CN116948837A - 根瘤菌gzhc2-2菌株招募根际真菌的方法及在缓解敏感大豆涝害胁迫的应用 - Google Patents
根瘤菌gzhc2-2菌株招募根际真菌的方法及在缓解敏感大豆涝害胁迫的应用 Download PDFInfo
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Abstract
本发明涉及农业微生物技术领域,具体公开了根瘤菌GZHC2‑2菌株招募根际真菌的方法及在缓解敏感大豆涝害胁迫的应用,所述根瘤菌(Rhizobiummultihospitium)GZHC2‑2菌株于2023年1月4日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCCNo:63032。此菌株具有帮助大豆抵抗涝害的能力。在涝害胁迫下接种该根瘤菌可以增加大豆的地上、地下生物量,根瘤数量和根瘤鲜重。同时诱导涝害敏感大豆品种富集有益根际真菌(如Dothideomyceta、Corticiales、Kondoaceae、Cryptococcaceae、Polyporales、Cladosporium)的能力。经过纯化分离根际真菌,被富集的有益真菌也可以显著增加涝害胁迫下大豆的地上地下生物量,根瘤数量和根瘤鲜重。本发明可以用于提高涝害胁迫下大豆的生长和增加产量,具有广阔的应用前景。
Description
技术领域
本发明属于微生物技术领域,具体公开了一种根瘤菌GZHC2-2菌株招募根际真菌的方法及在缓解敏感大豆涝害胁迫的应用。
背景技术
在自然条件下,植物经常暴露于短暂或长期的土壤涝害中,这长期以来被认为是一种主要的非生物胁迫。土壤涝害严重限制了许多植物物种的生长、发育和生存,这不仅发生在自然生态***中,而且也发生在农业***中。涝害是目前世界上许多国家和地区的重大自然灾害之一,世界上遭受涝害的土壤约占世界耕地面积的10%,涝害导致作物减产约20%。在一些作物生产的关键领域,如美国、中国、加拿大、巴西、法国和日本受到的影响尤为显著。我国的热带和亚热带地区因受到季风气候影响,降雨强度大且季节分布不均匀,在长江中下游及南方地区涝害发生频繁,严重影响作物产量。涝害对我国农业的发展造成了严重的影响,因此研究农作物的耐涝性对于提高涝害地区作物的产量具有重要意义。
大豆是世界上最重要的粮食和油料作物之一,也是我国的传统农产品之一。然而,长期的涝害不仅会降低大豆产量和品质,还会导致大豆的根系受损,甚至导致死亡。因此,探索更多有效的方法来帮助大豆抵抗涝害是非常必要的。
发明内容
为了弥补现有技术存在的不足,本发明的目的是根瘤菌(Rhizobiummultihospitium)接种在涝害敏感品种大豆上招募耐涝害根际真菌(Dothideomyceta、Corticiales、Kondoaceae、Cryptococcaceae、Polyporales、Cladosporium)的方法抵抗涝害胁迫的应用。
一种根瘤菌GZHC2-2菌株招募根际细菌的方法,所述根瘤菌(Rhizobiummultihospitium)GZHC2-2菌株于2023年1月4日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No.63032,保藏地址为广州市先烈中路100号大院59号楼5楼广东省科学院微生物研究所;
S1.所述根瘤菌GZHC2-2菌株在根瘤菌培养基YMA(Yeast mannitol agar)中培养;
S2.所述根瘤菌GZHC2-2菌株培养温度为25-35℃、100-150rpm/min培养72-108h;
S3.所述根瘤菌GZHC2-2菌株接种至涝害敏感大豆品种根部;
S4.对接种十天后所述涝害敏感大豆品种的根部进行收集,对所述S3中根际土壤DNA进行ITS扩增子测序;
S5.对S4中的根际土壤中的真菌分离培养并进行测序;
S6.对S5中相对丰度显著增加根际土壤中的真菌进行筛选。
S1.所述根瘤菌GZHC2-2菌株在根瘤菌培养基YMA(Yeast mannitol agar)中培养;
S2.所述根瘤菌GZHC2-2菌株培养温度为25-35℃、100-150rpm/min培养72-108h;
S3.所述根瘤菌GZHC2-2菌株接种至涝害敏感大豆品种根部;
S4.对接种十天后所述涝害敏感大豆品种的根部进行收集,对所述S3中根际土壤DNA进行ITS扩增子测序;
S5.对S4中的根际土壤中的真菌分离培养并进行测序;
S6.对S5中相对丰度显著增加根际土壤中的真菌进行筛选。
优选的,所述根瘤菌GZHC2-2菌株的接种量为2×104-2×106个/株。
优选的,所述S6中相对丰度显著增加根际真菌至少包括有Dothideomyceta、Corticiales、Kondoaceae、Cryptococcaceae、Polyporales、Cladosporium中的任一一种或任意组合。
一种接种根瘤菌GZHC2-2菌株招募根际真菌的方法,在缓解敏感大豆涝害胁迫适应性方面的应用。
与现有技术相比,本发明具有以下优点及有益效果:
本发明中根瘤菌(Rhizobium multihospitium)具有高效的定植能力和较强的耐涝害能力。涝害敏感大豆在涝害胁迫下在接种根瘤菌(Rhizobium multihospitium)生长良好,显著增加了根际真菌(Dothideomyceta、Corticiales、Kondoaceae、Cryptococcaceae、Polyporales、Cladosporium)的丰度,经过纯化分离以上根际真菌,其均可以显著增加涝害胁迫下的根长和地上地下生物量。该根瘤菌可作为缓解大豆耐涝害的有效环境友好型菌剂,对增加大豆在华南地区涝害胁迫土壤的适应性有显著意义。
附图说明
图1为接种GZHC2-1,GZHC2-2,GZHC2-3,GZHC2-4对涝害敏感大豆的生长影响。
图2为接种GZHC2-2菌株后,基于ITS基因测序筛选出的根际富集的微生物物种。
图3为分离出的6个真菌菌株对大豆耐涝害的影响。
图4为混合接种6个菌株对大豆根系转录组的影响。
具体实施方式
以下结合具体实施例和附图来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。除非特别说明,本发明所用试剂和材料均为市购。
本发明实施例所用材料与试剂:PrepMan Ultra Kits核酸萃取剂,购于美国ThermoFisher公司;基因组DNA提取试剂盒:上海生工生物有限公司。
此处需要说明的是,本申请文件中的招募一词,解释为通过接种根瘤菌GZHC2-2菌株后,可对大豆根系的微生物进行富集。
实施例1根瘤菌(Rhizobium multihospitium)GZHC2-2菌株分离与鉴定
1、菌株分离
根瘤菌(Rhizobium multihospitium)分离于耐涝害品种“华春2号”,分离出6株根瘤菌。将根瘤菌置于根瘤菌培养基YMA(Yeast mannitol agar)中(pH为7.2),在28℃、135rpm/min条件下传代培养96小时。随后挑取单菌落在YMA培养基中培养48h后,再以种子液1%接种量接种于YMA培养基中,培养48h,随后在3500rpm/min,4℃离心收集菌体,用pH=7.2的PBS缓冲液洗涤2次后,将菌体重悬于2mLPBS溶液中备用。
2、分子鉴定:
使用核酸萃取剂对GZHC2-2菌株的DNA进行提取,提取后的DNA通过真菌ITS引物扩增。正向引物(ITS1F)序列如SEQ ID NO.2所示:5’-CTTGGTCATTTAGAGGAAGTAA-3’,反向引物(ITS2R)序列如SEQ ID NO.3所示:5’-GCTGCGTTCTTCATCGATGC-3’,扩增后对PCR产物纯化和测序,采用Applied Biosystems 3500基因分析仪进行核酸测序。序列如SEQ ID NO.1所示。
根据ITS的扩增测序,经NCBI中的BLAST进行同源性分析比对,得到菌株GZHC2-2与Rhizobium multihospitium序列相似度为100%,因此,最终鉴定菌株为Rhizobiummultihospitium。
实施例2根瘤菌的接种
将涝害敏感大豆(冀豆17号)种子经酒精消毒后播种在花盆中。盆高30厘米,顶径40厘米,底径30厘米,装土5公斤,置于华南农业大学(113°35′N,23°15′E)的温室内(昼夜温差分别为20℃和28℃)。试验采用完全随机区组设计,在酸性土壤中接种或不接种从HC2号中分离出来的4株根瘤菌。土壤含水量保持在田间容水量的80%。每盆播种8粒大小一致的种子,并在播种后第8天将种子稀释到4粒。对于每个处理,有6个重复(盆),每盆有4株幼苗。播种大豆后待其生长至花期,进行淹水处理:取与种植用花盆等量的相同规格的花盆作为隔水用花盆。将塑料膜裁剪成合适大小的方片,紧贴着隔水用花盆内壁覆盖花盆,用以阻隔水分,防止水分从花盆底下流失,保证淹水处理效果。塑料膜铺好后需要高出花盆1~2cm。确认无破孔漏洞后,在种植用花盆外能直接套入外层隔水用花盆,两花盆之间能紧密贴合。添加水量至淹没土壤表面以上2cm,每日2次定时定量加水,观察并记录生长情况,于水淹后3天后戳破花盆低漏洞处的塑料膜恢复排水,观察4株根瘤菌对大豆耐涝害的效果(图1),结果表明菌株2效果最为明显。收集GZHC2-2菌株处理及对照的根际土壤,用于扩增子测序。为了收集根际土壤,摇动根系以除去附着在根系上的疏松土壤。然后将根系和附着的土壤转移到一个装满磷酸盐缓冲盐水的烧杯中。收集根际土壤混合离心后10克,-80℃保存,用于微生物DNA提取。
实施例3根瘤菌对根际真菌的招募效果
使用Fast DNA SPIN Kit for Soil(MP Biomedicals,SantaAna,CA)提取土壤总DNA。利用正向引物(ITS1)序列如SEQ ID NO.4所示:5′-TCCGTAGGTGAACCTGCGG-3′,反向引物(IST4)序列如SEQ ID NO.5所示:5′-TCCTCCGCTTATTGATATGC-3′。每个PCR反应包含4μL缓冲液,2μL dNTPs(2mM),1μL正/反引物(10μM),10ng DNA和10μLddH2O。然后在ABI 7900***中按照程序进行PCR。95℃30秒;30个循环,95℃15秒,55℃10秒,72℃5分钟。然后,使用QIAquick凝胶提取试剂盒(Qiagen),对PCR产物进行纯化。所有样品以等摩尔浓度汇集,并在Illumina MiSeq平台上进行测序(上海迈瑞生物医药科技有限公司)。真菌ITS基因序列的原始FASTQ文件用QIIME(v1.9.1)和USEARCH(v10.0)处理。引物和质量不高的序列(得分低于20,长度小于200bp)被修剪掉。然后将成对的ITS读数合并成一个文件。使用在扩增子序列变体(ASVs)上训练的Naive Bayes分类器进行分类分析。使用"edgeR"软件包,在R中执行广义线性模型,分析每两个处理之间的微生物和代谢物的差异,然后在火山图中进行可视化,并在P<0.001的显著性阈值。
实施例4根际微生物的分离及人工菌群构建
共从大豆的根际中分离出85株真菌。根据所分离的微生物及测序结果,我们筛选出6株真菌即(Dothideomyceta、Corticiales、Kondoaceae、Cryptococcaceae、Polyporales、Cladosporium),结果表明6株真菌单独接种或混合接种菌有提高大豆抗涝害的能力。
进一步研究被富集的株真菌的生长促进机制,我们对用混合接种菌的大豆根系都进行了RNA-seq分析。结果表明,菌群处理明显增加了控制类固醇生物合成过程,细胞代谢和氧化-还原过程的基因的丰度。
以上所述仅为本发明的较佳实施例,其并非用以限定本发明,凡在不脱离本发明的精神和原则范围内,所做出的任何修改、等同替换、改进等,均应包含在本发明的保护范围内。
Claims (4)
1.一种根瘤菌GZHC2-2菌株招募根际真菌的方法,其特征在于:所述根瘤菌(Rhizobiummultihospitium)GZHC2-2菌株于2023年1月4日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCCNo.63032;
S1.所述根瘤菌GZHC2-2菌株在根瘤菌培养基YMA(Yeastmannitolagar)中培养;
S2.所述根瘤菌GZHC2-2菌株培养温度为25-35℃、100-150rpm/min培养72-108h;
S3.所述根瘤菌GZHC2-2菌株接种至涝害敏感大豆品种根部;
S4.对接种十天后所述的涝害敏感大豆品种根部进行收集,对所述S3中根际土壤DNA进行ITS扩增子测序;
S5.对S4中的根际土壤中的真菌分离培养并进行测序;
S6.对S5中相对丰度显著增加根际土壤中的真菌进行筛选。
2.根据权利要求1所述的一种根瘤菌GZHC2-2菌株招募根际真菌的方法,其特征在于:所述根瘤菌GZHC2-2菌株的接种量为2×104-2×106个/株。
3.根据权利要求1所述的一种根瘤菌GZHC2-2菌株招募根际真菌的方法,其特征在于:所述S6中相对丰度显著增加根际真菌至少包括有Dothideomyceta、Corticiales、Kondoaceae、Cryptococcaceae、Polyporales、Cladosporium中的任一一种或任意组合。
4.权利要求1或3任一所述的一种接种根瘤菌GZHC2-2菌株招募根际真菌的方法,在缓解敏感大豆涝害胁迫适应性方面的应用。
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