CN117106668A - 一株耐盐碱促生天津假单胞菌p522及其应用 - Google Patents
一株耐盐碱促生天津假单胞菌p522及其应用 Download PDFInfo
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Abstract
本发明公开一株耐盐碱促生天津假单胞菌P522及其应用,属于农业资源与环境微生物领域。本发明筛选得到的耐盐碱促生天津假单胞菌P522菌株能在盐碱土中稳定定殖,同时该菌株还具有产ACC脱氨酶、产吲哚乙酸(IAA)、产铁载体、解无机磷、形成生物膜等性能,有效丰富了旱作作物促生菌的菌种资源。该菌株能显著促进盐碱旱作作物种子发芽和幼苗生长,具有良好的实际应用价值。
Description
技术领域
本发明涉及农业微生物应用技术领域,特别是一株耐盐碱促生天津假单胞菌P522及其应用。
背景技术
我国盐碱地总面积约9900万公顷,将盐碱地转变为可利用耕地以及充分利用盐碱性退化耕地是盐碱地区农业的重中之重。
多数盐碱地区水资源缺乏,因此以旱作粮油作物和蔬菜为主。盐碱地区旱作作物在生产中直播时和幼苗移栽后面临的首要问题是盐碱下种子发芽率低以及幼苗存活率低,进而引起盐碱旱作作物不同程度播种无效和幼苗死亡或僵苗,需要重新播种或移栽幼苗,对作物生产带来很大的农事负担,经济利益下降严重。
土壤中的微生物与作物具有互作关系,可在作物种子发芽和幼苗生长上发挥显著的促进作用,进而提高最终产量与经济效益。但目前,尚未有针对盐碱旱作作物种子发芽和幼苗生长的促生功能菌。假单胞菌属隶属于细菌域,变形菌门,γ-变形菌纲,假单胞菌目,假单胞菌科。目前属内已报道有180多个种和15个亚种,属内微生物理化特性差别极大,有的是人体病原菌,有的是农业用有益菌,有的尚未发现其研究价值。中国专利CN110029077A公布了一种耐盐促生菌菌株Y4及其应用,该橄榄盐单胞菌菌株Y4可促进番茄幼苗生长,但未显示对种子发芽率的促进效果,未显示对番茄以外作物的促生效果。CN115612638A公布了一株罗氏假单胞菌OOR2-11菌株及其应用,CN113881606A公布了一种耐盐促生的假单胞菌RL-WG26菌株及其应用,这两个专利显示出对水稻幼苗和产量的促进效果,并不针对种子发芽和旱作作物。CN108660098A公布了一株具有耐盐防病促生的枯草芽孢杆菌HG-15及其菌剂制备和应用,未展示出在盐碱土上对作物种子发芽和幼苗生长的影响。CN 110117560A公布了一株增强作物耐盐能力的根际促生菌及其微生物肥料和应用,该专利并不以菌液为促生剂,而以含促生菌的微生物肥料为促生剂。同时,天津假单胞菌的应用尚未见报道。
针对目前盐碱地区旱作作物直播种子发芽率低和移栽幼苗受盐碱抑制严重,有必要筛选出可盐碱土下稳定定殖,具有多种促生生理生化特性,能显著促进旱作作物种子发芽和幼苗生长的功能菌株。
发明内容
本发明要解决的技术问题是克服目前尚未有促进盐碱旱作作物发芽与苗期生长的促生功能菌,提供一株可在盐碱土中稳定定殖,并促进盐碱蔬菜、油菜和大豆播种后发芽和幼苗移栽后生长的菌株。
本发明上述目的通过以下技术方案实现:
首先,本申请一株耐盐碱促生天津假单胞菌(Pseudomonas tianjinensis)菌株P522,其保藏编号为CGMCC NO:27872。
本发明从盐碱旱作作物种植区根际土中分离纯化得到了一株天津假单胞菌,并将其自命名为P522菌株,该菌株具有产ACC脱氨酶、产吲哚乙酸(IAA)、产铁载体、解无机磷、形成生物膜等多种促生功能,对盐碱土壤种植下的旱作作物具有显著的促发芽和幼苗促生作用,可有效提高种子发芽率和幼苗存活率。申请人将该菌株于2023年7月13日保藏于中国普通微生物菌种保藏管理中心(China General Microbiological Culture CollectionCenter,CGMCC),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,邮政编码:100101。其保藏号为CGMCC NO:27872。
其次,本申请提供了上述保藏号为CGMCC NO:27872的天津假单胞菌菌株P522在如下1)-4)至少一种中的应用:
1)提高盐碱旱作作物种子出芽率;上述旱作作物包括蔬菜、油菜和大豆中的至少一种;
2)促进盐碱旱作作物幼苗生长;上述旱作作物包括蔬菜、油菜和大豆中的至少一种;
3)制备盐碱旱作作物种子促芽剂;上述旱作作物包括蔬菜、油菜和大豆中的至少一种;
4)制备盐碱旱作作物幼苗促生剂;上述旱作作物包括蔬菜、油菜和大豆中的至少一种。
优选的,上述提高盐碱旱作作物种子出芽率是指,在播种前,以菌株P522的菌液浸种2~10h。进一步,上述菌液中,菌株P522的菌含量为1×106~1×108CFU/mL。
优选的,上述促进盐碱旱作作物幼苗生长是指,在作物幼苗移栽后20天内,以菌含量为1×106~1×108CFU/mL的菌株P522的菌液灌根1~2次,每次10~100mL/棵。
本申请中,上述菌株P522的菌液的制备方法为:将菌株P522接种到牛肉膏蛋白胨琼脂培养基中,30℃培养2天,所得液体即为菌株P522菌液。
第三,本申请还提供了含有上述保藏号为CGMCC NO:27872天津假单胞菌菌株P522的盐碱地作物促芽剂或幼苗促生剂。
第四,本申请提供了一种促进盐碱旱作作物种子发芽的方法,其具体步骤为:,在旱作作物播种前,以菌株P522的菌液浸种2~10h。进一步,上述菌液中,菌株P522的菌含量为1×106~1×108CFU/mL。上述旱作作物包括油菜、大豆、西蓝花、大蒜、青菜、生菜、洋葱、辣椒、番茄中的至少一种。
第五,本申请提供了一种促进盐碱旱作作物幼苗生长的方法,即在旱作作物幼苗移栽后20天内,以菌株P522的菌液灌根1~2次。进一步,上述菌液中,菌株P522的菌含量为1×106~1×108CFU/mL。上述旱作作物包括蔬菜、油菜和大豆中的至少一种。
术语“盐碱地”一般是指根据国标GB/T 42828.3-2023所规定的“土壤中含有较多可溶性盐分而使非耐盐性植物不能正常生长的土地”。本申请中,术语“盐碱地”特指可溶性盐(即土壤中可溶解于水的无机盐总和)含量为2‰-6‰的盐碱地。
与现有技术相比,本发明具有以下有益效果:
本申请首次发现并保藏了一株具有耐盐碱促生作用的天津假单胞菌P522,该菌株可以用于土壤盐碱胁迫下促进旱作作物种子发芽和幼苗生长的作用。P522菌株具有产ACC脱氨酶、产吲哚乙酸(IAA)、产铁载体、解无机磷、形成生物膜等多种促生功能。P522菌株筛选于盐碱土壤中,可在盐碱土壤中稳定定殖并发挥作用。应用于盐碱土壤旱作作物如蔬菜、油菜和大豆时可显著促进种子发芽率和幼苗生长。本申请进一步利用该菌株作为活性成分制备了盐碱地作物促芽剂或幼苗促生剂,具有广泛的应用前景,为应用微生物菌剂、微生物菌肥来提高盐碱旱作作物发芽和幼苗存活与生长提供了理论依据和技术支撑。
附图说明
图1为P522菌在NA培养基上的菌落形态照片;
图2为P522菌液浸种促进盐碱土油菜发芽实景照片;
图3为P522菌液浇灌促进盐碱土油菜苗生长实景照片;
图4为培养皿盐胁迫下P522菌液添加对西蓝花发芽率、茎长和根长的影响;
图5为培养皿4‰盐胁迫下P522菌液添加促进西蓝花发芽实景照片;
图6为P522菌液浇灌促进盐碱土生菜幼苗生长实景照片;
图7为P522菌液浇灌促进田间盐碱土大蒜苗生长实景照片。
具体实施方式
以下结合具体实施例对本发明涉及的技术做进一步说明。
实施例涉及的培养基:
NA培养基(牛肉膏蛋白胨琼脂培养基):牛肉膏5g,蛋白胨10g,NaCl 5g,琼脂20g,水1L,pH 7.2-7.4。121℃灭菌30min。
NB液体培养基(牛肉膏蛋白胨液体培养基):牛肉膏5g,蛋白胨10g,NaCl 5g,水1L,pH 7.2-7.4。121℃灭菌30min。
实施例1耐盐碱促生天津假单胞菌P522的分离、筛选与鉴定
申请人在江苏省盐城市盐碱旱作经济作物产区采集长势好的油菜和大豆根际土壤样品,取10克土壤样品置于装有90mL无菌水的三角瓶中28℃下180r/min震荡30min,随后取1mL土壤悬液置于装有9mL无菌水的试管中摇匀,再如此逐步稀释三次后取100uL液体涂布在含有5%NaCl含量的NA培养基上,30℃下培养60h后,挑取不通形态的菌株,进行纯化、编号与培养。
将纯化出来的菌株进行产IAA、生物膜形成、解无机磷、ACC脱氨酶活、产铁载体等特性测定。IAA测定参考文献“ACritical Examination ofthe Specificity oftheSalkowski Reagent for Indolic Compounds Produced by PhytopathogenicBacteria.Applied and environmental microbiology,Feb.1995,793–796.”;生物膜测定参考文献“菲降解菌Pseudomonas sp.JM2-gfp细胞特性对生物膜形成能力的影响及其在植物根表的定殖,微生物学通报,2021,48(11):4019-4029”;解无机磷测定参考文献“麦田土壤解无机磷细菌的分离、筛选及其解磷效果,生物学杂志,2010,27(5):19-21”;ACC脱氨酶活测定参考:Methods for isolating and characterizing ACC deaminase-containingplant growth-promoting rhizobacteria.Physiologia plantarum 2003,118:10–15.”;产铁载体测定参考文献“产铁载体PGPR菌筛选及其对病原菌的拮抗作用,植物保护,2011,37(1):59-64”。这些检测方法均为本领域的常规方法。
根据各项指标综合筛选出一株菌株,该菌株IAA48h时产生量为9.34(μg/ml),生物膜48h时形成厚度为34.6um,7d液培后解无机磷可达104.2ug/mL,ACC脱氨酶活为48.38umol/(mg*h),铁载体7d后可溶性指数为3.54。申请人将该菌株自命名为P522菌株,其在NA培养基上的菌落形态如图1所示。
将P522菌株在NB培养基进行液体培养,所得菌液采用细菌DNA提取试剂盒提取DNA,再采用细菌16S rDNA通用引物(正向引物27F(SEQ ID NO.1):5’-AGAGTTTGATCCTGGCTCAG-3’;反向引物1492R(SEQ ID NO.2):5’-GGTTACCTTGTTACGACTT-3’)进行PCR扩增后,将扩增PCR产物送至生工生物工程(上海)股份有限公司官方进行测序,并将所得16S rDNA序列(如SEQ ID NO.3所示)上传至EzBioCloud(https://www.ezbiocloud.net/)进行同源性比对,鉴定该菌株为天津假单胞菌(Pseudomonastianjinensis)。
申请人将该菌株于2023年7月13日保藏于中国普通微生物菌种保藏管理中心(China General Microbiological Culture Collection Center,CGMCC),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,邮政编码:100101。保藏号为CGMCC NO:27872。
实施例2天津假单胞菌P522在盐碱土中稳定定殖
将实施例1筛选获得的菌株P522在NB液体培养基中培养,30℃,160r/min培养2d,获得含菌量为8.7×108CFU/mL的P522菌液(在具体实施中,也可以通过加水调整菌液中的菌含量)。在盐城多个旱作作物种植区采耕作土壤,然后取用适量菌液分别与这些土壤拌匀,使得土壤天津假单胞菌P522浓度为107CFU/g,保湿50天25-28℃培养,并在培养第20天和第50天对土壤进行假单胞菌P522计数,结果见表1。pH测定方法依据NY/T 1377-2007,可溶性盐测定方法依据NY/T1121.16-2006,有机质测定方法依据LY/T 1237-1999。
表1不同性状盐碱土下外源添加P522后土培20天和50天定殖量
由表1可见,P522菌在东台油菜盐碱土、响水西蓝花盐碱土、亭湖大豆盐碱土、射阳大蒜盐碱土种都能较好的定殖。
实施例3天津假单胞菌P522在盐碱土下对油菜种子的促芽效果
在江苏省东台市近海油菜产区取盐碱化严重的0-20cm耕层土,该土壤可溶性盐达6.0‰,油菜直播后发芽易受盐害而不发芽或发芽后死亡。
本实施例以塑料盆装该0-20cm盐碱耕层土500g后,设置两个处理,即CK和P522处理组。CK处理即空白对照,播种10粒油菜种子。P522处理为播种以108CFU/mL的P522菌液(实施例2制备)浸种4h的油菜种子10粒。播种后两个处理均盖土75g,播种后以常规方式管理。
播种10天后实景如图2所示。可见该盐碱化土壤显著抑制油菜发芽,CK处理油菜发芽率为16.7%,而P522处理油菜发芽率为36.6%。而且P522处理可显著促进油菜生物量。因此P522菌液浸种能显著改善油菜在盐碱土上的发芽率。
实施例4天津假单胞菌P522在盐碱土下对油菜幼苗促生长效果
在东台市近海油菜产区选取盐碱化严重的耕层土,该土壤可溶性盐达4.2‰,塑料盆装盐碱土500g后,设置两个处理,即CK和P522处理。CK处理即空白对照,移栽5cm株高油菜苗。P522处理为移栽5cm株高油菜苗时浇灌107CFU/mL菌液50mL/株一次,培养5天后浇灌107CFU/mL菌液50mL/株一次。培养10天后,用SPAD-502Plus手持式叶绿素测定仪测定叶绿素含量,挖取所有处理的油菜植株,称重茎叶部分为地上生物量,称重根系为地下生物量。本实施例移栽的油菜苗是以常规方法栽培获得的油菜苗,未经P522菌液浸种处理。
表2不同处理在盐碱土下对油菜苗生长的影响
表2结果显示,CK处理油菜地上和地下生物量为2.38g和0.56g,而P522处理为4.76g和0.90g。培养10天后实景如图3所示,可见P522菌液浇灌能显著促进油菜苗生长。
实施例5天津假单胞菌P522在对西蓝花的促芽效果
在培养皿上进行盐胁迫下P522菌对西蓝花的促芽试验。盐胁迫浓度有4‰和6‰NaCl,培养皿放置滤纸后添加10mL4‰和6‰NaCl溶液。每个盐浓度下均设置CK、107、108处理。CK、107、108分别代表不添加菌液、添加1mL107CFU/mL菌液(实施例2制备的菌液以灌溉水稀释)、添加1mL108CFU/mL菌液(实施例2制备)。
放置20粒西蓝花种子后25℃培养60h,茎长、根长和发芽率数据见图4。图4中,A、B、C分别为茎长、根长、发芽率的统计结果。4‰盐胁迫下107和108处理分别提高茎长74%和71%,分别提高根长63%和49%,分别提高发芽率9.6和10.5个百分点。6‰盐胁迫下107和108处理分别提高茎长74%和71%,分别提高根长154%和73%,分别提高发芽率11.5和9.2个百分点。
图5为4‰盐胁迫下CK和107处理对西蓝花出芽影响的实景图,可见107处理改善发芽效果极其显著。
实施例6天津假单胞菌P522在盐碱土下对生菜幼苗促生长效果
在江苏省响水县叶菜产区选取盐碱化严重的表层土,该土壤可溶性盐达4.3‰,生菜苗易受盐害。设置CK和P522两个处理。CK处理即空白对照,移栽芽长2cm生菜幼苗。P522处理为移栽2cm生菜幼苗后浇灌根部2×107CFU/mL菌液一次,浇灌量为30mL每棵。移栽10天后实景如图6。本实施例移栽的生菜幼苗是以常规方法栽培获得的,未经P522菌液浸种处理。
该土壤显著抑制生菜苗的生长,CK处理生菜幼苗死亡率为75%,而P522死亡率为37.5%。因此P522菌液灌根能显著改善生菜苗的定植存活率。
实施例7天津假单胞菌P522在田间试验条件下促进盐碱土大蒜苗生长
在江苏省射阳县临海镇种植大蒜的区域进行田间试验,该地区在大蒜茬因降水较少返盐严重,可溶性盐可达3‰以上,苗期大蒜生长易受盐害。设置两个处理,即无菌液和菌液处理,无菌液浇灌处理即空白对照,菌液处理为鳞茎播种前用0.6×107CFU/mL(实施例2制备的菌液以灌溉水稀释)菌液浸种8h,出芽20天后浇灌鳞茎处0.4×107CFU/mL菌液一次,浇灌方式为行浇6L每100棵。大蒜播种50天后实景如图7,两组处理叶绿素、生物量检测结果如表3所示。
表3无菌液浇灌和菌液浇灌对大蒜生长的影响
由图7和表3可见,与无菌液浇灌相比,菌液浇灌两次可明显促进盐碱土大蒜生长,具体表现在增加叶绿素含量47.5%,增加地上生物量22.3%,增加地下生物量39.6%。
Claims (9)
1.一株耐盐碱促生天津假单胞菌(Pseudomonas tianjinensis)菌株P522,其保藏编号为CGMCC NO:27872。
2.如权利要求1所述天津假单胞菌菌株P522在如下1)-4)至少一种中的应用:
1)提高盐碱旱作作物种子出芽率;
2)促进盐碱旱作作物幼苗生长;
3)制备盐碱旱作作物种子促芽剂;
4)制备盐碱旱作作物幼苗促生剂。
3.根据权利要求2所述的应用,其特征在于,所述碱旱作作物包括蔬菜、油菜和大豆中的至少一种。
4.根据权利要求2所述的应用,其特征在于,所述提高盐碱旱作作物种子出芽率是指,在播种前,以菌含量为1×106~1×108 CFU/mL菌株P522的菌液浸种2~10h。
5.根据权利要求2所述的应用,其特征在于,所述促进盐碱旱作作物幼苗生长是指,在作物幼苗移栽后20天内,以菌含量为1×106~1×108 CFU/mL菌株P522的菌液灌根1~2次。
6.根据权利要求3所述的应用,其特征在于,所述蔬菜包括西蓝花、大蒜、青菜、生菜、洋葱、辣椒、番茄中的至少一种。
7.一种促进盐碱旱作作物种子发芽的方法,其特征在于,其具体步骤为:在旱作作物播种前,以菌含量为1×106~1×108 CFU/mL菌株P522的菌液浸种2~10h。
8.一种促进盐碱旱作作物幼苗生长的方法,其特征在于,其具体步骤为:旱作作物幼苗移栽后20天内,以以菌含量为1×106~1×108 CFU/mL菌株P522的菌液灌根1~2次。
9.一种含有如权利要求1所述天津假单胞菌菌株P522的盐碱耕地作物促芽剂或幼苗促生剂。
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