CN116814489A - 耐碱促生菌bh-7及其在黄瓜栽培中的应用 - Google Patents
耐碱促生菌bh-7及其在黄瓜栽培中的应用 Download PDFInfo
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Abstract
本发明提供了一种耐碱促生菌BH‑7及其在黄瓜栽培中的应用，属于农业微生物技术领域，耐碱促生菌BH‑7的分类命名为嗜碱芽孢杆菌（Bacillus alcalophilus）BH‑7，保藏于中国典型培养物保藏中心，保藏地址：中国武汉武汉大学，保藏日期为2023年6月12日，保藏编号为CCTCC NO:M20231004。该菌株具有较高的耐碱性和良好的促生特性，具有解钾、产铁载体、合成生长素IAA的能力，在碱胁迫下能够显著提高黄瓜的耐碱促生能力，改善土壤养分，提高黄瓜幼苗的抗逆性，可以有效用于盐碱地黄瓜种植，提升黄瓜产量和品质。
Description
技术领域
本发明属于农业微生物技术领域，具体涉及一种耐碱促生菌BH-7及其在黄瓜栽培中的应用。
背景技术
对于植物来说，耐盐碱的特性对于生长发育和产量表现重要，因为高盐、高碱环境下的生长条件对植物生理代谢会产生影响，导致生长发育不良、减产甚至死亡。生物肥料在盐碱地生物改良中扮演着重要角色，例如微生物如盐碱菌等也具有耐盐碱的特性，能够在高盐、高碱的环境下生存和繁殖，对于一些盐碱化土壤的改良和利用具有一定的作用。
目前在黄瓜栽培中，专门针对黄瓜的高效耐碱促生微生物较少。因此，筛选适用于盐碱地黄瓜栽培的高效耐碱促生微生物，对提升黄瓜产量和品质有重要意义。
发明内容
有鉴于此，本发明的目的在于提供一种耐碱促生菌BH-7及其在黄瓜栽培中的应用，该菌株具有较高的耐碱性和良好的促生特性，可以有效促进黄瓜的生长，进而有利于提高其产量和品质。
本发明采用的技术方案如下：
本发明提供了一种耐碱促生菌BH-7，分类命名为嗜碱芽孢杆菌(Bacillusalcalophilus)BH-7，保藏于中国典型培养物保藏中心，保藏地址：中国武汉武汉大学，保藏日期为2023年6月12日，保藏编号为CCTCC NO:M20231004。
本发明从宁夏石嘴山宝丰镇盐碱地采集土壤样品，从中分离出一株具有耐碱促生能力的菌株，经过理化性质鉴定及16S rDNA序列分析，鉴定该菌株为嗜碱芽孢杆菌(Bacillus alcalophilus)，命名为嗜碱芽孢杆菌(Bacillus alcaloph ilus)BH-7，耐碱程度pH＝8.5，其促生特性有：解钾、产铁载体、合成生长素IAA。
本发明还提供所述耐碱促生菌BH-7在碱胁迫下促进黄瓜幼苗生长的应用。
本发明还提供所述耐碱促生菌BH-7在碱胁迫下改良土壤养分的应用。
本发明还提供所述耐碱促生菌BH-7在碱胁迫下提高黄瓜幼苗抗逆性的应用。
本发明还提供所述耐碱促生菌BH-7作为促进黄瓜生长的微生物液体肥料的应用，所述微生物液体肥料中含有所述耐碱促生菌BH-7作为有效成分。
优选的，所述耐碱促生菌BH-7的活菌数为108cfu/mL。
本发明的有益效果：本发明的耐碱促生菌BH-7的最大耐碱程度为pH＝8.5，具有解钾、产铁载体、合成生长素IAA的能力。该菌株能够在碱胁迫下增加黄瓜幼苗的株高、茎粗、叶面积、干重和鲜重，提高了黄瓜的耐碱促生能力；能够显著降低碱胁迫下土壤的电导率、酸碱度，提升土壤速效氮、全氮、全磷含量，有效改善了土壤养分；能够降低碱胁迫下黄瓜幼苗的丙二醛含量，提升抗氧化酶活性，从而减缓碱害，有助于提高黄瓜幼苗的抗逆性。
附图说明
图1为耐碱促生菌BH-7的菌落形态图。
图2为耐碱促生菌BH-7的系统发育树。
图3为耐碱促生菌BH-7解钾检测结果。
图4为耐碱促生菌BH-7产铁载体检测结果。
图5为耐碱促生菌BH-7合成生长素IAA检测结果。
图6为耐碱促生菌BH-7的盆栽促生效果示意图。
图7为碱胁迫下耐碱促生菌BH-7对黄瓜幼苗丙二醛含量的影响。
图8为碱胁迫下耐碱促生菌BH-7对黄瓜幼苗抗氧化酶活性的影响。
具体实施方式
以下结合实施例对本发明作进一步的详细阐述。
本发明提供了一种耐碱促生菌BH-7，分类命名为嗜碱芽孢杆菌(Bacillusalcalophilus)BH-7，保藏于中国典型培养物保藏中心，保藏地址：中国武汉武汉大学，保藏日期为2023年6月12日，保藏编号为CCTCC NO:M20231004。
耐碱促生菌BH-7的筛选：本发明从宁夏石嘴山宝丰镇盐碱地采集土壤样品，装入干净的采样袋，做好标记，于4℃冰箱中保存备用。将采集到的土样称取10g置于90mL灭菌水的250mL三角瓶中，于摇床180r/min震荡30min后，放入80℃恒温水浴锅中静置30min，用1mL移液器从上清液中吸取1mL样品加入盛有9mL无菌水的试管中充分混匀，然后用移液器从此试管中吸取1mL样品加入另一盛有9mL无菌水的试管中，混合均匀，依此类推制成10-1，10-2，10-3，10-4，10-5不同稀释度的溶液后，各吸取0.1mL分别在含10％氯化钠的NA培养基平板上涂布均匀，倒置在30℃恒温箱中培养2-3d，挑取单菌落接于NA培养基上，待长出菌苔后在4℃冰箱保存备用，筛选得到所述耐碱促生菌，命名为BH-7。
试验用到的BH-7菌液制备：在50mL三角瓶中分装10mL的LB液体培养基，封口置于120℃灭菌锅灭菌15min，挑取一环纯菌接种在冷却的培养基中，置于30℃，200r/min摇床内培养24h待用。以菌体OD600值为指标检测BH-7菌株的最适培养基以及发酵条件。
试验用到的培养基：NA培养基，LB培养基，PDA培养基，ADF液体培养基，NBRIP溶磷培养基，阿须贝培养基，甲基红试验培养基，葡萄糖氧化发酵试验培养基，淀粉水解试验培养基，纤维素水解试验培养基，明胶液化试验培养基，铬天青(CAS)培养基，硅酸盐细菌培养基，上述培养基均采用常规方法制备。
用Na2CO3和NaHCO3调节NA培养基pH值及配制碱水，Na2CO3和NaH CO3配比如下表1：
表1：Na2CO3和NaHCO3配比
实施例1、BH-7菌株的鉴定
(1)菌体形态与菌落特征
参看图1，BH-7菌落较小，乳白色，边缘整齐，表面光滑湿润，不透明，幼龄菌体为不规则杆状，端圆。
(2)生理生化特征
表2：生理生化结果
如表2生理生化检测结果可知，BH-7菌株具有氧化酶活性和接触酶活性，葡萄糖氧化发酵为阳性，其余皆为阴性反应。
(3)BH-7菌株的16S rDNA序列分析
进一步的，将分离纯化得到的BH-7菌株通过16Sr DNA序列分析，BH-7菌株的16SrDNA序列如下：
AGACTATACTGCAGTCGAGCGGACCAAAGGGAGCTTGCTCCTGGAGG
TTAGCGGCGAACGGGTGAGTAACACGTGGGCAACCTGCCTGTAAGACTGG
GATAACATCGAGAAATCGGTGCTAATACCGGATAATAAAAAGAACTGCATG
GTTCTTTTTTGAAAGATGGTTTCGGCTATCACTTACAGATGGGCCCGCGGC
GCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGC
CGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGAC
TCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGA
CGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAACTCTGT
TGTTAGGGAAGAACAAGTGCCGTTCGAAAGGGCGGCACCTTGACGGTACC
TAACGAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTA
GGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGT
CTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGG
AAACTGGGAGACTTGAGTACAGAAGAGGAGAGTGGAATTCCACGTGTAG
CGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTC
TCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGG
ATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTAGG
GGTTTCGACGCCCTTAGTGCCGAAGTTAACACATTAAGCACTCCGCCTGGG
GAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCAC
AAGCAGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCA
GGTCTTGACATCCTTTGACCACCCTAGAGATAGGGCTTTCCCCTTCGGGGG
ACAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGT
TGGGTTAAGTCCCGCAACGAGCGCAACCCTTGACCTTAGTTGCCAGCATTC
AGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGG
GATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTAC
AATGGATGGTACAAAGGGTTGCGAAGCCGCGAGGTGAAGCCAATCCCAGA
AAGCCATTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCCG
GAATTGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGC
CTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCG
GTGGGGTAACCTTTTGGAGCCAGCCGCATAAGCGATTTCG。测序结果在NC BI数据库中进行比对，进行了系统发育研究，参看图2，基于同源性比对分析结果，鉴定该菌株为嗜碱芽孢杆菌(Bacillus alcalophilus)，命名为嗜碱芽孢杆菌(Bacillus alcalophilus)BH-7。
实施例2、嗜碱芽孢杆菌(Bacillus alcalophilus)BH-7的耐碱性试验
挑取BH-7单菌落分别接种于pH值为7、7.5、8、8.5、9、9.5的NA培养基上，待长出菌苔后在4℃冰箱保存。结果表明该菌株最大耐碱程度为pH＝8.5。
实施例3、嗜碱芽孢杆菌(Bacillus alcalophilus)BH-7的促生特性检测
(1)解钾能力定性测定
将BH-7菌株在LB液体培养基培养24小时，10000rpm离心5min，吸取10uL菌悬液均匀涂布于硅酸盐细菌培养基平板上，30℃培养若干天，观察其有无油滴状菌体生长，3次重复。
(2)产铁载体能力测定
将BH-7菌株在LB液体培养基培养24小时，10000rpm离心5min，吸取10uL菌悬液接到铬天青CAS蓝色检测平板6mm的无菌滤纸片上，30℃培养若干天，观察其周围有无黄绿色的晕圈出现，3次重复。
(3)合成生长素IAA能力测定
用接种环挑取单菌落接种到含L-色氨酸的(100mg/L)LB液体培养基中，每菌株3个重复，30℃培养1d。取100μL菌液滴加在白色陶瓷板上，加入同体积的Salkowski比色剂，混匀，同时取100μL空白LB液体培养基与同体积Salko wski比色剂混合作为空白处理。在常温避光反应30min后观察颜色变化。若产生红色反应则说明有IAA的产生且红色越深说明IAA产量越高。
最终结果参看图3至图5，嗜碱芽孢杆菌(Bacillus alcalophilus)BH-7具有解钾、产铁载体、合成生长素IAA的能力。
实施例4、嗜碱芽孢杆菌(Bacillus alcalophilus)BH-7对黄瓜的耐碱促生测试
待黄瓜幼苗长出一心一叶后移栽进放有蛭石的小盆钵进行培养，待幼苗培养到三叶一心时，用筛选出的BH-7菌株进行灌根处理，每株灌溉50mL，灌溉浓度为108cfu/mL，对照组分别为清水和碱水灌溉，每株同样灌溉50mL，在BH-7灌溉3天后，所有处理进行pH＝8的碱水处理，每个处理进行三次重复，每个重复30株。待出现碱害后测定其株高、茎粗、叶面积及干鲜重。并称取0.5g不同处理黄瓜幼苗新鲜叶片，使用硫代巴比妥酸法测定丙二醛(MDA)含量，采用愈创木酚法测定过氧化物酶(POD)活性。
处理1：50mL清水
处理2：50mL 108cfu/mLBH-7菌株重悬液
处理3：50mL pH＝8的碱水
处理4：50mL pH＝8的碱水+50mL 108cfu/mLBH-7菌株重悬液
结果参看图6和表3，与清水对照组相比(处理1)，加入BH-7菌株后(处理2)，黄瓜幼苗的株高、茎粗、叶面积及干鲜重分别增加了30.77％，6.81％，9.97％，23.68％和20.20％，表明BH-7菌具有促生效果；碱胁迫下(处理3)，加入BH-7菌株后(处理4)，缓解了碱害对黄瓜幼苗生长的抑制，其株高、茎粗、叶面积及干鲜重相比对照组分别增加了24.32％，8.04％，146.85％，92.68％和92.53％，表明BH-7菌能够在碱胁迫下促进黄瓜幼苗的生长。
表3：BH-7菌株对黄瓜幼苗的促生效果
从表4可以看出，在碱胁迫下(处理3)，加入BH-7菌株能够降低土壤酸碱度(pH值)及土壤电导率(EC值)，分别降低了5.16％、3.85％，从而缓解碱害；同时BH-7菌株在碱胁迫下能够提高土壤速效氮和全氮及全磷含量，分别提升1.66％、16.28％、11.96％，改善土壤养分状况。
表4：BH-7菌株对基质理化性质的影响
丙二醛(MDA)是膜脂过氧化的最终分解产物，其含量可以反映植物遭受逆境伤害的程度。植物在正常情况下，MDA含量很低，而在逆境情况下，会随着时间的推移和逆境的积累，MDA含量显著增高，植物遭受逆境伤害越严重。植物在逆境条件下会产生大量活性氧类而危及植物正常的生理活动，而过氧化物酶(POD)能及时清除植物在正常代谢过程中或逆境胁迫下产生的过多超氧化物自由基，以维持细胞内有益而无害的低水平活性氧，保护植物细胞免遭氧化损伤，提高植物抗逆性。
参看图7，在正常环境中(处理1)，黄瓜幼苗MDA含量为0.7708umol/g，加入BH-7菌株处理后，降低了黄瓜幼苗MDA含量，降低了21.00％；碱胁迫下(处理3)，黄瓜幼苗MDA含量较高，为3.8070umol/g，而加入BH-7菌株处理后，MDA含量降低至1.3926umol/g，降低了63.42％。参看图8，在正常环境中，黄瓜幼苗POD含量为9094.58U/g，加入BH-7菌株处理后，POD含量为13909.35U/g，提升了52.94％；碱胁迫下(处理3)，黄瓜幼苗POD含量较低，POD含量为534.98U/g，而加入BH-7菌株处理后，POD含量显著增加至13766.69U/g，增加了2473.31％。上述结果表明BH-7菌株对黄瓜幼苗MDA和POD含量有影响，且能够在碱胁迫下有效降低MDA的积累，提高POD活性，从而缓解碱害。
Claims (6)
1. 一种耐碱促生菌BH-7，其特征在于：所述耐碱促生菌BH-7的分类命名为嗜碱芽孢杆菌（Bacillus alcalophilus）BH-7，保藏于中国典型培养物保藏中心，保藏地址：中国武汉武汉大学，保藏日期为2023年6月12日，保藏编号为CCTCC NO:M20231004。
2.如权利要求1所述的耐碱促生菌BH-7在碱胁迫下促进黄瓜幼苗生长的应用。
3.如权利要求1所述的耐碱促生菌BH-7在碱胁迫下改良土壤养分的应用。
4.如权利要求1所述的耐盐促生防病菌B21在碱胁迫下提高黄瓜幼苗抗逆性的应用。
5.如权利要求1所述的耐碱促生菌BH-7作为促进黄瓜生长的微生物液体肥料的应用，所述微生物液体肥料中含有所述耐碱促生菌BH-7作为有效成分。
6.如权利要求5所述的应用，其特征在于：所述耐碱促生菌BH-7的活菌数为108cfu/mL。
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