CN117535171A - 盐胁迫诱导大豆招募的剑菌Ensifer sp.GMS14及其应用 - Google Patents
盐胁迫诱导大豆招募的剑菌Ensifer sp.GMS14及其应用 Download PDFInfo
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Abstract
本发明提供了一种盐胁迫诱导大豆招募的剑菌Ensifer sp.GMS14及其应用，属于微生物技术领域。本发明提供的盐胁迫诱导大豆招募的剑菌Ensifer sp.GMS14，保藏编号为CGMCC No.27824，于2023年07月07日保藏于中国普通微生物菌种保藏管理中心。该剑菌在盐胁迫条件下能够显著增加大豆株重和根重，提高氮磷元素含量；显著降低大豆根系Na+含量，缓解盐胁迫的伤害。该剑菌的获得能够丰富现有微生物制剂的种类，同时有效解决了盐碱土壤贫瘠、作物生长缓慢的问题。
Description
技术领域
本发明属于微生物技术领域，尤其涉及到一种盐胁迫诱导大豆招募的剑菌Ensifer sp.GMS14及其应用。
背景技术
土壤盐碱化对全球土壤健康和可持续农业构成了严重威胁。根据粮食及农业组织调查，超过10亿公顷的土地受到盐渍化的影响，由于全球变暖、工业污染、缺水和不合理农艺措施等，盐渍化土壤面积每年都在增加。对盐碱地进行改良利用是缓解耕地压力、守住耕地红线的重要途径，具有重要的经济和社会价值。
大豆是我国主要油料作物，在我国农业生产中占有重要地位，其特有的固氮作用，能够改善土壤肥力，提高盐碱地的利用率。利用盐碱地后备耕地资源种植大豆，已成为现阶段大豆种植产业发展的重点方向之一。然而大豆对盐分敏感，在盐胁迫下大豆的产量降低。越来越多的研究表明，根际微生物群在促进植物生长和提高植物耐盐性方面发挥着重要作用，也是土壤修复过程中常用的生物接种剂。虽然许多微生物菌剂已被应用于盐碱地土壤改良中，并且取得了一定的成效，然而，大豆根际微生物组对盐胁迫的响应及其在盐碱地复垦中的潜在应用却鲜有报道。
发明内容
本发明提供了一种盐胁迫诱导大豆招募的剑菌Ensifer sp.GMS14及其应用，该剑菌在盐胁迫条件下能够显著增加大豆株重和根重；显著降低大豆根系Na+含量，缓解盐胁迫的伤害。该剑菌的获得能够丰富现有微生物制剂的种类，有效解决了现有菌剂环境适应性不强、耐盐度不高的问题。
为了达到上述目的，本发明提供了一种盐胁迫诱导大豆招募的剑菌Ensifersp.GMS14，保藏编号为CGMCC No.27824，于2023年07月07日保藏于中国普通微生物菌种保藏管理中心。
作为优选，其核苷酸序列如SEQ ID NO:1所示。
本发明还提供了一种根据上述技术方案所述的剑菌Ensifer sp.GMS14在提高大豆耐盐胁迫能力中的应用。
作为优选，在盐胁迫条件下，所述剑菌Ensifer sp.GMS14能够显著增加大豆株重和根重，并降低大豆根系Na+含量。
作为优选，所述剑菌Ensifer sp.GMS14在每盆接种浓度为107CFU/g时，与未接种剑菌Ensifer sp.GMS14处理相比，盐胁迫下大豆株重和根重分别增加了34.35％和43.47％。
作为优选，所述剑菌Ensifer sp.GMS14使用4ml/kg浓度进行大豆拌种田间试验时，与未接种剑菌Ensifer sp.GMS14处理相比，接种剑菌Ensifer sp.GMS14的植株的结瘤数量和重量分别增加了0.8倍和2.8倍，大豆根瘤中总氮含量增加1.95倍。
本发明还提供了一种盐碱地大豆抗盐生物菌剂，以上述技术方案所述的剑菌Ensifer sp.GMS14为主要有效成分。
与现有技术相比，本发明的优点和积极效果在于：
本发明提供了一种由盐胁迫诱导大豆招募的剑菌Ensifer sp.GMS14，该菌在盐胁迫条件下能够显著增加大豆株重和根重；显著提高氮磷元素含量；降低大豆根系Na+含量，从而缓解盐胁迫伤害。该剑菌的获得能够丰富现有微生物制剂的种类，同时有效解决了盐碱土壤贫瘠、作物生长缓慢的问题。。
附图说明
图1为本发明提供的大豆根际微生物群落组成示意图，A为微生物群落在门和属水平上的分布图；B为微生物群落LEfSe富集分析图；
图2为本发明提供的菌株GMS14与其他模式菌株的系统进化关系图；
图3为本发明提供的菌株GMS14在YMA培养基上的菌落形态图；
图4为本发明提供的菌株GMS14的盐度耐受范围；
图5为本发明提供的盐胁迫下接种菌株GMS14对大豆株高(A)、根长(B)、根重(C)和株重(D)的影响，***表示P<0.001；
图6为本发明提供的接种菌株GMS14对大豆叶片(A-C)和根系(D-F)Na+、K+和Na+/K+含量的影响，*表示P<0.05；***表示P<0.001；
图7为本发明提供的接种菌株GMS14对大豆总氮(A-C)和总磷(D-F)的影响，*表示P<0.05；**表示P<0.01；
图8为本发明提供的接种菌株GMS14对田间大豆生长的影响，其中：(A)生长表型；(B)根瘤表型；(C)株高；(D)每株植物的根瘤总数；(E)每株植物的根瘤重量；(F)根瘤总氮含量；Mock表示未接种菌剂，**表示P<0.01；***表示P<0.001。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
YMA培养基(g/L)：酵母提取物20.0，甘露醇5.0，K2HPO4 5.0，MgSO4 0.5，NaCl0.186，CaCO3 1.2，琼脂粉20.0，pH值6.8-7.2。
YMB培养基(g/L)：酵母提取物20.0，甘露醇5.0，K2HPO4 5.0，MgSO4 0.5，NaCl0.186，CaCO3 1.2，pH值6.8-7.2。
盐度测试培养基(g/L)：酵母粉1.0，甘露醇10.0，KH2PO4 0.5，MgSO4 0.2，NaCl0.1，色氨酸0.1，pH值6.8-7.2，不同浓度氯化钠0-50g。
PKO无机磷培养基(g/L)：葡萄糖10.0，(NH4)2SO4 0.5，MgSO4·7H2O 0.1，NaCl 0.2，KCl 0.2，FeSO4·7H2O 0.003，MnSO4·4H2O 0.03，Ca3(PO4)2 5.0，酵母粉0.5，琼脂20.0，pH值7.0-7.2。
有机磷细菌培养基：购买自青岛海博生物技术有限公司。
改良CAS琼脂培养基：购自北京酷来搏科技有限公司。
其它生化试剂及普通化学试剂均为进口或国产分析纯。
实施例1盐胁迫大豆根际微生物组鉴定
本试验以大豆品种中黄13为试验材料，消毒后的种子放在湿润的滤纸上催芽三天。每盆装5kg土壤，播种后置于田间自然环境下培养。待大豆出苗后，分别用0、150mM NaCl进行处理。盐胁迫15天后采集根际土壤样品，并进行16S rRNA基因扩增子测序。
在盐胁迫条件下，大豆根际土壤中变形菌门(Proteobacteria)、放线菌门(Actinobacteriota)和厚壁菌门(Firmicutes)的相对丰度发生了显著变化(图1A)。在属水平上，盐胁迫后大豆根际土中芽孢杆菌属(Bacillus)的相对丰度降低，而类诺卡氏菌属(Nocardioides)、新鞘氨醇菌属(Novosphingobium)和剑菌属(Ensifer)的相对丰度增加(图1A和B)。
实施例2细菌分离培养与分子鉴定
将根际土样品与0.85％生理盐水充分混合(1:9，w/v)。静置后吸取100μL上清液加入到900μL生理盐水中，梯度稀释至10-4。分别吸取10-2和10-4浓度的稀释液100μL涂布于酵母甘露醇琼脂(YMA)培养基上。在28℃下恒温培养3d，根据菌落颜色、形态、表面光滑度等挑取不同的单菌落，经2-3次划线纯化，接种于YMB液体培养基，振荡培养24h至对数生长期，吸取菌液与50％甘油1:1混匀，于-80℃冰箱冷冻保存。
提取细菌基因组DNA，使用通用引物27F和1492R对细菌16S rRNA基因序列进行扩增，27F和1492R如下所示：
27F：5’-AGAGTTTGATCCTGGCTCAG-3’；
1492R：5’-GGTTACCTTGTTACGA CTT-3’。
其中：PCR反应体系为：2×Taq Master Mix 12.5μL、正反向引物(10μM)各1μL、DNA模板1μL、ddH2O 9.5μL，总体系25μL。
扩增条件为：95℃预变性5min；95℃变性1min、55℃退火1min、72℃延伸1.5min，循环30次；最后72℃延伸10min。
将PCR产物送往青岛睿博生物技术有限公司进行测序，所得细菌序列经校对后上传至EzBioCloud数据库(https://www.ezbiocloud.net/)进行比对。比对完成后分别下载同源性最高的模式菌株序列，使用MEGA5.0进行CLUSTALX多重比对，采用邻接法(Neighbor-Joining)构建系统发育树，自举值(Bootstrap)为1000。经比对，菌株GMS14的16S rRNA基因序列与剑菌属(Ensifer)细菌聚为一簇(图2)；因此，初步认定菌株GMS14为剑菌属细菌Ensifer sp.，其16S rRNA基因序列如SEQ ID NO:1所示，保藏编号为CGMCC No.27824，于2023年07月07日保藏于中国普通微生物菌种保藏管理中心。
实施例3菌种选择与定向应用
3.1剑菌GMS14的菌落形态
在YMA培养基上培养48h后，菌落特征为乳白色、湿润、有粘液、表面光滑、边缘整齐(图3)。
3.2剑菌GMS14的耐盐能力和促生指标
(1)耐盐能力测定：配置不同NaCl浓度(0-5％)的培养基，高压灭菌后分装于96孔培养板中，按照1％的接种量接种GMS14菌液，24h后用酶标仪测定培养液的OD值。
(2)溶磷活性测定：将10μL OD600＝0.6的菌液接种到PKO无机磷和有机磷细菌培养基上，28℃培养3天，观察菌落周围是否形成透明圈，用增溶指数(SI)评估菌株的增溶作用，计算公式如下：溶解指数(SI)＝(晕+菌落直径)/菌落直径。
(3)铁载体产生能力评价：将GMS14菌株接种到CAS琼脂培养基，30℃培养7d，观察细菌菌落周围的颜色变化，颜色从蓝色变为橙色或者深黄色，表明菌株可以产生铁载体。
(4)胞外多糖产生能力：将菌株GMS14接种至NB培养基，过夜培养后，静置48h(OD600＝1.5)。随后取200μL菌液与200μL苯酚(5％)、1mL浓硫酸混匀，在波长485nm下检测OD值。同时，取200μL不同浓度的葡萄糖做标准曲线，将菌液的OD485值带入标准曲线，计算胞外多糖产生量。
结果发现，菌株GMS14在3.5％的NaCl浓度下仍然生长，其中最适NaCl浓度为1.0％(图4)，具有溶解有机磷的活性；且具有较强的产生长素能力(86.67mg/L)；能够产生胞外多糖(0.31mg/mL)；不产ACC脱氨酶，不具有溶解无机磷和产铁载体能力。
3.3剑菌GMS14提高大豆在盐胁迫条件下的生长
(1)农艺性状
将剑菌GMS14接种至YMB培养基，28℃，180r/min下摇床培养24h。4℃，6000r/min离心5min，用无菌水重悬至OD600＝0.5。大豆种子用3％次氯酸钠溶液消毒处理，随后放在湿润的滤纸上催芽三天。每个小盆装120g蛭石，每盆放入4颗萌发的大豆种子，置于人工气候室培养。种植大豆时每盆接种浓度为107CFU/g，间隔2d再接种1次。第2次接种完毕3d后用150mM NaCl进行胁迫处理，对照组浇灌等量无菌水，每组设置6个重复。培养15天后测量大豆的株高、根长、株重和根重。
结果发现，盐胁迫下菌株GMS14显著增加大豆株重和根重(图5C和D)，分别增加了34.35％和43.47％；显著提高大豆株高，为14.05％(图5A)，根长变化不显著(图5B)，说明菌株GMS14通过促进大豆生长和增加根系生物量帮助其提高耐盐性。
(2)钠钾离子
称取烘干后的叶片和根系样品约0.05g，放入玻璃管中，加入1mL硝酸，110℃消解成澄清透明的溶液，转移至5mL离心管，加蒸馏水定容至5mL。吸取适量定容后溶液过0.45μm滤膜，得到2mL待测液，利用Optima 8000型等离子发射光谱仪进行检测。
结果发现，盐胁迫下接种GMS14显著降低了叶片和根系中Na+含量，同时降低了根系Na+/K+比值(图6)，推测GMS14可能通过提高根系对K+吸收来抑制Na+在根中过量累积，从而缓解盐胁迫伤害。
(3)总氮浓度
烘干后的叶片、茎和根系样品用研磨仪磨成粉末，称取2.5g左右，放在75μL通用软银容器内包好，用元素分析仪测定总氮浓度。
结果发现，在盐胁迫条件下，接种GMS14显著提高了大豆根系和三叶中总氮浓度，其中根系总氮浓度提高了13.36％，叶片中总氮浓度提高了17.85％，且大豆根系的氮素吸收能力显著提高了34.78％(图7A-C)。
(4)总磷浓度
称取0.1g左右烘干叶片放入玻璃管中，加入4mL硝酸，120℃消解2h，放凉后用去离子水稀释10倍，混匀备用。采用钒钼黄吸光光度法测定叶片总磷浓度。
结果发现，与对照组CK相比，盐胁迫下接种GMS14后，大豆各部位总磷浓度均显著提高，其中根系提高了191.45％，叶片提高了40.13％，且大豆根系对磷的吸收能力提高了41.90％(图7D-F)。
实施例4剑菌GMS14的田间大豆应用
通过田间试验，进一步评价菌株GMS14对盐渍土中大豆产量的影响。将GMS14菌液(4ml/kg)加水与大豆种子混匀搅拌，对照组为等量清水进行拌种，拌好的种子平摊风干，当天播种完毕。
结果发现对照组的大豆植株矮小、叶片发黄，接种GMS14的植株表现出显著的生长优势(图8A和C)。另外，GMS14显著提高了大豆的结瘤能力(图8B)，与未接种GMS14处理相比，接种GMS14植株的结瘤数量和重量分别增加了0.8倍和2.8倍(图8D和E)，同时提高了大豆固氮能力，接种GMS14的大豆根瘤中总氮含量增加了1.95倍(图8F)。
综上盆栽和田间试验均表明，菌株GMS14明显提高了大豆的耐盐性，为实现大豆的盐碱地种植提供了技术支持。
Claims (7)
1.盐胁迫诱导大豆招募的剑菌Ensifer sp.GMS14，其特征在于，保藏编号为CGMCCNo.27824，于2023年07月07日保藏于中国普通微生物菌种保藏管理中心。
2.根据权利要求1所述的剑菌Ensifer sp.GMS14，其特征在于，其核苷酸序列如SEQ IDNO:1所示。
3.根据权利要求1或2所述的剑菌Ensifer sp.GMS14在提高大豆耐盐胁迫能力中的应用。
4.根据权利要求3所述的应用，其特征在于，在盐胁迫条件下，所述剑菌Ensifersp.GMS14能够显著增加大豆株重和根重，并降低大豆根系Na+含量。
5.根据权利要求4所述的应用，其特征在于，所述剑菌Ensifer sp.GMS14在每盆接种浓度为107CFU/g时，与未接种剑菌Ensifer sp.GMS14处理相比，盐胁迫下大豆株重和根重分别增加了34.35％和43.47％。
6.根据权利要求3所述的应用，其特征在于，所述剑菌Ensifer sp.GMS14使用4ml/kg浓度进行大豆拌种田间试验时，与未接种剑菌Ensifer sp.GMS14处理相比，接种剑菌Ensifersp.GMS14的植株的结瘤数量和重量分别增加了0.8倍和2.8倍，大豆根瘤中总氮含量增加1.95倍。
7.盐碱地大豆抗盐生物菌剂，其特征在于，以权利要求1或2所述的剑菌Ensifersp.GMS14为主要有效成分。
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