防治或抑制作物青枯病的气单胞菌的报道。
现有青枯病生防菌筛选是通过室内平板拮抗初筛选、温室生测复筛选获得,所得菌体对病原菌均具有显著的生长抑制效果,但这也导致此类生防菌长期使用,会增强病原菌的抗药性,影响对青枯病的防治效果。
公开于背景技术部分的信息仅仅旨在增加对本申请的总体背景的理解,而不应当被视为承认或以任何形式暗示该信息构成已为本领域普通技术人员所公知的现有技术。
发明内容
本申请针对上述技术问题提供了一种用于烟草青枯病防治的嗜水气单胞菌ZTYL3及其应用,菌株ZTYL3发挥控病作用不以产生抗生素为主,可能通过生态位竞争、诱导植株抗病、调节根际微生物群落等机制防病,有利于提高生防菌剂适用安全性。
本申请提供了一种用于烟草青枯病防治的嗜水气单胞菌ZTYL3,嗜水气单胞菌(Aeromonas hydrophila)保藏证号:CCTCC No:M2019922。
优选的,该菌能利用的碳源包括:N-乙酰-D-葡糖胺、吐温40、D-水杨苷、明胶、肌苷、α-D-葡糖、D-海藻糖、甘油、L-苹果酸、D-甘露醇、D-半乳糖、蔗糖、L-精氨酸、β-甲酰-D-葡糖苷、D-果糖-6-磷酸、L-乳酸、D-甘露糖、D-果糖、L-丝氨酸、D-葡糖-6-磷酸、3-甲酰葡糖、D-麦芽糖、N-乙酰-D-半乳糖胺、糊精、D-丝氨酸、D-葡糖酸、L-天冬氨酸、L-丙氨酸、L-组胺、L-谷氨酸、氨基乙酰-L-脯氨酸、柠檬酸、丙酸、粘液酸、果胶、溴-丁二酸、丙酮酸甲酯、乙酸、D-果糖、L-半乳糖醛酸内酯、D-乳酸甲酯、葡糖醛酰胺、糖质酸、肌醇、D-葡糖醛酸、L-焦谷氨酸、D-天冬氨酸;
优选地,该菌不能利用的碳源有:甲酸、蜜三糖、乙酰乙酸、N-乙酰神经氨酸、D-山梨醇、奎宁酸、p-羟基-苯乙酸、水苏糖、L-鼠李糖、γ-氨基-丁酸、α-D-乳糖、D-半乳糖醛酸、龙胆二糖、N-乙酰-β-D-甘露糖胺、D-纤维二糖、D-松二糖、D-苹果酸、α-羟基-丁酸、D-阿拉伯醇、L-果糖、α-酮-戊二酸、β-羟基-D,L丁酸、α-酮-丁酸、蜜二糖;
优选地,该菌在含有1%NaCl、1%乳酸钠、D-丝氨酸、pH 5、pH 6、丁酸钠、万古霉素、利福霉素SV、四唑蓝、林肯霉素、盐酸胍、硫酸四癸钠的条件下生长较好;
优选地,该菌在含有4%NaCl、8%NaCl、二甲胺四环素、亚碲酸钾、四唑紫、梭链孢酸、氨曲南、氯化锂、溴酸钠、萘啶酮酸、醋竹桃霉素的条件下生长缓慢。
嗜水气单胞菌(Aeromonas hydrophila)菌株ZTYL3不通过拮抗或抑制烟草罗尔斯通氏菌(Ralstonia nicotianae)、茄科罗尔斯通氏菌(Ralstonia solanacearum)或假茄科罗尔斯通氏菌(Ralstonia pseudosolanacearum)或蒲桃罗尔斯通氏菌(Ralstoniasyzygii)来防治青枯病。
本申请的另一方面还提供了一种烟草青枯病生防菌剂,包括:如上述菌株。
该菌剂可由本领域技术人员根据本申请的教导和启发,出于实际生产需要,结合微生物工艺领域常用技术手段选择合适的辅料加以调配,将本申请保藏编号为CCTCC No:M2019922的嗜水气单胞菌(Aeromonas hydrophila)菌株ZTYL3制成各种符合各类符合工艺生产要求的剂型产品,例如,粉剂、片剂、液体剂等。
本申请的菌剂剂型不限于粉剂,本领域技术人员根据本申请的教导和启发,出于实际生产需要,结合微生物工艺领域常用技术手段(例如,《制剂技术百科全书》、《药物制剂技术》等),选择合适的辅料加以调配,将本申请保藏编号为CCTCC No:M 2019922的嗜水气单胞菌(Aeromonas hydrophila)菌株ZTYL3制成各种符合各类符合工艺生产要求的其他剂型产品,例如,片剂、液体剂、喷雾剂、颗粒剂等。
优选地,生防菌剂还包括:辅料。
优选地,辅料选自:溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、黏合剂、崩解剂、填充剂、润滑剂、润湿剂、渗透压调节剂、稳定剂、助流剂、矫味剂、防腐剂、助悬剂、包衣材料、芳香剂、抗黏合剂、整合剂、渗透促进剂、pH值调节剂、缓冲剂、增塑剂、表面活性剂、发泡剂、消泡剂、增稠剂、包合剂、保湿剂、吸收剂、稀释剂、絮凝剂、反絮凝剂、助滤剂、释放阻滞剂中至少一种。
优选地,生防菌剂的剂型为粉剂。
优选地,生防菌剂的中菌株ZTYL3的菌浓度大于等于107CFU/mL,更优选地,为107~109CFU/mL。
优选地,生防菌剂制备方法:离心菌株ZTYL3发酵液,收集菌体,按1:1的质量比混合菌体与硅藻土,并添加占混合物料总质量5‰的蔗糖,干燥、粉碎得到生防菌剂的粉剂。
优选地,干燥操作为在阴凉干燥通风处晾干,干燥后物料水分含量达10±2%,进行粉碎。
优选地,生防菌剂的使用方法为:将生防菌剂或其稀释形式对烟株进行灌根处理
优选地,生防菌剂稀释形式为发酵稀释液或菌粉悬浮液。
优选地,烟草青枯病的病原菌为烟草罗尔斯通氏菌(Ralstonia nicotianae)、茄科罗尔斯通氏菌(Ralstonia solanacearum)或假茄科罗尔斯通氏菌(Ralstoniapseudosolanacearum)或蒲桃罗尔斯通氏菌(Ralstonia syzygii)中至少一种。
本申请的另一方面还提供了一种如上述菌株的发酵方法,包括以下步骤:菌株ZTYL3的种子液接种于发酵培养基中进行有氧发酵。
优选地,发酵培养基为葡萄糖5g/L,豆粕粉20g/L,骨蛋白胨2.5g/L,玉米淀粉15g/L,酵母膏2.5g/L,硫酸镁0.5g/L,磷酸氢二钾1g/L,pH7.3。
优选地,菌株ZTYL3的种子液的接种比例为种子液:发酵培养基体积比的1:100。
优选地,发酵条件为:保持120r/min搅拌、温度在37℃上下浮动0.5℃、通气量15m3/h、发酵过程中罐压在0.05Mpa至发酵结束;
优选地,发酵时间为40h。
具体地,将菌株ZTYL3的种子液按1:100比例转接至含有150L发酵培养基中发酵(葡萄糖5g/L,豆粕粉20g/L,骨蛋白胨2.5g/L,玉米淀粉15g/L,酵母膏2.5g/L,硫酸镁0.5g/L,磷酸氢二钾1g/L,pH7.3)。
发酵工艺参数如下:(1)灭菌:空消条件为:0.15MPa,123℃,1h;实消条件为:0.15MPa,123℃,35min;(2)接种:灭菌完成培养液温度下降至37℃时开始接种,接种比例为菌种/培养液1:120;(3)发酵:转速:保持120r/min至发酵结束;通气量:保持15m3/h时保持至发酵结束;整个过程保持罐压在0.05Mpa;因设备原因允许通气量在短时间内浮动。温度:整个发酵过程保持温度在37℃,上下可浮动0.5℃。发酵时间:运行发酵40h。
本文中,发明内容、具体实施方式部分记载的生防菌、嗜水气单胞菌ZTYL3、ZTYL3、菌株ZTYL3、ZTYL3菌株、嗜水气单胞菌均指:本申请的保藏编号为CCTCC No:M 2019922的嗜水气单胞菌(Aeromonas hydrophila)菌株ZTYL3。
本申请能产生的有益效果包括:
1)本申请所提供的用于烟草青枯病防治的嗜水气单胞菌ZTYL3及其应用,所得嗜水气单胞菌(Aeromonas hydrophila)ZTYL3的防效评价,不以拮抗效果为评价指标,直接以控病能力为指标,经平板拮抗能力测定发现菌株ZTYL3对罗尔斯通氏菌的无抑菌能力,说明菌株ZTYL3的控病机理与传统的拮抗菌不同,说明菌株ZTYL3发挥控病作用不以产生抗生素为主,可能通过生态位竞争、诱导植株抗病、调节根际微生物群落等机制防病。嗜水气单胞菌(Aeromonas hydrophila)ZTYL3为作物青枯病的防治提供新的生防资源。
2)本申请所提供的用于烟草青枯病防治的嗜水气单胞菌ZTYL3及其应用,在田间施用菌株ZTYL3防治烟草青枯病时不会产生罗尔斯通氏菌对菌株ZTYL3或菌株代谢产物的耐受性,长期使用该生防菌,能在获得防治青枯病效果的同时,避免产生抗药性。施用菌株ZTYL3具有较好的安全性。
附图说明
图1为本申请实施例1对嗜水气单胞菌ZTYL3培养48h所得菌落形态和菌株细胞形态图,其中a)培养皿照片;b)放大3倍;c)细胞形态图;
图2为本申请实施例3对嗜水气单胞菌ZTYL3基于全基因组构建的系统发育树图。
图3为本申请实施例3的粉剂ZTYL3防治烟草青枯病的效果图。
图4为本申请实施例3的嗜水气单胞菌ZTYL3与罗尔斯通氏菌QBRS-1对峙培养菌落形态对比照片。
本申请提供的嗜水气单胞菌(Aeromonas hydrophila)ZTYL3的保藏信息如下:保藏编号:CCTCC NO:M 2019922;分类命名:Aeromonas hydrophila ZTYL3;保藏日期:2019年11月12日;保藏单位:中国典型培养物保藏中心;保藏地址:中国、武汉、武汉大学。
具体实施方式
下面结合附图与实施例对本申请作进一步的详细说明,但不以任何方式对本申请加以限制,基于本申请教导所作的任何变换或改进,均落入本申请的保护范围。
实施例
以下实施例中所用物料和仪器,如无特殊说明均为商业渠道获得;所用检测方法如无特殊说明,均为现有方法。
1、生物材料的来源
一、以下各实施例中所用烟草材料为公知公用的烟草品种红花大金元,由申请人实验室保存,也可商购获得。
二、以下实施例中所用罗尔斯通氏菌来源:
烟草罗尔斯通氏菌(Ralstonia nicotianae)RST为“Liu Jun-Ying,Zhang Jian-Feng,Wu Han-Lian,et al.Proposal to classify Ralstonia solanacearum phylotypeI strains as Ralstonia nicotianae sp.nov.,and a genomic comparison betweenmembers of the genus Ralstonia.Frontiers in Microbiology,2023,14:1135872.doi:10.3389/fmicb.2023.1135872.”一文中记载的RST菌株。该菌已完成基因组测序,序列提交至GenBank数据库,Bioproject Number为PRJNA594457,GenBank assembly accession号为GCA_018243235.1,该菌已于2022年7月14日保藏于广东省微生物菌种保藏中心,保藏号为GDMCC 1.3533。
蒲桃罗尔斯通氏菌(Ralstonia syzygii)LLRS-1为“Can-Hua Lu,Jun-Ying Li,Meng-Ge Mi,et al.Complete Genome Sequence of Ralstonia syzygiisubsp.indonesiensis Strain LLRS-1,Isolated from Wilted Tobacco inChina.Phytopathology,2021,111:12:2392-2395”一文中记载的LLRS-1菌株。
茄科罗尔斯通氏菌(Ralstonia solanacearum)FQY_4为“Cao Yi,Tian Baoyu,LiuYanxia,et al.Genome Sequencing of Ralstonia solanacearum FQY_4,Isolated froma Bacterial Wilt Nursery Used for Breeding Crop Resistance.GenomeAnnouncements,2013,1(3):e00125-13.”一文中记载的FQY_4菌株。
烟草罗尔斯通氏菌(Ralstonia nicotianae)BSRS-1、QBRS-1、PEJG01均为申请人实验室保存的菌株,申请人承诺自本申请申请日起20年内免费向公众发放用于验证本申请的效果。
2、以下各实验例采用的培养基及培养育苗方法:
一、采用的培养基
LB液体培养基包含1%胰蛋白胨、0.5%酵母浸出物、1%氯化钠和0.5%蔗糖;
CG培养基包含0.1%酸水解酪蛋白、0.25%葡萄糖和2%蛋白胨;
CGA含0.1%酸水解酪蛋白、0.25%葡萄糖和2%蛋白胨、1.5%琼脂;
寡营养培养基CN含0.1%酪蛋白氨基酸、0.1%营养肉汤、1.5%琼脂;
TZC培养基含1%蛋白胨、25%葡萄糖、0.1%酪素水解物、1.5%琼脂及0.005%的氯化三苯基四氮唑(TTC)。
二、漂浮育苗
以感病烟草品种红花大金元为生测对象,漂浮育苗培育烟苗至4~5叶期。
三、病原菌培养
从-80℃超低温冰箱活化罗尔斯通氏菌RST于TZC培养基[1%蛋白胨、0.25%葡萄糖、0.1%酪素水解物、1.5%琼脂及0.005%的氯化三苯基四氮唑(TTC)]表面,置于28℃恒温培养箱培养36~48h;
挑取具有较宽白边、流动性较强、中间呈粉红色或浅红色稀液状的典型菌落,接种于盛有100mL CG液体培养基(1%蛋白胨、0.25%葡萄糖、0.1%酪素水解物)的三角瓶内,置于28℃、225r/min恒温振荡培养24h;
取100μL稀释至10-7,取100μL 10-5、10-6、10-7稀释液涂布TZC平板,并于48h后观察菌落形态及计数菌落数,计算培养的菌液含有的菌体量。
实施例1分离菌株ZTYL3
一、诱集、分离与培养
去除土壤样品(采集自云南省玉溪市红塔区植烟土壤,采集人卢灿华,采集时间2019年5月)中杂质和较大的块状物后,装入直径120mm的玻璃培养皿中,土层厚度约为1.5cm,用蒸馏水采用滴定瓶将土壤润湿;
制备微生物诱集装置:首先在直径50mm、孔径为0.45μm的微孔滤膜边缘处涂布胶水,将不锈钢平底垫圈置于微孔滤膜上;然后向垫圈内腔加入3mL固体培养基(1.2%结冷胶和1.0%维生素);再用胶水涂布金属垫圈上表面,盖上另一孔径为0.45μm微孔滤膜;
将微生物诱集装置置于湿润的所采土壤上,轻轻压实装置,确保微孔滤膜与土壤充分接触,再用剩余土壤将装置完全覆盖,采用装有蒸馏水的滴定瓶,再次润湿土壤;
盖好培养皿,用封口膜将培养装置封好,并置于30℃培养箱中培养7d,期间观察土壤湿度,若土壤湿度低则用无菌水补足;
从培养箱中取出经过诱集的培养装置,将固体培养基捣碎,加入3mL无菌水放置10min后,梯度稀释至10-4;
取10-4、10-5菌液涂布于寡营养培养基CN(含0.1%酪蛋白氨基酸,0.1%营养肉汤,1.5%琼脂),每个梯度涂布5皿,于超净工作台吹干,并置于30℃培养箱中培养7d;
通过上述实验,从CN培养皿上挑取再次划线于CN培养基,获得纯菌ZTYL3;挑取ZTYL3的单一菌落接种于盛有2.5mL LB液体培养基的试管,置于28℃225r/min恒温振荡培养48h,所得菌株的菌落形态如图1所示。
实施例2菌株ZTYL3鉴定
一、培养特性与形态特征:采用常规细菌鉴定参照文献《常见细菌系统鉴定手册》(东秀珠等编著.科学出版社.2001年)。
菌株ZTYL3培养特性与形态特征:在NA培养基于28℃培养,24h即可见圆形菌落形成,菌落初期浅,后期淡灰白色、略带浅红色、边缘光滑、中间凸起。菌株ZTYL3在LB、NB、PDB培养基中均能生长。菌株ZTYL3在LB液体培养基中于20~35℃条件下均能生长,以25℃最佳。挑取菌株置于显微镜下镜检,细菌呈杆状,鞭毛多束。细菌革兰氏染色呈阴性。
二、采用Biolog GEN III板分析菌株ZTYL3的化合物代谢特征。
1、化合物代谢特征:
菌株ZTYL3在各碳源和生长抑制条件下的生长情况如下,能利用的碳源包括:N-乙酰-D-葡糖胺、吐温40、D-水杨苷、明胶、肌苷、α-D-葡糖、D-海藻糖、甘油、L-苹果酸、D-甘露醇、D-半乳糖、蔗糖、L-精氨酸、β-甲酰-D-葡糖苷、D-果糖-6-磷酸、L-乳酸、D-甘露糖、D-果糖、L-丝氨酸、D-葡糖-6-磷酸、3-甲酰葡糖、D-麦芽糖、N-乙酰-D-半乳糖胺、糊精、D-丝氨酸、D-葡糖酸、L-天冬氨酸、L-丙氨酸、L-组胺、L-谷氨酸、氨基乙酰-L-脯氨酸、柠檬酸、丙酸、粘液酸、果胶、溴-丁二酸、丙酮酸甲酯、乙酸、D-果糖、L-半乳糖醛酸内酯、D-乳酸甲酯、葡糖醛酰胺、糖质酸、肌醇、D-葡糖醛酸、L-焦谷氨酸、D-天冬氨酸;
不能利用的碳源有:甲酸、蜜三糖、乙酰乙酸、N-乙酰神经氨酸、D-山梨醇、奎宁酸、p-羟基-苯乙酸、水苏糖、L-鼠李糖、γ-氨基-丁酸、α-D-乳糖、D-半乳糖醛酸、龙胆二糖、N-乙酰-β-D-甘露糖胺、D-纤维二糖、D-松二糖、D-苹果酸、α-羟基-丁酸、D-阿拉伯醇、L-果糖、α-酮-戊二酸、β-羟基-D,L丁酸、α-酮-丁酸、蜜二糖;
在含有1%NaCl、1%乳酸钠、D-丝氨酸、pH 5、pH 6、丁酸钠、万古霉素、利福霉素SV、四唑蓝、林肯霉素、盐酸胍、硫酸四癸钠的条件下生长较好;
而在含有4%NaCl、8%NaCl、二甲胺四环素、亚碲酸钾、四唑紫、梭链孢酸、氨曲南、氯化锂、溴酸钠、萘啶酮酸、醋竹桃霉素的条件下生长缓慢。
2、基于Gen III数据库的菌株鉴定
Gen III鉴定结果表明,菌株ZTYL3与数据库中的嗜水单胞菌(Aeromonashydrophila)相似度最高为0.589,其余较为相似的种包括Vibrio cholerae O1/O139(0.184)、Aeromonas jandaei(0.139)、Vibrio metschnikovii(0.088)。
三、采用16S rDNA和基因组序列鉴定菌株ZTYL3
1、分子鉴定方法如下:细菌基因组DNA的提取试剂盒,方法参见试剂盒说明书。用通用引物F27/R1492进行PCR扩增16S rDNA序列,常规条件扩增,扩增产物经胶回收后,连接于载体pEAZY-T5 Zero载体,热激转化大肠杆菌感受态细胞DH5α,挑取菌落以M13F/M13R为引物进行菌落PCR鉴定。阳性克隆送上海英骏生物技术有限公司进行序列测定。运用Ezbiocloud数据库(https://www.ezbiocloud.net/),分析与菌株ZTYL3相近的模式种,初步确认菌株的属一级分类地位。
2、运用基因组测序技术获取菌株的全基因组。运用TYPE数据库进行比对,计算专利菌株与亲缘关系最近的种的DNA分子杂交值(dDDH),最终确定菌株的分子分类地位,如图2和表2所示。
表2.ZTYL3与单胞菌属细菌的基因组相似性比较分析
16S rDNA序列分析表明菌株ZTYL3与嗜水气单胞菌嗜水亚种(Aeromonashydrophila subsp.hydrophila)ATCC 7966T相似性最高,为99.93%,与嗜水气单胞菌蛙亚种(A.hydrophila subsp.ranae)LMG19707T、A.media CECT 4232T的序列相似性分别为99.86%和99.73%,上述结果表明菌株ZTYL3属于单胞菌属(Aeromonas)。基因组测序后,经Type(Strain)Genome Server(https://tygs.dsmz.de/)数据库分析,结果表明菌株ZTYL3与嗜水气单胞菌(Aeromonas hydrophila)ATCC 7966T相似性最高,为73.6%(dDDH4),其次为Aeromonas hydrophila subsp.ranae CIP 107985T(71.6%),大于新种的鉴定阈值70.0%;菌株ZTYL3与Aeromonas aquariorum CECT 7289T、Aeromonas dhakensis CIP107500T的DDH(d4)值分别为50.4%、50.2%;菌株ZTYL3与其他单胞菌的dDDH值小于50%。基于菌株ZTYL3及其近缘种基因组构建系统发育树,结果表明菌株ZTYL3与嗜水气单胞菌(A.hydrophila)的标准菌株ATCC 7966T及Aeromonas hydrophila subsp.ranae CIP107985T的亲缘关系最近,三者聚为一支(图2)。上述结果说明菌株ZTYL3为嗜水气单胞菌。
通过上述鉴定结果,确认菌株ZTYL3为嗜水气单胞菌(Aeromonas hydrophila)的一个株系,命名为ZTYL3,并将该菌株送保藏,其保藏信息如下:保藏编号:CCTCC NO:M2019922;保藏日期:2019年11月12日;保藏单位:中国典型培养物保藏中心;保藏地址:中国、武汉、武汉大学。
实施3菌株ZTYL3在防治青枯病方面的应用
以下各实施例中青枯病是指烟草青枯病,由烟草罗尔斯通氏菌(Ralstonianicotianae)、假茄科罗尔斯通氏菌(Ralstonia solanacearum)或假茄科罗尔斯通氏菌(Ralstonia pseudosolanacearum)或蒲桃罗尔斯通氏菌(Ralstonia syzygii)至少任一种病菌侵染引发的疾病。
1、生防菌ZTYL3防治烟草青枯病的防效评价:
防效评价分为室内生测初筛选、复筛选,具体操作如下:
步骤S1,生测初筛
烟苗处理:漂浮育苗法培育烟苗至4~5叶期,用苗前1d从育苗池中取出备用烟苗晾干漂盘,次日用无菌刀片在烟苗根部距烟株中心1.5cm的两侧造伤,将烟苗分为试验组和对照组,每组2株烟苗;
生防菌预处理:将试验组烟株根部基质中接种1mL供试生防菌细菌,以只接种LB培养液培养基的烟苗设为对照组;
烟苗悬根培养:在28℃恒温人工气候室内的培养架上放置一张尺寸比培育烟苗的漂浮盘略大的厚塑料布,并在塑料布的4角和中心共放置5个一次性培养皿作为支撑,将漂浮盘置于其上培养1d,其间分早、中、晚三次用喷水器具浇水,浇水量以漂盘孔内的水不下滴为宜;
病原菌接种:生防菌预处理1d后,对试验组和对照组接种0.5mL烟草罗尔斯通氏菌RST 10倍稀释液,在28℃恒温人工气候室内继续培养15~20d,其间分早、中、晚三次用喷水器具浇水保湿;
观察记录:观察试验组和对照组发病情况,当对照组发病率>80%时,记录实验组烟株发病情况,烟株健康记为1,烟株发病但未枯死赋值为0.5,烟株枯死赋值为0,每株供试细菌处理的烟株的数值为两株烟株赋值的总和,选择供试细菌中赋值最高的菌株为潜力生防菌,用于室内复筛选。
步骤S2,生测复筛
步骤S2生测复筛,除以下试验不同外,其余操作步骤与步骤S1生测初筛相同。
1)供试菌株为步骤S1生测初筛获得的潜力生防菌;
2)生测复筛中增加处理烟株数量,处理组和对照组均处理8株烟苗;
3)分别于接种罗尔斯通氏菌后10、20d调查各处理组烟株的病害发生情况,如表1所示。
表1.室内菌株ZTYL3防治烟草青枯病的效果
实验结果表明,在初筛选中,经菌株ZTYL3处理的两株烟均健康,故赋值2.0;在温室复筛选中,接种10、20dpi分别有8、5株烟株健康,而对照处理的烟株分别有5、0株健康。上述结果说明ZTYL3具有较好的防治效果,可用作温室大棚盆栽评价其防效。
2、生防菌ZTYL3防治烟草青枯病的温室大棚盆栽实验
试验设置:于温控28~30℃温室大棚进行,试验设置生防菌处理组、对照组(灌根等体积LB培养基+自来水),每处理3次重复,每重复10株烟株;
供试菌株培养:室内筛选获得的生防菌ZTYL3用LB液体培养基(1%胰蛋白胨、0.5%酵母浸出物、1%氯化钠和0.5%蔗糖)培养,在225r/min30℃摇培48h,即获得菌剂ZTYL3;
试验选择实验室保存的来自云南省玉溪市(LLRS-1)、临沧市(BSRS-1)、普洱市(PEJG01)及福建省(FQY_4)的4株罗尔斯通氏菌为病原菌,病原罗尔斯通氏菌用CG液体培养基培养,在225r/min 30℃摇培24h;
烟苗移栽:采用漂浮育苗方式培育的烟苗,烟苗为二次剪叶的烟苗,约培育50d,移栽时将红壤与有机质按3:1混匀后移栽烟苗;
接种:移栽后将250mL生防菌菌株ZTYL3发酵液稀释25倍,每株烟灌根200mL稀释液(稀释液中的ZTYL3菌浓度为107-108CFU/mL);
对照组包括:以等体积LB稀释液处理;
以化学药剂噻菌铜(农药登记证号PD20086024,浙江龙湾化工有限公司生产)用自来水稀释500倍后每株烟灌根200mL;
次日将CG培养基摇陪24h的罗尔斯通氏菌稀释100倍,每株烟接种100mL罗尔斯通氏菌稀释液(接种浓度约为107CFU/mL)。
调查统计:接种后每7d调查一次病情指数,共调查3~6次。烟草青枯病的发病率、病情指数和防治效果按下式计算:
发病率=发病植株/调查植株总数×100%;病情指数=[Σ(病情级数×此级菌株数)/(最高级数×总株数)]×100;
防治效果=(对照病情指数-处理病情指数)/对照病情指数×100%。
病情指数按照中华人民共和国烟草行业标准烟草病害分级及调查方法调查(GB/T23222-2008),病害分级如下:全株无病为0级,病侧二分之一以下叶片凋萎为1级,病侧二分之一至三分之二叶片凋萎为3级,病侧三分之二以上叶片凋萎为5级,病株叶片全部凋萎为7级,病株基本枯死为9级。
试验所得结果参见表3
表3.菌剂ZTYL3对不同罗尔斯通氏菌的防治效果
上述结果表明,经菌剂ZTYL3处理的烟株在观察期内病情指数均低于清水对照组和药剂噻菌铜处理组,且菌剂ZTYL3对不同地理来源的供试罗尔斯通氏菌均有防治效果,防效在35.66~78.95%,其中对来自普洱(PEGJ01)和玉溪(LLRS-1)的罗尔斯通氏菌引发的青枯病防治效果较好,分别达59.28%和78.95%。菌剂ZTYL3对来自福建的罗尔斯通氏菌FQY_4的防效与对照药剂噻菌铜相近。上述结果说明菌剂ZTYL3对不同地理来源的罗尔斯通氏菌引发的青枯病均具有较好的防治效果。
3、生防菌剂ZTYL3对烟草青枯病的温室防效评价
本实验与“2、生防菌ZTYL3防治烟草青枯病的温室大棚盆栽实验”的区别如下:
1、种子液培养:分别挑取一环生防菌ZTYL3接种于含有500mL种子培养液(蛋白胨10.0g/L、牛肉浸膏3.0g/L、氯化钠5.0g/L、琼脂15.0g/L,pH 7.0)的500mL离心管内,置于28℃恒温摇床培养15h。
2、发酵:将生防菌ZTYL3的种子液按1:100比例转接至含有150L发酵培养基中发酵(葡萄糖5g/L,豆粕粉20g/L,骨蛋白胨2.5g/L,玉米淀粉15g/L,酵母膏2.5g/L,硫酸镁0.5g/L,磷酸氢二钾1g/L,pH7.3)。
发酵工艺参数如下:(1)灭菌:空消条件为:0.15MPa,123℃,1h;实消条件为:0.15MPa,123℃,35min;(2)接种:灭菌完成培养液温度下降至37℃时开始接种,接种比例为菌种/培养液1:120;(3)发酵:转速:保持120r/min至发酵结束;通气量:保持15m3/h时保持至发酵结束;整个过程保持罐压在0.05Mpa;因设备原因允许通气量在短时间内浮动。温度:整个发酵过程保持温度在37℃,上下可浮动0.5℃。发酵时间:运行发酵40h。
3、制粉:将发酵液(约167.43亿CFU/mL)进行离心,收集离心菌体作为原料按1:1的质量比与硅藻土混合并添加占混合物料总质量5‰的蔗糖作为营养物质,将混合物料置于阴凉干燥通风处晾干,当水分含量达10%左右,进行粉碎后制得ZTYL3粉剂(52亿CFU/g)。
4、防治:每棵烟苗施用2.5g菌剂,兑水50mL,得到的菌浓度为2.6亿CFU/mL的菌粉悬浮液进行灌根,每重复15株,设置3次重复,设置清水为对照;次日接种烟草罗尔斯通氏菌QBRS-1,每株0.1OD;每周调查一次病害等级,计算病情指数。
试验结果如图3所示,由图3可知,随着接种天数的增加,对照组病情逐渐加重,而菌剂ZTYL3处理的烟株发病较轻,在接种后24d时病情指数曲线下面积为352.6±124.6,而对照组则为1219±86.26。以病情指数曲线下面积计算,菌剂ZTYL3处理组的防效为71.07%,具有较好防效。
4、菌株ZTYL3平板对峙拮抗罗尔斯通氏菌
采用平板对峙培养法,测量菌株的抑菌带。
操作步骤如下:分别将CG培养基培养24h的罗尔斯通氏菌QBRS-1、RST、LLRS-1和BSRS-1梯度稀释至10-4;取100μL稀释液置于CGA培养基表面,用涂布株涂布均匀,置于超净工作吹干;挑取菌株ZTYL3、拮抗菌贝莱斯芽孢杆菌(Bacillus velezensis)WY2(保藏地点:北京市朝阳区大屯路,中国科学院微生物研究所;保藏单位名称:中国微生物菌种保藏管理委员会普通微生物中心;保藏编号:CGMCC No.6662)的单菌落接种于含有罗尔斯通氏菌的培养基表面,培养2~3d后观测各菌株的抑菌情况,有抑菌效果的菌株用四点法对峙培养并测量菌株的抑菌圈和抑菌带,所得结果图4和表4所示。
表4.菌株ZTYL3对不同烟草青枯病菌的拮抗能力
罗尔斯通氏菌 | 抑菌圈/cm | 抑菌带/cm |
RST | 0.00±0.00 | 0.00±0.00 |
LLRS-1 | 0.00±0.00 | 0.00±0.00 |
BSRS-1 | 0.00±0.00 | 0.00±0.00 |
QBRS-1 | 0.00±0.00 | 0.00±0.00 |
试验结果表明,菌株ZTYL3对罗尔斯通氏菌RST、LLRS-1、BSRS-1和QBRS-1均无拮抗作用。上述结果说明生防菌ZTYL3不以抑菌作用控制青枯病,可能通过非拮抗的生态位竞争、诱导抗性、调节根际微生态结构而发挥作用。
尽管参照前述实施例对本申请进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换,凡在本申请的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本申请的保护范围之内。
Claims (10)
1.一种用于烟草青枯病防治的嗜水气单胞菌ZTYL3,其特征在于,嗜水气单胞菌Aeromonas hydrophila菌株ZTYL3保藏证号:CCTCCNo:M 2019922。
2.根据权利要求1所述的用于烟草青枯病防治的嗜水气单胞菌ZTYL3及其应用,其特征在于,该菌能利用的碳源包括:N-乙酰-D-葡糖胺、吐温40、D-水杨苷、明胶、肌苷、α-D-葡糖、D-海藻糖、甘油、L-苹果酸、D-甘露醇、D-半乳糖、蔗糖、L-精氨酸、β-甲酰-D-葡糖苷、D-果糖-6-磷酸、L-乳酸、D-甘露糖、D-果糖、L-丝氨酸、D-葡糖-6-磷酸、3-甲酰葡糖、D-麦芽糖、N-乙酰-D-半乳糖胺、糊精、D-丝氨酸、D-葡糖酸、L-天冬氨酸、L-丙氨酸、L-组胺、L-谷氨酸、氨基乙酰-L-脯氨酸、柠檬酸、丙酸、粘液酸、果胶、溴-丁二酸、丙酮酸甲酯、乙酸、D-果糖、L-半乳糖醛酸内酯、D-乳酸甲酯、葡糖醛酰胺、糖质酸、肌醇、D-葡糖醛酸、L-焦谷氨酸、D-天冬氨酸;
优选地,该菌不能利用的碳源有:甲酸、蜜三糖、乙酰乙酸、N-乙酰神经氨酸、D-山梨醇、奎宁酸、p-羟基-苯乙酸、水苏糖、L-鼠李糖、γ-氨基-丁酸、α-D-乳糖、D-半乳糖醛酸、龙胆二糖、N-乙酰-β-D-甘露糖胺、D-纤维二糖、D-松二糖、D-苹果酸、α-羟基-丁酸、D-阿拉伯醇、L-果糖、α-酮-戊二酸、β-羟基-D,L丁酸、α-酮-丁酸、蜜二糖;
优选地,该菌在含有1%NaCl、1%乳酸钠、D-丝氨酸、pH 5、pH6、丁酸钠、万古霉素、利福霉素SV、四唑蓝、林肯霉素、盐酸胍、硫酸四癸钠的条件下生长较好;
优选地,该菌在含有4%NaCl、8%NaCl、二甲胺四环素、亚碲酸钾、四唑紫、梭链孢酸、氨曲南、氯化锂、溴酸钠、萘啶酮酸、醋竹桃霉素的条件下生长缓慢。
3.一种烟草青枯病生防菌剂,其特征在于,包括:如权利要求1或2所述嗜水气单胞菌菌株ZTYL3。
4.根据权利要求3所述的生防菌剂,其特征在于,生防菌剂还包括:辅料;
优选地,辅料选自:溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、黏合剂、崩解剂、填充剂、润滑剂、润湿剂、渗透压调节剂、稳定剂、助流剂、矫味剂、防腐剂、助悬剂、包衣材料、芳香剂、抗黏合剂、整合剂、渗透促进剂、pH值调节剂、缓冲剂、增塑剂、表面活性剂、发泡剂、消泡剂、增稠剂、包合剂、保湿剂、吸收剂、稀释剂、絮凝剂、反絮凝剂、助滤剂、释放阻滞剂中至少一种;
优选地,生防菌剂的剂型为粉剂。
5.根据权利要求3所述的生防菌剂,其特征在于,生防菌剂的中菌株ZTYL3的菌浓度大于等于107CFU/mL。
6.根据权利要求3所述的生防菌剂,其特征在于,生防菌剂制备方法:离心菌株ZTYL3发酵液,收集菌体,按1:1的质量比混合菌体与硅藻土,并添加占混合物料总质量5‰的蔗糖,干燥、粉碎得到生防菌剂的粉剂;
优选地,干燥操作为在阴凉干燥通风处晾干,干燥后物料水分含量达10±2%,进行粉碎。
7.根据权利要求3所述的生防菌剂,其特征在于,生防菌剂的使用方法为:将生防菌剂或其稀释形式对烟株进行灌根处理;
优选地,生防菌剂稀释形式为发酵稀释液或菌粉悬浮液。
8.根据权利要求3所述的生防菌剂,其特征在于,烟草青枯病的病原菌为烟草罗尔斯通氏菌(Ralstonia nicotianae)、茄科罗尔斯通氏菌(Ralstonia solanacearum)或假茄科罗尔斯通氏菌(Ralstonia
pseudosolanacearum)或蒲桃罗尔斯通氏菌(Ralstonia syzygii)中至少一种。
9.一种如权利要求1或2所述的嗜水气单胞菌菌株ZTYL3的发酵方法,其特征在于,包括以下步骤:菌株ZTYL3的种子液接种于发酵培养基中进行有氧发酵。
10.根据权利要求9所述的发酵方法,其特征在于,发酵培养基为葡萄糖5g/L,豆粕粉20g/L,骨蛋白胨2.5g/L,玉米淀粉15g/L,酵母膏2.5g/L,硫酸镁0.5g/L,磷酸氢二钾1g/L,pH7.3;
优选地,菌株ZTYL3的种子液的接种比例为种子液:发酵培养基体积比的1:100;
优选地,发酵条件为:保持120r/min搅拌、温度在37℃上下浮动0.5℃、通气量15m3/h、发酵过程中罐压在0.05Mpa至发酵结束;
优选地,发酵时间为40h。
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