CN116286500A - 一种盐生植物内生的耐盐贝莱斯芽孢杆菌及用途 - Google Patents
一种盐生植物内生的耐盐贝莱斯芽孢杆菌及用途 Download PDFInfo
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Abstract
本发明涉及一种盐生植物内生的耐盐贝莱斯芽孢杆菌及用途,属于农业生物防治领域,具体公开了从盐生植物——藜(Chenopodium album L.)中分离的耐盐贝莱斯芽孢杆菌Bacillus velezensis。所述的贝莱斯芽孢杆菌为Bacillus velezensis编号ES‑120,已保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC NO. 25530,经测试该芽孢杆菌对苹果腐烂病、棉花黄萎病、南瓜蔓枯病、黄瓜炭疽病、小麦根腐病、大豆根腐病、小麦赤霉病、水稻立枯病以及香蕉枯萎病均具有显著的拮抗作用,抑菌对象具广谱性;为农作物病害的生物防治提供了新的微生物资源。此外,该菌株具有固氮,溶磷、产铁载体以及高产植物激素(吲哚乙酸,IAA)的能力,并且其拥有繁殖速度快、稳定性高、耐高温、无毒性的特点。因此,贝莱斯芽孢杆菌菌株ES‑120存在着生防、促生功能开发利用的潜力。
Description
技术领域
本发明属于农业生物防治领域,具体涉及一种盐生植物内生的耐盐贝莱斯芽孢杆菌及用途。
背景技术
农作物真菌源病害种类繁多、影响大,对我国农业生产产生重大影响,严重的制约着我国农业的可持续发展。化学类农药虽然是一种有效控制农作物真菌类病害的有效方法,但是存在污染环境、产生耐药性、生态失衡及食品安全等问题。因此,减少化学类农药的应用,寻找和开发能够替代化学类农药的生物防治是必行之路。生物防治具有环境兼容性好、残留少、选择性高、对人畜安全、不易产生抗药性等优点,成为替代化学农药的首选,备受青睐。生物防治的实施为响应农业部提出的到2020年实现农药化肥使用零增长的政策提供重要举措。
目前,具有生物防治功效的细菌类微生物的来源主要集中于芽孢杆菌属(Bacillus spp.)、假单胞杆菌属(Pseudomonas spp.)、溶杆菌属(Lysobacter spp.)、链霉菌属(Streptomyces spp.)以及其它一些有益细菌。芽孢杆菌属菌株在生物防治领域中应用广泛,荧光假单胞菌类群对于植物病害的防治。此外,链霉菌属的菌株产生的众多农用抗生素在动植物病害防治中发挥着重要作用。
芽孢杆菌类群特殊的生理结构(芽孢),使其广泛分布于空气、河流和土壤等普通环境中,以及盐湖,沙漠以及特殊植物等特殊生境。芽孢杆菌菌类群作为一类广泛应用于农作的生防菌株,可以通过与病原菌竞争营养和生态位点、分泌抗菌物质抑制病原菌的生长以及激发植物系统抗病性来防治植物病害,如苏云金芽孢杆菌产生的杀虫晶体蛋白应用于虫害防治,甲基营养型芽孢杆菌具有植物促生防病功效,此外地衣芽孢杆菌、深褐芽孢杆菌、解淀粉芽孢杆菌均具有生防功效。
贝莱斯芽孢杆菌(Bacillus velezensis)作为芽孢杆菌属的一个新种成员,近些年被广泛的应用于生物防治领域,对于诸多植物病害具有良好的作用,其主要分离源主要集中于土壤,各类健康植物等,但来源于盐生植物的贝莱斯芽孢杆菌尚未有报道。
发明内容
本发明目的在于,提供一种盐生植物内生的耐盐贝莱斯芽孢杆菌及用途,该贝莱斯芽孢杆菌为Bacillus velezensis,编号ES-120,已保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为:CGMCC NO.25530,保藏地址:中国北京经研究表明:该菌种对苹果腐烂病(苹果黑腐皮壳菌,Valsa mali)、棉花黄萎病(大丽轮枝菌,Verticilliumdahliae)、南瓜蔓枯病(瓜拟多隔孢菌Stagonosporopsis cucurbitacearum)、黄瓜炭疽病(瓜炭疽病菌,Colletotrichum orbiculare)、小麦根腐病(麦根腐平脐蠕孢菌,Bipolarissorokiniana)、大豆根腐病(立枯丝核菌,Rhizoctonia solani)、小麦赤霉病(禾谷镰孢菌,Fusarium graminearum)、水稻立枯病(尖孢镰孢菌,Fusarium oxysporium)以及香蕉枯萎病(尖孢镰孢菌,Fusarium oxysporium)都具有均具有显著的拮抗作用,抑菌对象具广谱性;为农作物病害的生物防治提供了新的微生物资源。此外,该菌株具有固氮,溶磷、产铁载体以及高产植物激素(吲哚乙酸,IAA)的能力,并且其拥有繁殖速度快、稳定性高、耐高温、无毒性的特点。因此,贝莱斯芽孢杆菌菌株ES-120存在着生防、促生功能开发利用的潜力。
本发明所述的一种盐生植物内生的耐盐贝莱斯芽孢杆菌,该菌株为贝莱斯芽孢杆菌Bacillus velezensis,编号ES-120,已保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC NO.25530,保藏时间2022年8月15日,该菌株分离于盐生植物——藜(Chenopodium album L.)。
所述盐生植物内生的耐盐贝莱斯芽孢杆菌在拮抗农作物致病真菌中的应用。
所述植物致病菌为苹果腐烂病(苹果黑腐皮壳菌,Valsa mali)、棉花黄萎病(大丽轮枝菌,Verticillium dahliae)、南瓜蔓枯病(瓜拟多隔孢菌Stagonosporopsiscucurbitacearum)、黄瓜炭疽病(瓜炭疽病菌,Colletotrichum orbiculare)、小麦根腐病(麦根腐平脐蠕孢菌,Bipolaris sorokiniana)、大豆根腐病(立枯丝核菌,Rhizoctoniasolani)、小麦赤霉病(禾谷镰刀菌,Fusarium graminearum)、水稻立枯病(尖孢镰刀菌,Fusarium oxysporium)、以及香蕉枯萎病(尖孢镰刀菌,Fusarium oxysporium)。
用于鉴定菌株ES-120的分子标记,由SEQ ID No.1所示的核苷酸序列组成。
贝莱斯芽孢杆菌(Bacillus velezensis)菌株ES-120的16S rRNA基因序列:GGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACC
本发明所述的一种盐生植物内生的耐盐贝莱斯芽孢杆菌,该贝莱斯芽孢杆菌Bacillus velezensis,菌株编号为ES-120,分离植物采集时间:2019年8月;采集地点:玛纳斯湖湿地();菌株分离时间:2019年9月
附图说明
图1为本发明菌株ES-120的菌落形态及菌体形态图;
图2为本发明温度37℃下菌株ES-120的生长曲线图;
图3为本发明基于菌株ES-120的16S rRNA基因构建系统发育树(邻位归并法)图;
图4为本发明菌株ES-120与菌属的基因组系统发育树图;
图5为本发明A菌株ES-120的基因组圈图;B,菌株ES-120与之贝莱斯芽孢杆菌(Bacillus velezensis)NRRL B-41580T和暹罗芽孢杆菌(Bacillus siamensis)KCTC13613T基因组的泛基因组分析;C,菌株ES-120与两株参比标株基因组的共线性分析图;
图6为本发明菌株ES-120对植物病原真菌的抑制作用图;
图7为本发明菌株ES-120对不同专化型尖孢镰刀菌(Fusarium oxysporium)的抑制作用图;
图8为本发明菌株ES-120植物益生作用筛选结果照片;
图9为本发明菌株ES-120不同处理对不同植物病原菌的抑制图;
图10为本发明菌株ES-120不同处理方式对植物病害的防治效果图。
具体实施方式
下面通过具体实施例对本发明进行详细地说明。
实施例1
菌株的分离、鉴定与发酵培养:
1、菌株ES-120贝莱斯芽孢杆菌(Bacillus velezensis)的分离:
本发明中编号为ES-120的菌株贝莱斯芽孢杆菌(Bacillus velezensis)分离于盐生植物藜属(Chenopodium album L.),首先,新鲜盐生植物——藜(Chenopodium albumL.)进行次氯酸钠植株表面消毒,整株分解成较小碎片,制备组织匀浆,随后利用稀释涂布平板法进行植物内生微生物的分离,取200μL植物组织匀浆(10-3、10-4和10-5)含盐的Tryptose soya agar(TSA)固体培养基:胰蛋白胨15g/L,大豆蛋白胨5g/L,氯化钠30g/L,琼脂粉18-20g/L,每天观察菌落生长情况,根据菌落颜色、形态、干湿、透明度及边缘等特征挑取有代表性的单菌落纯化培养,获得的纯培养菌株利用20%甘油-20℃保存;选用苹果腐烂病(Valsa mali)与棉花黄萎病(Verticillium dahliae)病原真菌作为平板指示菌,选取PDA培养基,利用平板对峙法方法对拮抗菌株进行筛选,筛选出一株抑菌效果较好的细菌,将该菌命名为ES-120。
2、菌株ES-120的分类鉴定:
(1)形态特征:菌株的ES-120贝莱斯芽孢杆菌(Bacillus velezensis)的菌落形态为圆形,表明光滑湿润,边缘整齐,菌落颜色成乳白色(Marine agar 2216E),菌株的ES-120的电镜照片形态为短杆状,长约1μm,宽约0.5μm,细胞内包含丰富的细胞内容物(图1)。
(2)生理生化特性:菌株ES-120贝莱斯芽孢杆菌(Bacillus velezensis)在Marineagar 2216E、TSA、LB(Lysogeny Broth agar)、R2A等培养基上均可生长,该菌株生长的温度范围为4-37℃,最适pH值范围为5-8,其耐盐能力较好,可在NaCl浓度0-10%(w/v)的范围生长,生理生化指标API 20NE结果显示:菌株ES-120贝莱斯芽孢杆菌(Bacillus velezensis)具有硝酸盐还原、β-葡萄糖甙酶、明胶水解、葡萄糖同化、阿拉伯糖同化、甘露糖同化、N-乙酰-葡萄糖胺同化、麦芽糖同化、苹果酸同化等活性;API ZYM的结果显示:菌株ES-120贝莱斯芽孢杆菌(Bacillus velezensis)具有碱性磷酸盐酶、脂酶(C4)、类磷脂酶(C8)、白氨酸芳氨酶、胰蛋白酶、胰凝乳蛋白酶、酸性磷酸酶、萘酚-AS-BI-磷酸水解、β-糖醛酸甙酶、α-葡萄糖甙酶、β-葡萄糖甙酶等活性。此外,该菌株还具有过氧化氢酶、蛋白酶和几丁质酶活性。Biolog GEN III的结果显示,菌株ES-120贝莱斯芽孢杆菌(Bacillus velezensis)可以利用海藻糖、纤维二糖、龙胆二糖、蔗糖、水苏糖、棉子糖、乳糖、蜜二糖、水杨苷、N-乙酰-D-甘露糖胺、甘露糖、果糖、半乳糖、D/L-岩藻糖、1%乳酸钠、丝氨酸、肌醇、果糖-6-磷酸、明胶、组氨酸、盐酸胍、半乳糖酸內酯、D-葡萄糖醛酸、葡糖醛酰胺、奎宁酸、γ-氨基丁酸,丁酸钠;且该菌株可以在pH 6.0,8%NaCl条件下生长,并对利福霉素、林可霉素、氯化锂、亚碲酸钾、氨曲南等具有抗性。利用LB培养基对菌株ES-120贝莱斯芽孢杆菌(Bacillus velezensis)生长曲线进行测定,结果表明,菌株ES-120于3小时后进入对数生长期,第8小时进入平台期(图2)。
(3)系统发育地位分析:利用细菌通用引物27F和1492R扩增菌株ES-120贝莱斯芽孢杆菌(Bacillus velezensis)的16S rRNA基因序列并测序,获得长度为1344bp的目的片段;经EZBioCloud在线数据库比对,该菌株隶属于Bacillus属,亲缘关系最近的两个种分别为暹罗芽孢杆菌(Bacillus siamensis)和贝莱斯芽孢杆菌(Bacillus velezensis),其16SrRNA基因相似度均为99.9%。随后构建基于16S rRNA基因的系统进化树,结果表明:菌株ES-120与暹罗芽孢杆菌(Bacillus siamensis)和贝莱斯芽孢杆菌(Bacillus velezensis)聚为一枝(图3);为了进一步确定该菌株的分类学地位,对菌株ES-120的基因组进行测定,通过构建基因组系统发育树发现,菌株ES-120贝莱斯芽孢杆菌(Bacillus velezensis)仍然与之亲缘关系最近的贝莱斯芽孢杆菌(Bacillus velezensis)和暹罗芽孢杆菌(Bacillus siamensis)聚为一枝(图4);基因组结果显示:该菌株的基因组由23个片段(contigs)组成,大小为3,867,476bp,GC含量为46.4%(图5A);通过计算菌株ES-120与之亲缘关系较近成员的平均核苷酸一致性可以判断出该菌株与贝莱斯芽孢杆菌(Bacillusvelezensis)为同一种(98.3%);对菌株ES-120,贝莱斯芽孢杆菌(Bacillus velezensis)和Bacillus siamensis进行泛基因组分析表明,三株菌株共有基因为5313个,其中三株菌株共享的核心基因数目为2539个,菌株ES-120贝莱斯芽孢杆菌(Bacillus velezensis)特有的可变基因为277个,这表明这三株菌在基因层面存在一定的差异(图5B);同时基因组共线性分析也显示菌株ES-120与暹罗芽孢杆菌(Bacillus siamensis)和贝莱斯芽孢杆菌(Bacillus velezensis)之间均存在一定的差异(图5C);上述结果表明:菌株ES-120与贝莱斯芽孢杆菌(Bacillus velezensis)NRRL B-41580T属于同一物种的不同菌株;
3、贝莱斯芽孢杆菌(Bacillus velezensis)菌株ES-120的生防促生的功能:
(1)贝莱斯芽孢杆菌(Bacillus velezensis)菌株ES-120对多种植物病原真菌的抑制验证:利用平板对峙法方法对菌株ES-120拮抗病原真菌的能力进行验证。首先,将菌株ES-120分别点接至PDA培养基平板十字等分线的中间位置,保留上部为对照,随后将8种植物病原真菌(苹果腐烂病(苹果黑腐皮壳菌,Valsa mali)、棉花黄萎病(大丽轮枝菌,Verticillium dahliae)、南瓜蔓枯病(瓜拟多隔孢菌,Stagonosporopsiscucurbitacearum)、黄瓜炭疽病(瓜炭疽病菌,Colletotrichum orbiculare)、小麦根腐病(麦根腐平脐蠕孢菌,Bipolaris sorokiniana)、大豆根腐病(立枯丝核菌,Rhizoctoniasolani)、小麦赤霉病(禾谷镰刀菌,Fusarium graminearum)、水稻立枯病(尖孢镰刀菌,Fusarium oxysporium)以及香蕉枯萎病(尖孢镰刀菌,Fusarium oxysporium))接种于平板中间,进行对峙平板培养后观察,结果表明,菌株ES-120对测试病原真菌均呈现较好的抗菌活性(图6);
(2)菌株ES-120贝莱斯芽孢杆菌(Bacillus velezensis)对拟南芥的生防验证:
利用拟南芥验证贝莱斯芽孢杆菌(Bacillus velezensis)菌株ES-120对尖孢镰刀菌(Fusarium oxysporium)Fo5176(拟南芥专化型)的生防效果。体外拮抗试验表明:菌株ES-120贝莱斯芽孢杆菌(Bacillus velezensis)平板菌落与上清对尖孢镰刀菌Fo5176的菌丝体具有抑制作用。与此同时,菌株ES-120贝莱斯芽孢杆菌(Bacillus velezensis)对香蕉专化型尖孢镰刀菌(Fusarium oxysporium)Fo302(香蕉转化型)也表现出良好的活性(图7a);拟南芥模拟染病试验结果表明:与单独接种病原真菌Fo5176相比,同时接种菌株Fo5176和ES-120时,拟南芥植株疾病发生率显著降低(图7b);
(3)菌株ES-120贝莱斯芽孢杆菌(Bacillus velezensis)的植物促生特性:
利用固氮培养基ASHBY和NBF对菌株ES-120贝莱斯芽孢杆菌(Bacillusvelezensis)进行测试,菌株能具有一定的固氮能力,菌株ES-120贝莱斯芽孢杆菌(Bacillus velezensis)还有中等强度的铁载体产生能力,中等强度的溶磷作用(图8a),通过Salkowski比色法测定菌株ES120产生吲哚-3-乙酸(IAA)的能力,结果表明:菌株ES-120贝莱斯芽孢杆菌(Bacillus velezensis)具有产IAA的特征,OD530高达0.859-1.083,且产量相对较高,综合来讲菌株ES-120贝莱斯芽孢杆菌(Bacillus velezensis)具有一定潜力的植物促生作用。
进一步验证菌株ES-120贝莱斯芽孢杆菌(Bacillus velezensis)对模式植物拟南芥的促生能力,利用拟南芥进行促生试验,分别测量拟南芥植株干/湿重量与根长,发现菌株ES-120贝莱斯芽孢杆菌(Bacillus velezensis)处理后可以显著促进拟南芥根部生长(图8b),在菌株ES-120贝莱斯芽孢杆菌(Bacillus velezensis)的培养上清液处理后,拟南芥的湿/干重均显著增加;
4、菌株ES-120贝莱斯芽孢杆菌(Bacillus velezensis)微生物菌剂的制备与使用方法优化:
(1)菌株ES-120贝莱斯芽孢杆菌(Bacillus velezensis)微生物菌剂制备与活性验证:
利用LB培养基对菌株ES-120贝莱斯芽孢杆菌(Bacillus velezensis)进行发酵培养,培养条件如下:选用LB培养基作为发酵培养基:
种子液制备:将菌株接入装有50mL LB培养基的250mL药瓶,温度37℃,180rpm,培养6个小时,使其处于对数期,备用;
发酵液制备:将种子液以2%的比例接入装有500mL LB液体培养基的1L摇瓶,37℃,180rpm,培养24个小时,获得菌株ES-120发酵培养液。
ES-120菌剂制备:将获得的发酵液离心,收集ES-120菌体,然后利用0.22μm细菌滤器过滤,制备无菌上清液,随后将无菌上清液小部分加入到收集的菌体中,制备高密的微生物菌剂。
无菌上清液浓缩液制备:将无菌上清液进行低温冷冻干燥,将获得固体干燥物,收集整合,按体积比甲醇:水=1:1加入溶解液进行固体物质溶解,获得菌株ES-120发酵产物溶液;抗菌试验结果表明:菌株ES-120发酵液作为潜在的微生物菌剂,同样具有良好的生防作用;与此同时,无菌上清液同样对8种植物病原真菌均表现出良好抗植物病原真菌特性(图9);
(2)贝莱斯芽孢杆菌(Bacillus velezensis)菌株ES-120微生物菌剂使用方法:
为了更为全面的了解菌株ES-120微生物菌剂的使用方式,对其施用方式展开摸索;获得ES-120的菌株发酵菌剂分别采用浸根,将植物幼苗浸入1-5×106CFU/mL的菌株发酵液5min,随后幼苗移入无菌土壤和灌根植物幼苗至于无菌土壤中,随后每株植物根部注入1-5×106CFU/g菌株发酵液制备的菌剂两种方式对模式植物拟南芥进行处理,接种48小时后接入病原真菌尖孢镰刀菌拟南芥专化型(Fusarium oxysporium Fo5176)的孢子悬浮液,7d-10d对处理效果进行分析,结果表明,灌根方式对拟南芥植株的生物防护作用更佳(图10)。因此,后期菌剂田间应用可以采用干制粉剂,调整至合适工作浓度,并以灌根的方式应用于大田。
Claims (3)
1.一种盐生植物内生的耐盐贝莱斯芽孢杆菌,其特征在于:该菌株为贝莱斯芽孢杆菌Bacillus velezensis,编号ES-120,已保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC NO. 25530,保藏时间2022年8月15日,该菌株分离于盐生植物——藜Chenopodium album L.。
2.如权利要求1所述的盐生植物内生的耐盐贝莱斯芽孢杆菌在拮抗农作物致病真菌中的应用。
3.如权利要求2所述的农作物致病真菌为苹果腐烂病、棉花黄萎病、南瓜蔓枯病、黄瓜炭疽病、小麦根腐病、大豆根腐病、小麦赤霉病、水稻立枯病以及香蕉枯萎病。
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CN117903986A (zh) * | 2024-01-18 | 2024-04-19 | 东北农业大学 | 贝莱斯芽孢杆菌、菌剂、植物促生长剂、生物肥料与应用 |
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