CN116333905B - 一株具有促生和产酸功能的巨大芽孢杆菌及其应用 - Google Patents
一株具有促生和产酸功能的巨大芽孢杆菌及其应用 Download PDFInfo
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Abstract
本发明公开了一株具有促成和产酸功能的巨大芽孢杆菌及其应用,本发明的巨大芽孢杆菌(Bacillus megaterium)CNBG PGPR 16生长快,稳定性好,可在大体积装置中放大培养,在产酸方面,巨大芽孢杆菌CNBG PGPR 16在液体产酸培养基中培养1d即出现较明显的显色反应,在固体产酸培养基中培养2天后可明显观察到变色圈,其产酸能力较强。同时,本发明的巨大芽孢杆菌CNBG PGPR 16施用于蓝莓,可以显著降低蓝莓土壤的pH值,对蓝莓枝条长度、粗度、叶面积、地上部鲜重和干重等指标都有着显著的提升,说明该巨大芽孢杆菌在减少土壤pH的同时,能够提高蓝莓植株的各项指标,促进蓝莓植株的生长。
Description
技术领域
本发明属于生物技术领域,具体涉及一株具有促生和产酸功能的巨大芽孢杆菌及其应用。
背景技术
蓝莓(Vaccinium spp.)属杜鹃科越桔属植物又称越桔、蓝浆果,蓝莓果实色泽美观,风味独特,含有丰富的维生素、矿物质和抗氧化物质,具有较高的营养价值和医疗保健作用,被国际粮农组织列为健康食品之一(韩明三等,2010;Lisa et al.,2011)。蓝莓果实多为蓝紫色,柔软多汁,酸甜适度,风味纯美,具有特殊的香气,富含维生素C,有较高的食用价值。蓝莓果实中含有丰富的营养成分,它不仅具有良好的营养保健作用,还具有防止脑神经老化、强心、抗癌软化血管、增强人机体免疫、解除眼睛疲劳、改善视力、抗衰老、防止尿道感染、增强记忆力、抗癌等功能,近年来,随着人们对蓝莓营养价值和保健价值的认识,国内外对蓝莓的需求与日俱增,使其成为21世纪最具发展潜力的水果之一。
蓝莓适宜生长在pH为4.5~5.2的砂质壤土中,根系没有主根,呈纤维状,无根毛。土壤pH值过高容易导致蓝莓发生缺铁失绿症,光合作用受到抑制(宋雷等,2015);而土壤pH值过低时,土壤中的铁、铝活性离子增加,抑制蓝莓根系的生长。此外,蓝莓的栽培基质要求疏松透气,排水性能好,湿润但不积水。目前国内常见大田土壤改良技术是施用东北草炭、松树皮、稻壳、珍珠岩等。施草炭为了增加土壤有机质,增加土壤保水保肥性,但由于东北草炭分解过快,时常达不到这样的改良效果,而且草炭资源有限,价格较贵;施用松树皮一是因为松树皮分解慢,可以增加土壤通气性,二是松树皮分解过程基本保持酸性,三是最终分解后增加土壤有机质。然后这种方法成本较高,降低土壤pH成为蓝莓栽培中最为重要的环节。
微生物可以分泌多种生理活性物质,直接或间接地调节根系环境、刺激植物生长。因此,施用具有产酸功能的菌剂来降低蓝莓土壤pH值,是一个符合现代农业和未来农业发展的重要途径。同时,大多数菌种进入土壤后由于难与土著微生物竞争,很难存活。芽孢杆菌因具有芽孢和较强的耐热抗逆性,在发酵工艺、剂型加工、贮存、繁殖等方面具有优势。巨大芽孢杆菌是芽孢杆菌属的常见菌种,但目前发现具有实际利用价值的巨大芽孢杆菌还仅仅是极少数的一部分,巨大芽孢杆菌生长营养要求低,培养条件简便,适合工业化生产,目前已有部分上市的肥料产品加入了巨大芽孢杆菌,但是目前报道的筛选的巨大芽孢杆菌很多只有单一功能,而且不具备产酸功能。因此,筛选特殊功能的巨大芽孢杆菌,开发有效的微生物肥料,改善土壤环境对绿色农业发展具有重要意义。
发明内容
发明目的:本发明所要解决的技术问题是提供了一株具有促生和产酸能力的巨大芽孢杆菌CGMCC No.23672。
本发明还要解决的技术问题是提供了所述巨大芽孢杆菌在蓝莓种植方面的应用。
技术方案:为解决上述技术问题,本发明提供了一株巨大芽孢杆菌,所述巨大芽孢杆菌CNBG-PGPR-16于2021年10月28日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.23672,分类命名为巨大芽孢杆菌(Bacillus megaterium),保藏地址: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。
本发明内容还包括一种微生物复合制剂,所述微生物复合制剂包含所述的巨大芽孢杆菌(Bacillus megaterium)CNBG-PGPR-16。
本发明内容还包括所述的巨大芽孢杆菌(Bacillus megaterium)CNBG-PGPR-16或所述的微生物复合制剂在促进植物生长中的应用。
本发明内容还包括所述的巨大芽孢杆菌(Bacillus megaterium)CNBG-PGPR-16或所述的微生物复合制剂在生产酸性物质中的应用。
其中,所述植物包括但不仅限于蓝莓。
本发明内容还包括所述的巨大芽孢杆菌(Bacillus megaterium)CNBG-PGPR-16或所述的微生物复合制剂在调节环境中pH中的应用。
其中,所述环境包括培养基环境或土壤环境。
其中,所述培养基包括固体培养基或液体培养基。
其中,所述液体培养基包括葡萄糖1%、酵母粉1%、无水乙醇3%、0.04%溴甲酚紫5%、余量为水,所述固体培养基包括葡萄糖1%、酵母粉1%、无水乙醇3%、0.04%溴甲酚紫5%、琼脂2%,余量为水。
有益效果:与现有技术相比,本发明具有以下优点:本发明的巨大芽孢杆菌(Bacillus megaterium)CNBG-PGPR-16CGMCC No.23672生长快,稳定性好,可在大体积装置中放大培养,在产酸方面,巨大芽孢杆菌CNBG-PGPR-16在液体产酸培养基中培养1d即出现较明显的显色反应,在固体产酸培养基中培养2天后可明显观察到变色圈,其产酸能力较强。同时,本发明的巨大芽孢杆菌CNBG-PGPR-16施用于蓝莓,可以显著降低蓝莓土壤的pH值,对蓝莓枝条长度、粗度、叶面积、地上部鲜重和干重等指标都有着显著的提升,说明该巨大芽孢杆菌在减少土壤pH的同时,能够提高蓝莓植株的各项指标,促进蓝莓植株的生长。
附图说明
图1巨大芽孢杆菌CNBG-PGPR-16在平板上的菌落形态;
图2显微镜100倍镜下巨大芽孢杆菌CNBG-PGPR-16细胞形态;
图3巨大芽孢杆菌CNBG-PGPR-16系统进化树;
图4巨大芽孢杆菌CNBG-PGPR-16生长曲线;
图5巨大芽孢杆菌CNBG-PGPR-16在液体培养基中产酸能力测定;
图6巨大芽孢杆菌CNBG-PGPR-16在固体培养基中产酸能力测定
图7巨大芽孢杆菌CNBG-PGPR-16对蓝莓生长发育的影响。
具体实施方式
下列实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1巨大芽孢杆菌CNBG-PGPR-16的分离、鉴定
(1)样品稀释与涂布
取江苏省南京市江宁区谷里街道蓝卉庄园(N118°40′19″,E31°50′10″),蓝莓种植地土壤样品约0.5g于4.5mL无菌生理盐水中悬浮震荡,即为10-1梯度(十倍稀释),从该稀释液中取出0.5mL于4.5mL无菌生理盐水中悬浮震荡,即为10-2梯度(百倍稀释),以此类推,直至稀释至10-6梯度。分别从6个稀释梯度的稀释液中吸取100μL,涂布于营养琼脂NutrientAgar(NA)固体培养基平板上,平行制备2份,倒置,放于30℃恒温培养箱中培养24-36h,定时观察。
(2)菌株分离与纯化
将同一样品下所有长出菌落的平板取出,选出单菌落明显且数量适中的稀释度平板,将平板上形态不一样的菌落挑至新的NA平板上,并进行分区纯化,重复纯化直至单菌落出现,后再重复划线培养至少5次。
(3)染色与保藏
挑取待检测菌株的菌落于载玻片中央区域,经过草酸铵-结晶紫染液初染、碘液媒染、75%乙醇脱色、番红复染四个步骤后,显微镜镜检观察,记录染色结果;将完成纯化和染色的单菌落从NA固体培养基平板上挑至无菌NA液体培养基中,200r/m、30℃下培养24±2h,
震荡摇匀后,将60%无菌甘油∶菌液按V∶V=1∶1进行混合,分装于无菌保菌管中,标记并置于-80℃冰箱中保藏。
(4)菌株鉴定
将含有对应编号菌株的平板从4℃冰箱中取出,挑取单菌落为DNA模板,进行PCR扩增和16S rDNA测序与序列比对,具体操作如下:
①扩增体系50μL:
50μL体系有以下五部分组成:Taq酶Mix(艾科瑞生物有限公司,货号:AG11019)(25μL)、上游引物27F(1μL)、下游引物1492R(1μL)、DNA模板(1μL)、ddH2O(22μL)。其中27F序列为:5’-AGAGTT TGATCM TGG CTCAG-3’,1492R序列为:5’-GGYTAC CTT GTTACGACT T-3’。扩增出的片段长度为1400bp,扩增出的核苷酸序列见SEQ ID NO:1所示。
②扩增条件
预变性温度:105℃
第一步变性:95℃7min;95℃30s
第二步退火:55℃30s
第三步延伸:72℃90s
循环次数:95℃30s,33次循环
第四步最后延伸:72℃5min
第五步保持:12℃10min
③琼脂糖凝胶电泳
称取1g琼脂糖溶解于100mL TBE电泳缓冲液,微波加热至溶液澄清透明,加入1‰的GelRed核酸染料,摇晃均匀,静置至无气泡,缓慢倒入胶板,静置1h左右,取出凝固的胶块置于电泳槽中,在每个胶孔中加入4μL PCR扩增产物,120V 20min跑胶,取出胶块置于凝胶成像仪中,选择UV光照并拍照,记录胶块上1500bp处条带清晰的对应样品编号,将该样品的剩余PCR扩增产物置于4℃冰箱。
④DNA纯化
将4℃冰箱中贮藏的PCR扩增产物取出,使用AxyPrepTM PCR Cleanup Kit核酸纯化试剂盒纯化PRC产物。
⑤序列鉴定
将上述步骤得到的重悬DNA,送至南京擎科生物科技有限公司测序,将邮件返回的序列输入至NCBI网站的序列检索栏目中进行检索,比对结果,结果显示,本发明提供的菌株为巨大芽孢杆菌(Bacillus megaterium),命名为巨大芽孢杆菌(Bacillus megaterium)CNBG-PGPR-16。根据2020年发表于International Journal of Systematic andEvolutionary Microbiology的一篇论文,论文题目为Robust demarcation of 17distinct Bacillus species clades,proposed as novel Bacillaceae genera,byphylogenomics andcomparative genomic analyses:description of Robertmurrayakyonggiensis sp.nov.and proposal for an emended genus Bacillus limiting itonly to the members of the Subtilis and Cereus cladesof species(DOI 10.1099/ijsem.0.004475),将巨大芽孢杆菌(Bacillus megaterium)重新划分为普里斯特氏菌属(Priestiagen)分支下。
将该巨大芽孢杆菌CNBG-PGPR-16菌株于2021年10月28日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏地址: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,该菌株的保藏编号为:CGMCC No.23672,分类命名为巨大芽孢杆菌(Bacillus megaterium)。
保藏编号为CGMCC No.23672的巨大芽孢杆菌CNBG-PGPR-16的菌落形态如图1和图2,在NA平板培养基表面菌落较大,圆形,整体呈凸透镜状,奶黄色,表面光滑不透明,边缘锯齿状、质地均匀、乳质状。于油镜下观察到的CNBG-PGPR-16的菌体呈长杆状,长度为2.5μm~3.0μm、宽度为1.5μm~1.8μm,呈链状排布,周身无鞭毛,不能运动。革兰氏染色结果为紫色,即为革兰氏阳性菌。
通过比对本发明菌剂16S序列,制作系统进化树进行分析(图3),与阿氏普里斯特氏菌(Priestia aryabhattai)以及辣椒芽孢杆菌(Bacillus zanthoxyli)亲缘关系较近。
实施例2巨大芽孢杆菌CNBG-PGPR-16在NA中的生长曲线
挑取CNBG-PGPR-16单菌落至NA液体培养基中活化,按5%接种量活化2代后,按5%接种量接入第4代NA液体中,置于30℃下培养72h,从0h开始每隔0.5h测定菌液波长600nm处的OD值(吸光值),平行三次测定,以时间为横坐标,吸光值为纵坐标绘制生长曲线。由图4可知,菌株CNBG-PGPR-16在8h就进入对数中期,OD600值为1.115,随后增速放缓,至第28h左右到达稳定期,且稳定期持续稳定时间较长,第34h左右到达最大值,稳定期OD600值在1.55-1.61之间,表明本发明的菌株CNBG-PGPR-16生长速度快,稳定性好(图4)。
实施例3巨大芽孢杆菌CNBG-PGPR-16产酸能力测定
(1)液体培养基产酸能力测定
产酸显色液体培养基的制备:葡萄糖1%(质量分数);酵母粉1%(质量分数);无水乙醇3%(体积分数);0.04%(质量分数)溴甲酚紫5%(体积分数);用盐酸调节pH到6.8;120℃条件下灭菌30min,无水乙醇为灭菌后培养基凉至75℃左右后加入,分装50ml离心管备用。
挑取CNBG-PGPR-16单菌落至NA液体培养基中活化,按5%接种量活化2代后,按5%接种量接入分装好的产酸培养基中,置于30℃、180rpm条件下培养24h,培养结束后进行拍照观察,根据颜色变化程度确定其产酸能力。结果显示,相比于对照CK,本发明所述菌剂紫色变淡,具有明显的产酸能力(图5)。
(2)固体培养基产酸能力测定
产酸显色固体培养基的制备:葡萄糖1%(质量分数);酵母粉1%(质量分数);无水乙醇3%(体积分数);0.04%(质量分数)溴甲酚紫5%(体积分数);琼脂2%(质量分数);用盐酸调节pH到6.8;120℃条件下灭菌30min,无水乙醇为灭菌后培养基凉至75℃左右后加入,倒平板备用,若时间较长可放4℃冰箱保存。
挑取CNBG-PGPR-16单菌落至NA液体培养基中活化后,用移液枪吸取20μL菌液滴在产酸显色固体培养基中心,置于30℃条件下培养48h,培养基结束后进行拍照观察,变色圈大小表示产酸能力的强弱。结果显示,本发明所述菌剂周围变色圈明显,产酸能力强(图6)。
实施例4巨大芽孢杆菌CNBG-PGPR-16促进蓝莓生长情况评价
于江苏省南京市中山植物园蓝莓基地开展菌剂促进蓝莓生长实验。实验设置3个处理,处理1为不处理对照(CK);处理2为本发明的巨大芽孢杆菌CNBG-PGPR-16(Bm)。每个处理选择3盆长势一直的蓝莓组培苗进行浇菌实验,每个月浇菌一次,共计7次,每盆浇菌200ml浓度为107CFU的菌液,不处理浇200mlNA培养基。实验从3月10日开始进行,于10月10日进行采收,采收后测试指标有:株高、地上部鲜重、地下部鲜重、土壤pH、EC、有机质含量。
表1
从表1中可以看出,施用本发明巨大芽孢杆菌CNBG-PGPR-16的菌液后,土壤pH相比于对照出现明显的下降,土壤Ec值也有一定程度的下降,同时,对照菌对土壤pH并没有显著的影响;其次,本发明巨大芽孢杆菌CNBG-PGPR-16在对蓝莓枝条长度、粗度、叶面积、地上部鲜重和干重等指标都较对照有显著差异,说明该巨大芽孢杆菌CNBG-PGPR-16在产酸的同时,能够促进蓝莓植株的生长(图7)。
Claims (6)
1.一株巨大芽孢杆菌(Bacillus megaterium)CNBG-PGPR-16,其特征在于,所述巨大芽孢杆菌(Bacillus megaterium)CNBG-PGPR-16于2021年10月28日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.23672,分类命名为巨大芽孢杆菌(Bacillus megaterium)。
2.一种微生物复合制剂,其特征在于,所述微生物复合制剂包含权利要求1所述的巨大芽孢杆菌(Bacillus megaterium)CNBG-PGPR-16。
3.权利要求1所述的巨大芽孢杆菌(Bacillus megaterium)CNBG-PGPR-16或权利要求2所述的微生物复合制剂在促进植物生长中的应用。
4.根据权利要求3所述的应用,其特征在于,所述植物为蓝莓。
5.权利要求1所述的巨大芽孢杆菌(Bacillus megaterium)CNBG-PGPR-16或权利要求2所述的微生物复合制剂在生产酸性物质中的应用。
6.权利要求1所述的巨大芽孢杆菌(Bacillus megaterium)CNBG-PGPR-16或权利要求2所述的微生物复合制剂在降低土壤中pH中的应用。
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