CN109294945A - 一株小麦内生菌及其应用 - Google Patents
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Abstract
本发明属于微生物应用领域,具体涉及一株小麦内生菌及其应用。具体的技术特征为:一株小麦内生菌,于2018年8月6日保藏于CGMCC,保藏地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,保藏编号为:CGMCC No.13938。该菌可应用与植物病害防治,尤其可针对用于小麦病害及西瓜枯萎病的防治上。本发明提供了一种全新的地杆菌属菌种,在防治植物病害方面有明显作用,还可能存在其它未知作用。本发明提供的地杆菌对多种经济作物的健康生长和增产具有重要的推广价值,还对研究真菌对植物病害的作用具有研究意义和指导价值。
Description
技术领域
本发明属于微生物应用领域,具体涉及一株小麦内生菌及其应用。
背景技术
植物病害是植物栽培面临的主要问题,每年约造成全球作物总产量10%的损失。而80%以上的植物病害又是由真菌引起的,目前针对这类病害,主要依靠化学防治,即通过化学合成的杀菌剂消除和控制真菌病害。但化学杀真菌剂依赖于有限的几种作用模式,长期使用不仅需要不断增加用量,还容易使病菌产生抗性。过去40年,化学杀菌剂用量增加了3倍,严重加剧了环境污染。此外,化学杀菌剂也会杀死土壤中的益虫和有益微生物,并很可能进入食物链。
因此,寻找新的防治方法替代化学防治迫在眉睫,使用内生菌进行生物防治是一种可能的环境友好型植物病害防治策略。内生菌,是指在其生活史的一定阶段或全部阶段,生活于植物的各种组织和器官的细胞间隙或细胞内的细菌。植物与内生菌在长期的共同进化中,形成了一种微妙的关系,内生菌已经成为植物微生态系统的一部分,其存在可增加植物对恶劣环境的适应能力,促进系统生态平衡,保证植物的健康生长。
相对于其它细菌而言,采用内生细菌作为生防因子具有更大的优势:第一,内生细菌占有有利于生防的生态位:植物内生细菌分布于植物的不同组织中,有充足的营养物质,同时受到植物组织的保护作用,不易受外部恶劣环境如强烈日光、紫外线、风雨等的影响,具有稳定的生态环境,相对于其它外源细菌更易于发挥生防作用。第二,内生细菌可以经受住植物防卫反应的作用:病原物侵染植物时,无论感病植株还是抗病植株,寄主植物或多或少地产生一些抗菌物质如植保素、病程相关蛋白、酚类化合物等,而内生细菌与植物长期地生活在一起,对植物产生的抗菌毒性物质有了耐性,相对于其它细菌作为生防菌更具有竞争性。第三,内生细菌与病菌可以直接相互作用:内生细菌系统地分布于植物体根茎叶花果实种子等的细胞或细胞间隙中,它可以直接面对病菌的侵染,对病菌的致病因子或病菌本身发起攻击,降解病菌菌丝或致病因子,产生拮抗物质,或诱导植物产生ISR抑制病菌生长。第四,内生细菌可以作为外源基因的载体:植物内生细菌不仅能主动进入植物体内定殖和传导,还能作为外源基因导入植物的良好载体。
小麦是中国最重要的粮食作物之一,其产业发展直接关系到国家粮食安全和社会稳定。植物病害严重影响小麦的品质和产量,因此如何防治小麦病害是广泛的社会问题乃至国际问题。目前对农作物内生菌研究主要集中于水稻、土豆,玉米等,对小麦内生菌的研究较少。
因此提供一株具有特异性防治小麦病害的内生菌及其应用方法,对小麦在抑制真菌病害及增产上有重大意义。
发明内容
本发明的目的是提供一株对小麦病原真菌有拮抗作用的地杆菌属细菌新种,可以应用于小麦病害乃至其它植物镰刀菌病害的防治上。
为实现上述发明目的,本发明所采用的技术方案是:一株地杆菌,于2018年8月6日保藏于CGMCC,保藏地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,保藏编号为:CGMCC No.13938。
相应的,一株地杆菌,其16Sr DNA序列如SEQ ID NO 1所示。
相应的,所述的地杆菌在植物病害防治中的应用。
优选的,所述地杆菌在防治镰刀菌引起的植物病害中的应用。
优选的,所述应用的条件为:4~40℃、pH=6~9。
优选的,所述应用的条件为:28℃、pH=7。
本发明具有以下有益效果:
1、本发明提供了一种全新的地杆菌属菌种,在防治植物病害方面有明显作用,还可能存在其它未知作用。
2、本发明提供的地杆菌对赤霉菌等有明显抑制作用,可针对性防治西瓜枯萎病;尤其可针对性应用于小麦病害防治上;对多种经济作物的健康生长和增产具有重要的推广价值,还对研究真菌对植物病害的作用具有研究意义和指导价值。
附图说明
图1为CM134L-2菌的菌落形态图;
图2为CM134L-2菌的个体形态图;
图3为CM134L-2菌的系统发育树示意图;
图4为CM134L-2菌抑制赤霉病病原菌生长示意图;
图5为CM134L-2菌抑制西瓜枯萎病病原菌生长示意图;
具体实施方式
1、培养基准备:
(1)富集培养基:
1)溶液1:0.1%2-水柠檬酸钠、0.6%K2HPO4、1.4%KH2PO4、0.2%(NH4)2SO4;
2)溶液2:0.02%MgSO4·7H2O;
3)溶液3:1.1%α-几丁质粉末溶液;
4)将上述三种溶液按体积比为89:1:10比例混合。
(2)几丁质固体培养基:
溶液A 50ml,溶液B 50ml,100倍的微量金属溶液10ml,100倍的维生素混合溶液10ml,酵母提取物1.0g,胶体几丁质10g,琼脂18g,加蒸馏水至1000ml pH=7.0~7.2。其中,各溶液的具体组分如表1所示。
表1几丁质固体培养基成分
胶体几丁质的制备:称取10g片状几丁质于三角瓶中,加入200ml浓盐酸,在磁力搅拌器上搅拌过夜,然后用4℃的水进行水合沉淀,4000r/min离心10min用4℃纯水洗涤至pH为中性。
(3)胰蛋白胨大豆肉汤培养基(TSB):胰蛋白胨1.7%,大豆蛋白胨(0.3%),氯化钠0,5%,pH=7.0~7.2.
(4)胰蛋白胨大豆肉汤琼脂培养基(TSA):胰蛋白胨1.7%,大豆蛋白胨(0.3%),氯化钠0.5%,琼脂1.8%,pH=7.0~7.2.
(5)马铃薯葡萄糖琼脂培养基(PDA):马铃薯200g,葡萄糖20g,琼脂18g,蒸馏水1000ml,自然pH值,115℃灭菌30min。
1、CM134L-2菌的分离和筛选
本发明所使用的菌株由中国科学院成都生物研究所从四川省成都市双流县中农业示范基地(102°54′~104°53′E,30°05′~31°26′N)中的中科麦138号小麦叶中取样,采取的样品放入保鲜袋中,立即送入实验室处理。将采集的样品用自来水冲洗干净,再用无菌水洗涤,自然阴干;将阴干后的植物组织剪成2~3㎜的小段,用体积分数为2~3%的次氯酸钠溶液浸泡5min,取出后用无菌水冲洗5次,将最后一次的洗涤水涂布在PDA平板上,于37℃培养两天,若无菌生长,说明表面消毒彻底。
在菌株分离之前使用富集步骤:将10g小麦(Triticum aestivum)的表面灭菌叶组织与100ml富集培养基混合,并在28℃下培养7天。取富集培养的培养液300ul于3ml的灭菌后的TSB培养基里,按10-1、10-2、10-3、10-4、10-5、10-6梯度进行连续稀释,然后将最小稀释梯度的培养液取200ul涂布在几丁质固体培养基(平板)上,做三个平行。将涂布好的各平板置于28℃培养2~3天后,获得一株亮黄色菌落,命名为CM134L-2。然后再使用TSA(平板)进一步纯化2~3次该菌落,最后在含有20%(v/v)甘油作为保护剂的TSB中冻存在-80℃。
上述菌株已于2018年8月6日保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏编号为:CGMCC No.13938,保藏单位地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,邮编:100101。
2、CM134L-2菌的生理生化特征鉴定
(1)对该菌株进行一系列特征鉴定:参照《伯杰氏细菌鉴定手册》第八版及《常见细菌鉴定手册》中实验方法进行鉴定;其中,碳源利用情况采用Biolog全自动细菌鉴定系统,脂肪酸成分测定用GC-MS分析,酶学特性采用API ZYM试剂条鉴定,底物代谢采用API 20NE试剂条鉴定。参考菌株为:南阳地杆菌Q-4。具体结果如表2、3所示。
表2所述菌株的生理生化特征(API 20NE和常规鉴定实验结果)
实验 | CM134L-2 | Q-4 | 实验 | CM134L-2 | Q-4 |
葡萄糖 | - | - | 吲哚 | - | - |
乳糖 | + | + | 甲基红 | - | - |
L-阿拉伯糖 | + | - | V-P | - | + |
麦芽糖 | - | + | 玉米素 | + | - |
甘露糖 | + | + | 运动性 | + | + |
蔗糖 | + | - | 明胶 | - | - |
纤维二糖 | + | + | 硫化氢 | - | - |
山梨醇 | + | - | 卫矛醇 | + | - |
苹果酸 | + | + | 甘露醇 | - | - |
对硝基-D-甲基半乳糖 | + | w | 精氨酸水解 | - | - |
七叶灵 | + | + | 尿酶 | - | - |
柠檬酸 | - | - | 菌落颜色 | 亮黄色 | 黄白色 |
表3所述菌株的API ZYM酶反应结果
测定的酶 | CM134L-2 | Q-4 | 测定的酶 | CM134L-2 | Q-4 |
碱性磷酸盐酶 | + | + | 苯酚-AS-BI-磷酸水解酶 | + | - |
酯酶(C4) | W | + | α-半乳糖苷酶 | - | - |
类脂酯酶(C8) | W | - | β-半乳糖苷酶 | + | - |
类脂酶(C14) | - | - | β-糖醛酸苷酶 | - | - |
白氨酸芳胺酶 | + | + | α-葡萄糖苷酶 | + | - |
缬氨酸芳胺酶 | + | + | β-葡萄糖苷酶 | + | - |
胱氨酸芳胺酶 | + | + | N-乙酰-葡萄糖胺酶 | + | - |
胰蛋白酶 | + | W | α-甘露糖苷酶 | W | + |
胰凝乳蛋白酶 | + | W | β-岩藻糖苷酶 | - | + |
酸性磷酸酶 | + | - | 对照 | - | - |
表2、表3中,“-”表示阴性;“+”表示阳性;“W”表示弱阳性(弱阳性:与该酶发生反应,但反应较弱)。
(2)从表2可以看出,该菌株的生理生化特征为:该菌株在TSA、NA、R2A、LB培养基上生长良好,菌株可以利用葡萄糖、阿拉伯糖、甘露糖、对硝基-D-甲基半乳糖、纤维二糖、苹果酸盐,不产生脲酶、精氨酸双水解酶等。从表3 API ZYM试验条鉴定发现CM134L-2碱性磷酸盐酶、白氨酸芳胺酶、缬氨酸芳胺酶、胱氨酸芳胺酶、胰蛋白酶、胰凝乳蛋白酶、酸性磷酸酶、苯酚-AS-BI-磷酸水解酶、β-半乳糖苷酶、α-葡萄糖苷酶、β-葡萄糖苷酶、N-乙酰-葡萄糖胺酶等的酶反应为阳性,而类脂酶(C14)、α-半乳糖苷酶、β-糖醛酸苷酶、β-岩藻糖苷酶等反应为阴性,酯酶(C4)、类脂酯酶(C8)、α-甘露糖苷酶等反应呈弱阳性。
(3)通过培养和外观观察发现:该菌株最适生长条件为温度28℃,培养基采用TSA,28℃培养2d后,培养基上的菌落中间凸起,不透明,边缘规则,表面光滑,呈亮黄色状态。该菌株为革兰氏阴性菌,在显微镜下个体形态呈长杆状,无鞭毛、无芽孢生成。其菌落和个体形态分别如图1和图2所示。
(4)通过上述对于CM134L-2菌形态、培养特征观察及生理生化指标测定可以看出,CM134L-2菌虽然具有一些常见地杆菌属(Pedobacter sp.)的共性,但如表2、表3所示,该菌株与参考菌株在形态、底物利用、酶反应等方面具有一定的差异,表明菌株是一种典型的新菌种,综合鉴定为地杆菌属(Pedobacter sp.),从菌种分类角度将该菌种命名为CM134L-2。
3、CM134L-2菌的分子水平鉴定
利用细菌基因组快速提取试剂盒提取所述菌的DNA,引物选用细菌通用引物,细菌通用16S rRNA引物序列:
27F:5′-AGAGTTTGATC(C/A)TGGCTCAG-3′;
1492R:5′-TACGG(C/T)TACCTTGTTACGACTT-3′
所述引物27F/1492R、2×Tap PCR Master Mix均购自生工生物(上海)有限责任公司。PCR扩增程序:95℃预变性5min;95℃变性30s,55℃复性30s,72℃延伸90s,29个循环;最后72℃延伸5min。PCR产物经1.0%琼脂糖电泳,核酸染料染色后荧光可见光凝胶成像分析系统检测。得到的16SrRNA基因片段,通过胶回收纯化,pUC-Tm载体连接,DH5α转化,由金维智生物技术有限公司(苏州)测序分析。
所述菌的16S rRNA测序结果如SEQ ID NO 1所示。将该序列提交NCBI数据库对比鉴定,采用MEGA7.0软件选择模型,以菌株在NCBI工上查询的相近各个种的16SrRNA序列,采用Neighbour-joining方法构建系统发育树,并bootstrap进行分析,重复次数为1000次,所述系统发育树如图3所示。
如图3所示,菌株CM134L-2的16SrDNA序列与数据库Pedobacter nanyangensis Q-4T同源性最高,最高相似性为97.7%;与该属其他标准菌株序列比对最高同源性分别为97.4%(Pedobacter zeaxanthinifaciens TDMA-5T)。经比对,再次鉴定确认,CM134L-2菌确实是地杆菌属(Pedobacter sp)的新种。
4、CM134L-2菌的脂肪酸特征测定
通过气相色谱-质谱法测定CM134L-2菌的总脂肪酸含量和脂肪酸组成。所有菌株的数据都是在28℃培养箱中,在TSA中生长2d后提取测得的。模式菌株为:南阳地杆菌Q-4。结果如表4所示。
表4 CM134L-2菌的脂肪酸特征展示
其中,ND表示未检测出。
表中各值代表该脂肪酸占总脂肪酸的百分比。
Summed features*:是由两到三种脂肪酸组成的一组,这些脂肪酸被一起处理以用于MIDI系统的评估,包括两个具有离散的ECLs的峰值,以及那些没有单独报告ECLs的峰值。其中,Summed feature 1是C13:0 3-OH和/或C15:1 i H的组成;summed feature 3是C16:1ω6c和/或C16:1ω7c的组成;summed feature 5是s C18:2ω6,9c和/或ante-C18:0的组成;summed feature 9是iso-C17:1ω9c和/或C16:0 10-methyl的组成。
结果显示,CM134L-2菌的主要细胞脂肪酸组成为iso–C15:0,summed feature 3(C16:1ω7c和/或C16:1ω6c)和C16:0,与相似菌株脂肪酸种类、含量上均有差异。通过脂肪酸特征测试,再次证明CM134L-2菌确实是地杆菌属(Pedobacter sp)的新种。
5、CM134L-2菌的生长条件优选
(1)将CM134L-2菌株在不同温度、pH、盐度下,分别在TSB和TSA中进行培养。具体培养条件及在该培养条件下的生长情况如表5所示。
表5培养条件表
其中,“-”表示不生长;“+”表示生长;“++”表示生长良好;“+++”表示生长优。
(2)从表5可知,CM134L-2菌的最佳培养温度为28℃,最适生长pH为7.0,最适盐度为1%。
CM134L-2菌可在常见的TSA培养基中进行增殖培养。利用常规固体斜面培养、低温保藏的方法,每次传代可保藏3个月以上;以干燥冷冻法制做的长期保藏菌种,可保藏1年以上,以20%甘油管在-80℃条件下,可进行长期保藏。
6、CM134L-2菌的抑菌作用
(1)采用对峙培养法测定菌株对赤霉病(小麦病害之一)、西瓜枯萎病的病原菌进行抑制试验。其中,赤霉病病原菌为禾谷镰刀菌(Fusarium graminearum Schw),西瓜枯萎病病原菌为尖孢镰刀菌(Fusarium oxysporum)。
1)菌株的活化:取保存的CM134L-2菌种甘油管10ul涂布于TSA平板上落,于28℃下培养2d,然后挑取单菌落接种于TSB液体培养基里于28℃下培养2天;同时用接种环挑取植物病原真菌的菌丝划线于新的PDA培养基上,于37℃下培养5~7d。
2)挑取步骤1)中得到的病原真菌菌落,利用圆形打孔器取带有病原真菌的菌饼接种于PDA培养基正中央,然后在距真菌1~2cm位置的四周各接种上1)中的新鲜CM134L-2菌液100ul。37℃培养一周,用未接菌的培养基的作为对照,观察有无抑菌效果。
3)接种赤霉病的结果如图4所示;接种西瓜枯萎病的结果如图5所示。从图中可以看出,接种CM134L-2菌的产生了明显抑菌圈,说明CM134L-2菌对赤霉病和西瓜枯萎病的病原菌有明显的抑制作用。
序列表
<110> 中国科学院成都生物研究所
<120> 一株小麦内生菌及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1487
<212> DNA
<213> 地杆菌属(Pedobacter sp)
<400> 1
tacggctacc ttgttacgac ttagccccag ttatcggttt taccctagga cgctccttgc 60
ggttacgtac tttaggtacc cccaacttcc atggcttgac gggcggtgtg tacaaggccc 120
gggaacgtat tcaccgcgtc attgctgata cgcgattact agcgaatcca acttcacggg 180
gtcgagttgc agaccccgat ccgaactgtg aacggctttt tgagattggc acgacattgc 240
tgtctagctg ccctctgtac cgtccattgt agcacgtgtg tagccccgga cgtaagggcc 300
atgatgactt gacgtcgtcc cctccttcct ctctgtttgc acaggcagtc tgtctagagt 360
ccccaccatt acatgctggc aactagacat aggggttgcg ctcgttgcgg gacttaaccc 420
aacacctcac ggcacgagct gacgacagcc atgcagcacc tagtttcgtg tcccgaagga 480
cgcatccatc tctggatact tcactaactt tcaagcccgg gtaaggttcc tcgcgtatca 540
tcgaattaaa ccacatgctc ctccgcttgt gcgggccccc gtcaattcct ttgagtttca 600
cccttgcggg cgtactcccc aggtggaaca cttaacgctt tcgcttagcc gctgaccgtg 660
tatcgccaac agcgagtgtt catcgtttag ggcgtggact accagggtat ctaatcctgt 720
tcgatcccca cgctttcgtg cctcagcgtc aataggacca tagtaagctg ccttcgcaat 780
cggtgttctg tgacatatct atgcatttca ccgctacttg tcacattccg cctacctcta 840
gtccattcaa gcccatcagt atcaagggca ctgcgatggt tgagccaccg tctttcaccc 900
ctgacttaac aggccgccta cgcacccttt aaacccaata aatccggata acgcttggat 960
cctccgtatt accgcggctg ctggcacgga gttagccgat ccttattctt acggtacatt 1020
cagcttcctt cacgaagaaa ggtttattcc cgtacaaaag cagtttacaa cccggagggc 1080
cgtcttcctg cacgcggcat ggctggttca ggcttgcgcc cattgaccaa tattccttac 1140
tgctgcctcc cgtaggagtc tggtccgtgt ctcagtacca gtgtgggggg ccatcctctc 1200
agatccccta gccatcgtag ccttggtggg ccgttacccc gccaactagc taatggcacg 1260
catgcccatc cttctcccat aaatgtttga ccccgtcacg atgccgtgtc gtggtcttat 1320
gcggtattaa tccgaatttc ttcgggctat cccccagaaa agggtaggtt gcatacgcgt 1380
tacgcacccg tgcgccggtc tcagggaaag caagctctcc catacccctc gacttgcatg 1440
tattaggcct gccgctagcg ttcatcctga gccaggatca aactctc 1487
Claims (6)
1.一株地杆菌,其特征在于:于2018年8月6日保藏于CGMCC,保藏编号为:CGMCCNo.13938。
2.一株地杆菌,其特征在于:其16Sr DNA序列如SEQ ID NO 1所示。
3.权利要求1或2所述的地杆菌在植物病害防治中的应用。
4.根据权利要求3所述的地杆菌在植物病害防治中的应用,其特征在于:所述地杆菌在防治镰刀菌引起的植物病害中的应用。
5.根据权利要求3所述的地杆菌在植物病害防治中的应用,其特征在于:所述应用的条件为:4~40℃、pH=6~9。
6.根据权利要求5所述的地杆菌在植物病害防治中的应用,其特征在于:所述应用的条件为:28℃、pH=7。
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