CN116333928B - 一种碱蓬根际促生菌的分离及其应用 - Google Patents
一种碱蓬根际促生菌的分离及其应用 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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Abstract
本发明公开了一种碱蓬根际促生菌的分离及其应用,属于微生物技术领域,本发明选取碱蓬,对其根系土壤中的微生物进行筛选分离,筛选出具有促生功能的根际促生菌并命名为Sugl‑6,菌株的保藏编号为GDMCCNO:63071。将接种Sugl‑6菌株的紫花苜蓿进行盐胁迫处理,与对照组相比,其发芽率、株高、地上鲜重、地上干重、根长、根鲜重和根干重均有明显增加,说明Sugl‑6菌株具有良好的耐盐能力,具有很好的应用潜力。上述方案解决了紫花苜蓿在盐胁迫下不能萌发的问题,为紫花苜蓿在盐碱地种植以及盐碱地改良探索提供依据。
Description
技术领域
本发明涉及微生物技术领域,尤其涉及一种碱蓬根际促生菌的分离及其应用。
背景技术
土壤盐渍化严重影响农作物的生长发育及作物产量,是限制农业生产发展的重要因素之一。土壤盐渍化从多方面影响植物生长,其中包含抑制种子萌发、幼苗生长、植物形态的生长发育以及次生代谢物的形成。在土壤盐渍化日益严峻的背景下,如何制定有效的策略来改良土壤盐渍化问题成为重要环节。针对土壤盐渍化的治理和改良,目前的主要方式为建立完善的农田灌溉水利设施、施加化学改良剂减少土壤盐分的累积、种植耐盐植物提高盐土利用率等。通过生态、生物措施来改良盐渍化是热门方向,不仅促进土壤降低含盐量,提高农作物的产量,更重要的是投资成本低还节能环保,是农户优先选择的技术措施。
随着人们对生物措施的研究,根际微生物受到了人们的关注,而其中的植物根际促生细菌(plant growth promoting rhizobacteria,PGPR)可以提高植物对干旱、盐等逆境胁迫的抵抗能力,促进植物生长,已经成为一种很重要的生物改良措施。植物根际促生菌是一类可以分泌表面活性剂、植物生长激素等物质的有益菌类。具有一定耐盐能力的根际促生细菌对盐生植物生长起到一定的促进作用。利用植物根际促生菌的作用机制可以改善土壤成分,使得土壤肥力越来越适合植物生长,进一步提高植物修复的效率。
紫花苜蓿(Medicago sativa)是世界种植的一种多年生的豆科牧草,其生态适应性广,有“牧草之王”的美誉,不仅具有丰富的营养价值,适口性好,还具有较强的耐旱能力,是我国大面积种植的优质牧草,因此可以利用紫花苜蓿修复盐碱地土壤。但紫花苜蓿属于中等耐盐胁迫的植物,耐盐能力较弱,盐胁迫是限制紫花苜蓿生产的重要影响因子,在土壤pH>8.3的条件下,紫花苜蓿的种子不能萌发,在盐碱草地推广种植受到限制。
因此需要寻找一种耐盐碱的根际促生菌,以促进紫花苜蓿在盐胁迫下种子发芽和幼苗生长发育及生理代谢,为紫花苜蓿在盐碱地种植以及盐碱地改良探索提供依据。
发明内容
有鉴于此,本发明所要解决的技术问题为:为了挖掘和筛选能促进紫花苜蓿在盐胁迫下种子发芽和幼苗生长发育及生理代谢的PGPR菌株资源,并将其应用于植物修复土壤中,本发明目的之一是提供一种具有抗盐碱、促生功能的根际促生菌,以及该根际促生菌的分离方法,另一个目的是提供该菌株的应用。
本发明通过以下技术手段解决上述技术问题:
一种碱蓬根际促生菌,是源于碱蓬根系土壤的根际促生菌Sugl-6,为泛菌属,分类学名称为Pantoea sp.,于2022年12月22日保藏于广东省微生物菌种保藏中心,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,保藏编号为GDMCC NO:63071。
所述菌株的16S rRNA基因序列如序列1所示,具体为:
“5′-GTAGCACAGAGGAGCTTGCTCCTTGGGTGACGAGTGGCGGACGGGTGAGTAATGTCTGG GGATCTGCCCGATAGAGGGGGATAACCACTGGAAACGGTGGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTCACTATCGGATGAACCCAGATGGGATTAGCTAGTAGGCGGGGTAATGGCCCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGCGGTGCGGTTAATAACCGCGCCGATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGG GTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTTAAGTCAGATGTGAAATCCCCGGGCTTAACCTGGGAACTGCATTTGAAACTGGCAGGCTTGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTATATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTTCCCTTGAGGAGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCACGGAATTCGGCAGAGATGCCTTAGTGCCTTCGGGAACCGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGATTCGGTCGGGAACTCAAAGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCACAAAGTGCGTCGTAGTCCGGATCGGAGTCTGCAACTCGACTCCGTGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAG-3′”
进一步,所述菌株的菌落特征为:其培养初期菌落呈亮黄色,表面光滑,为扁平的圆形。
本发明的第二个目的是提供根际促生菌Sugl-6的分离方法,具体步骤如下:
(1)样品采集与富集培养:采集野生碱蓬,取根际土样于装有含盐液体培养基的锥形瓶中,用封口膜封住瓶口,置于摇床中进行富集培养24h;
(2)分离纯化:对菌液进行梯度稀释得到稀释液,吸取稀释液涂布于含盐培养基中,密封培养皿,于28℃恒温培养箱中培养60-96h,对长出的菌落进行初步筛选,挑取单菌落,经过2-4次平板划线纯化获得单一纯化菌株,将获得的菌株储存于-80℃冰箱。
本发明的第三个目的是提供根际促生菌Sugl-6的应用。
进一步,所述碱蓬根际促生菌在改善土壤生态中的应用。
更进一步,所述改善土壤生态具体为:土壤解磷、解钾的应用。
更进一步,所述改善土壤生态具体为:土壤固氮的应用。
进一步,所述的碱蓬根际促生菌还通过分泌铁载体和脱氨酶活性促进盐胁迫下植物种子萌发和幼苗生长的应用。
进一步,所述植物为紫花苜蓿。
有益效果:
本发明植物根际促生细菌Sugl-6能够分泌生长调节物质促进植物的生长,而且还具有解磷、解钾、固氮、产铁载体的能力,脱氨酶活性高,能提高作物营养。本发明将分离出的Sugl-6菌接种到紫花苜蓿上,观测其在不同浓度盐胁迫下,发现能够促进种子发芽和幼苗生长发育及生理代谢,为紫花苜蓿在盐碱地种植以及盐碱地改良探索提供依据。
附图说明
图1:Sugl-6菌株的菌体及菌落形态;
图2:凝胶电泳图;
图3:Sugl-6菌株的IAA活性检测;
图4:Sugl-6菌株对紫花苜蓿生物量的影响((a)Sugl-6菌株对紫花苜蓿株高的影响;(b)Sugl-6菌株对紫花苜蓿地上鲜重的影响;(c)Sugl-6菌株对紫花苜蓿地上干重的影响;(d)Sugl-6菌株对紫花苜蓿根长的影响;(e)Sugl-6菌株对紫花苜蓿根鲜重的影响;(f)Sugl-6菌株对紫花苜蓿根干重的影响)
具体实施方式
以下将结合具体实施例对本发明进行详细说明:
本发明提供了一种碱蓬根际促生菌,并提供了该促生菌的分离鉴定方法和应用,在进行碱蓬根际促生菌的分离鉴定前,需要先制备在分离过程中所用的培养基,其中所用原料均为市售,如实施例1所示:
实施例1:培养基的制备
(1)含盐液体培养基:称取氯化钠50g、牛肉膏5g、胰蛋白胨10g,调节pH至7.0-7.2,去离子水定容到1L。
(2)含盐固体培养基:称取氯化钠50g、牛肉膏5g、胰蛋白胨10g、琼脂20g,调节pH至7.0-7.2,去离子水定容到1L。
(3)LB培养基:称取氯化钠10g、酵母膏5g、胰蛋白胨10g、琼脂15g,1M氢氧化钠溶液调节pH至7.0-7.2,去离子水定容到1L。
(4)无机磷培养基(NPA):称取葡萄糖10.0g、硫酸镁0.3g、硫酸铵0.5g、硫酸锰0.03g、氯化钾0.3g、硫酸亚铁0.03g、氯化钠0.3g、磷酸钙10.0g、琼脂15.0g,用1M氢氧化钠溶液调节pH至7.1,去离子水定容到1L。
(5)有机磷培养基(OPA):称取葡萄糖10.0g、硫酸铵0.5g、硫酸镁0.3g、硫酸锰0.03g、氯化钾0.3g、硫酸亚铁0.03g、氯化钠0.3g、碳酸钙5.0g、卵磷脂0.2g、琼脂15.0g,1M氢氧化钠溶液调节pH至7.1,去离子水定容到1L
(6)固氮培养基:称取甘露醇10g、硫酸镁0.2g、硫酸钙0.1g、磷酸二氢钾0.2g、碳酸钙5g、琼脂20g,1M氢氧化钠溶液调节pH至7.3;去离子水定容到1L。
(7)解钾培养基:称取磷酸氢二钠2g、氯化铁0.005g、碳酸钙0.1g、硫酸镁0.5g、钾长石粉1g、蔗糖5g、琼脂18g,1M氢氧化钠溶液调节pH至7.1,去离子水定容到1L。
(8)CAS检测培养基:取1mmol/L硫酸镁2mL、1mmol/L氯化钙100μL、质量浓度为20%的蔗糖溶液1mL、10%酸水解酪素3mL、琼脂2.0g,在60℃时缓慢加入5mL磷酸盐缓冲液和5mLCAS染液。
实施例2:碱蓬根际土壤微生物的分离筛选与鉴定
1、分离
(1)样品采集与富集培养:在东北松嫩盐碱草地采集野生碱蓬,挖取植物根部,将其装入无菌袋中,用无菌刷刷取碱蓬根际周围土样,取5g根际土样于装有100mL含盐液体培养基的锥形瓶中,用封口膜封住瓶口,置于180r/min,28℃的摇床中,对其进行富集培养24h;
(2)分离纯化:富集培养后,对菌液进行梯度稀释,稀释倍数为10-2、10-3、10-4、10-5。吸取稀释液,采用稀释涂布平板法涂布于含盐固体培养基中,密封培养皿,倒置于28℃恒温培养箱,培养96h。观察菌落生长状态,待菌落长出后,根据菌落的形态、大小、颜色等进行初步筛选,挑取类型不一的单菌落,经过3次平板划线纯化,直至获得单一纯化菌株,将纯化获得的菌株用甘油储存于-80℃冰箱。
对实施例2分离纯化获得的菌株进行鉴定,其中包括形态特征、生理生化分析测定、分子学鉴定、功能性鉴定,如实施例3-6所示:
实施例3:菌株的形态特征
根据所分离菌株纯化培养的菌落特征如图1、表1所示:菌落呈亮黄色,表面光滑,为扁平的圆形。
表1
实施例4:菌株的生理生化分析鉴定
参照《常见细菌***鉴定手册》对菌株生理生化进行分析测定;
经菌株生理生化分析鉴定可知,Sugl-6革兰氏染色、甲基红、精氨酸双水解酶、鸟氨酸脱羧酶、明胶液化鉴定为阴性,荚膜、淀粉水解、接触酶、V-P测定、赖氨酸脱羧酶、柠檬酸盐利用、丙二酸盐、吲哚、乳糖、蔗糖、葡萄糖鉴定为阳性。
实施例5:菌株的分子学鉴定
提取DNA:将目的菌株接种到LB液体培养基中,28℃、150r/min摇床培养24h,试验采用试剂盒提取菌株的DNA(试剂盒选用中国康为世纪的细菌基因组DNA提取试剂盒),具体分离步骤如下:
S1:取细菌培养物1-5ml置于离心管中,12000rpm离心1分钟,尽量吸净上清;向沉淀中加入180μL Buffer GTL,振荡使菌体重悬,加入20μL Proteinase K,涡旋混匀,56℃孵育,直至溶液变清亮,孵育过程中每隔一段时间颠倒或震荡离心管使样本分散;
S2:加入200μL Buffer GL,涡旋震荡充分混匀,加200μL无水乙醇,涡旋震荡充分混匀。短暂离心,使管壁上的溶液收集到管底。将所得溶液(包括形成的沉淀)全部加入到已装入收集管的吸附柱中,若一次不能加完溶液,可分多次转入,12000rpm离心1分钟,弃废液,将吸附柱放回收集管中;
S3:向吸附柱中加入500μL Buffer GW1,12000rpm离心1分钟,倒掉收集管中的废液,将吸附柱放回收集管中,向吸附柱中加入500μL Buffer GW2,12000rpm离心1分钟,倒掉收集管中的废液,将吸附柱放回收集管中。
S4:12000rpm离心2分钟,倒掉收集管中的废液。将吸附柱置于室温数分钟,以彻底晾干,将吸附柱置于一个新的离心管中,向吸附柱的中间部位悬空加入50-200μL BufferGE,室温放置2-5分钟,12000rpm离心1分钟,收集DNA溶液,-20℃保存DNA。
以提取后的DNA作为模板,以16S rRNA通用引物(27F:AGTTTGATCMTGGCTCAG1492R:GGTTACCTTGTTACGACTT)为引物,进行PCR扩增菌株的序列测定。
(2)序列测定:
PCR反应体系(共50μL):DNA 1μL,2×Tsing KE Master Mix 25μL,模板1μL,引物各1μL,添加去离子水至体系总体积为50μL。
PCR反应程序:94℃预变性5min;94℃变性30s,55℃退火30s,72℃延伸90s,循环30次;72℃补延伸10min,4℃降温结束。
凝胶电泳:取2μL扩增好的PCR产物进行1%琼脂糖凝胶电泳(0.2g琼脂糖加入20ml1×TAE)进行条带检测,结果如图2所示。
电泳检测扩增产物后,送至北京睿博兴科生物技术有限公司进行测序。将测序序列结果输入NCBI网站中的BLAST项目进行比对分析,以相似性99%及以上为标准,进行菌种的分子学鉴定,结果表明,本发明菌株Sugl-6为泛菌属,分类学名称为Pantoea sp.,鉴定率为100%。
Sugl-6的16S rRNA序列如序列1所示,具体为:
“5′-GTAGCACAGAGGAGCTTGCTCCTTGGGTGACGAGTGGCGGACGGGTGAGTAATGTCTGG GGATCTGCCCGATAGAGGGGGATAACCACTGGAAACGGTGGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTCACTATCGGATGAACCCAGATGGGATTAGCTAGTAGGCGGGGTAATGGCCCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGCGGTGCGGTTAATAACCGCGCCGATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTTAAGTCAGATGTGAAATCCCCGGGCTTAACCTGGGAACTGCATTTGAAACTGGCAGGCTTGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTATATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTTCCCTTGAGGAGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCACGGAATTCGGCAGAGATGCCTTAGTGCCTTCGGGAACCGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGATTCGGTCGGGAACTCAAAGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCACAAAGTGCGTCGTAGTCCGGATCGGAGTCTGCAACTCGACTCCGTGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAG-3′”
实施例6:菌株的功能性鉴定
a:菌株的溶磷能力的测定
将实施例2中储存的菌株进行平板划线,菌液活化后,在超净工作台中,使用高压灭菌后的竹签挑取目的菌株,并将其接种至OPA和NPA上,采用五点接种法接种,将接种好的培养基封口倒置于28℃培养箱中培养一周,并观察菌株的生长情况和是否有透明圈存在。在一周后对菌株的透明圈直径(D)和菌落直径(d)进行测量,通过比较(D/d)的比值大小,对目的菌株的溶磷能力进行测定。
b:菌株解钾能力测定
将实施例2中储存的菌株进行平板划线,菌液活化后,在超净工作台中,使用灭菌后的竹签挑取目的菌株,并将其接种至解钾培养基中,采用五点接种法接种,将接种好的培养基封口倒置于28℃培养箱中培养一周,观察菌株生长状况,以及包含的透明圈大小,判断菌株的解钾能力。在一周后分别对菌株透明圈直径(D)和菌落直径(d)进行测量,通过比较(D/d)的比值大小,对目的菌株的解钾能力进行测定。
c:菌株固氮能力的测定
将实施例2中储存的菌株进行平板划线,菌液活化后,在超净工作台中,使用灭菌后的竹签挑取目的菌株,并将其接种至固氮培养基上,采用五点接种法接种,将接种好的培养基封口倒置于28℃培养箱中培养一周,观察菌株的生长情况,若菌株生长,则证明菌株具有固氮活性,对菌落直径(d)进行测量并记录,通过比较菌落直径大小,对目的菌株的固氮能力进行测定。
d:菌株铁载体性能的测定
将实施例2中储存的菌株进行平板划线,菌液活化后,分别接种至CAS琼脂培养基进行定性检测,封口倒置于30℃恒温箱培养一周,观察菌落外圈是否产生橘黄色透明圈,并测量所产生透明圈最大直径(D)与菌落直径(d),通过比较(D/d)植,比较菌株产铁载体能力强弱。
e:菌株分泌IAA能力的测定
(1)菌株分泌IAA能力的测定:将目的菌株接种至含有L—色氨酸的LB液体培养基中,放置于180r/min,28℃摇床富集培养24h。配制50mL的35%的高氯酸溶液,1mL的0.5mol/L氯化铁溶液,混合得到Salkowski反应液待用。取50μL菌悬液滴于白色陶瓷板上,加入等量Salkowski显色液进行显色反应,同时以加入等量IAA标准液作为阳性对照,在室温、避光条件下放置显色30min,颜色变红者表示能够产生IAA。
(2)菌液中IAA浓度的测定:活化目的菌株,将其接种至LB液体培养基中,放置于180r/min,28℃摇床富集培养24h。将培养后目的菌液进行4000r/min离心15min,各取等体积的4mL上清液与Salkowski比色液混合。室温遮光放置30min,测定OD530值。通过OD530与IAA浓度的标准曲线计算相应的IAA浓度值。
色氨酸(L-Trp)是植物生长激素吲哚乙酸(IAA)的合成前体,把菌株分别加入0、100、200和500ug/mL色氨酸的培养基中检测合成IAA的情况。以吲哚乙酸为标准样,计算菌株产生IAA量,结果如图3所示。
f:ACC脱氨酶活性测定
菌体蛋白含量测定采用比色法,以α-丁酮酸为标准样,紫外分光光度计测定OD450值,对照α-丁酮酸标准曲线和牛血清白蛋白测定标准曲线,计算菌株的ACC脱氨酶活性。
结果分析:
Sugl-6菌株在NPA培养基及OPA培养基中均可生长,在NPA培养基中平均菌落直径1.92cm,平均透明圈直径4.11cm,D/d为2.14,在OPA培养基中平均菌落直径1.33cm,平均透明圈直径4.09cm,D/d为2.15。在解钾筛选培养基中平均菌落直径1.44cm,平均透明圈直径2.15cm,D/d为1.49。在固氮筛选培养基中平均菌落直径1.69cm。在CAS检测培养基中平均菌落直径1.53cm,平均透明圈直径2.66cm,D/d为1.48。脱氨酶的活性为0.746U/mg。说明Sugl-6菌株具有解磷、解钾、固氮、分泌铁载体以及脱氨酶活性的特性。由图3可知,Sugl-6株菌可以在无L-Trp条件下产生IAA,Sugl-6产生IAA的含量随着培养基中L-Trp浓度的增加而呈现不同程度的显著增加,成正比例关系。
实施例7:苜蓿的发芽及盆栽试验
菌液制备:
将到的Sugl-6菌株接入LB液体培养基中,28℃、180r/min摇床培养24h,离心收集菌体,重悬于已灭菌的30mM硫酸镁溶液中,制成的Sugl-6菌悬液浓度OD600为0.50,备用。
(1)发芽试验:
样品选择与处理:挑选大小适宜,颗粒饱满的紫花苜蓿种子,用70%酒精消毒1min,再用质量分数为1%次氯酸钠消毒10min,得到消毒后的紫花苜蓿种子;
实验分组:
对照组:菌液接种:消毒过的紫花苜蓿种子浸于无菌水中1h;
实验组:菌液接种:消毒过的紫花苜蓿种子浸于Sugl-6菌液中1h;
试验方法:配制10mL去离子水、10mL 25mM氯化钠溶液、10mL 50mM氯化钠溶液、10mL 100mM氯化钠溶液作为处理液灭菌后分别转入已灭菌的空白培养皿中,每个空白培养皿内平铺3层滤纸。将消毒过的紫花苜蓿种子进行菌株接种,置于已灭菌的空白培养皿中。各组25℃培养1周后,测发芽率,结果如表2所示:
表2
(2)盆栽试验:
样品选择与处理:挑选大小适宜、颗粒饱满的紫花苜蓿种子,对其进行灭菌处理,处理方法同发芽试验相同。
实验分组:灭菌后将种子摆放在内置装有无菌滤纸的培养皿内,Sugl-6组每皿接入10mL Sugl-6菌液,对其进行48h浸种处理;Control组每皿接入10mL用无菌水作为对照,对其进行48h浸种处理;
将发芽后的紫花苜蓿种子装入已灭菌的园艺土壤的花盆中,每盆播种10颗紫花苜蓿种子,待其幼苗生长至三叶一心期开始进行菌液处理和盐胁迫处理,每种处理各三次重复。每隔3天盐胁迫处理一次,每次100mL,每隔5天向植株根部浇灌一次菌液,每次20mL,以维持菌群稳定存在,中间定期用自来水浇灌保持土壤水分;待紫花苜蓿生长一个月后对其进行生物量及各项生理指标的测量,结果如图4所示;
其中紫花苜蓿生物量测定方法如下:
a株高:取出紫花苜蓿植株,量取植株根颈部到顶部的距离;
b根长:取出紫花苜蓿植株,量取植株主根长度;
c地上鲜重:取出紫花苜蓿植株,剪去植株根部,截取地上部分植株,每5棵为一组,进行称量;
d地上干重:将截取的紫花苜蓿地上部分,每5棵为一组,用报纸包裹放入烘箱,先105℃杀青30min,然后65℃烘干至恒重,取出称量;
e根鲜重:将剪去的植株根部,每5棵为一组,进行称量;
f根干重:将剪去的植株根部,每5棵为一组,用报纸包裹放入烘箱,先105℃杀青30min,然后65℃烘干至恒重,取出称量。
由表1、图4可得:
表1为Sugl-6菌株对不同盐处理下紫花苜蓿发芽率的影响,可以看出实验组接种Sugl-6菌株的紫花苜蓿种子在0mM、25mM、50mM、100mM氯化钠溶液处理条件下,与对照组相比发芽率分别提高了3.63%、3.90%、10.98%、15.45%。
图4为Sugl-6菌株对不同盐处理下紫花苜蓿生长的影响,其中图4-a为Sugl-6菌株对不同盐胁迫下紫花苜蓿株高的影响,接种Sugl-6菌株后紫花苜蓿的株高在0mM、25mM、50mM、100mM氯化钠溶液胁迫条件下,与Control组相比,分别提高了24.08%、31.14%、39.73%、30.83%。
图4-b为Sugl-6菌株对不同盐胁迫下紫花苜蓿地上鲜重的影响,0mM、25mM、50mM、100mM氯化钠溶液胁迫条件下,接种Sugl-6菌株后与Control组相比,紫花苜蓿地上鲜重分别提高了15.47%、37.66%、31.32%、30.62%。
图4-c是Sugl-6菌株对不同盐胁迫下紫花苜蓿地上干重的影响,在0mM、25mM、50mM、100mM氯化钠溶液的胁迫条件下,接种Sugl-6菌株后与Control组相比,紫花苜蓿地上干重分别提高了28.45%、35.16%、31.76%、38.89%。
图4-d为Sugl-6菌株对不同盐胁迫下紫花苜蓿根长的影响;在0mM、25mM、50mM、100mM氯化钠溶液的胁迫条件下,接种Sugl-6菌株后与Control组相比,紫花苜蓿根长分别提高了34.57%、53.36%、56.01%、44.93%。
图4-e为Sugl-6对不同盐胁迫下紫花苜蓿根鲜重的影响,在0mM、25mM、50mM、100mM氯化钠溶液的胁迫条件下,接种Sugl-6菌株后与Control组相比,紫花苜蓿根鲜重分别提高了9.93%、13.39%、13.79%、43.68%。
图4-f为Sugl-6对不同盐胁迫下紫花苜蓿根干重的影响,在0mM、25mM、50mM、100mM氯化钠溶液的胁迫条件下,接种Sugl-6菌株后与Control组相比,紫花苜蓿根干重分别提高了36.73%、43.01%、62.26%、64.52%。
可见,Sugl-6菌株能促进盐胁迫下植物种子萌发和幼苗生长,尤其是促进紫花苜蓿在盐胁迫下种子发芽和幼苗生长发育及生理代谢,使得紫花苜蓿在盐碱草地后续的推广种植中不再受到限制。
以上实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的宗旨和范围,其均应涵盖在本发明的权利要求范围当中。本发明未详细描述的技术、形状、构造部分均为公知技术。
Claims (3)
1.一种碱蓬根际促生菌,其特征在于,所述根际促生菌已保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC NO:63071。
2.如权利要求1所述的碱蓬根际促生菌在改善土壤生态中的应用,其特征在于,所述改善土壤生态具体为:土壤解磷、解钾以及土壤固氮。
3.如权利要求1所述的碱蓬根际促生菌在促进盐胁迫下植物种子萌发和幼苗生长的应用,其特征在于,所述植物为紫花苜蓿。
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