CN114381408B - 具有耐旱性能的蚕豆根瘤菌及其应用 - Google Patents
具有耐旱性能的蚕豆根瘤菌及其应用 Download PDFInfo
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- CN114381408B CN114381408B CN202210126700.XA CN202210126700A CN114381408B CN 114381408 B CN114381408 B CN 114381408B CN 202210126700 A CN202210126700 A CN 202210126700A CN 114381408 B CN114381408 B CN 114381408B
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- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
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- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
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- Virology (AREA)
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- Wood Science & Technology (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了具有耐旱性能的蚕豆根瘤菌及其应用。本发明所分离的具有耐旱性能的蚕豆根瘤菌菌株系从蚕豆宿主中获得,对该菌株经形态,理化性能以及全基因组分析鉴定,该菌株属于细菌界,变形菌门,α‑变形菌纲,根瘤菌目,根瘤菌科,根瘤菌属的新菌株系,其微生物保藏编号是CGMCC No.24092。本发明的蚕豆根瘤菌通过PEG模拟干旱胁迫、根瘤菌回接蚕豆人工控水盆栽干旱试验以及雨养农业区大田接种验证试验证明本发明提供的蚕豆根瘤菌具有耐旱性、高效结瘤、固氮能力强等特性,接种该根瘤菌能有效提高蚕豆抗旱能力,增加蚕豆的生物量以及产量,在提高豆科作物的抗逆性、促进生长以及提高其产量等方面具有应用前景。
Description
技术领域
本发明涉及根瘤菌(Rhizobium),尤其涉及从蚕豆宿主中分离获得的具有耐旱性能的蚕豆根瘤菌及其在提高豆科作物抗旱能力及促进生长或提高其产量中的应用,属于蚕豆根瘤菌分离及其应用领域。
背景技术
根瘤菌(Rhizobium)是一类能够固定大气中游离态的氮与豆科植物建立共生关系的α-和β-变形菌,蚕豆-根瘤菌构成的共生固氮体系可为蚕豆提供生长所需80%的氮源,一个生长季节能够固定并提供约200kg N/ha,远远高于大豆。然而根瘤菌与豆科植物之间有很强的选择性,只有接种与豆科作物相匹配的根瘤菌,才能形成有效共生固氮体系,生物固氮作用才得以充分发挥。干旱缺水虽使得土著根瘤菌的数量相对较少,结瘤能力弱,生物固氮作用在该地区不能得到充分发挥,但在这种环境中却能孕育出能够耐干旱的根瘤菌,研究表明接种根瘤菌能够改善干旱胁迫在内的非生物胁迫条件下植物的生长状况,使宿主植物结瘤固氮,提高其抗旱性。大豆、苜蓿、白三叶等大宗豆科作物和牧草根瘤菌接种提高抗逆性的研究屡屡报道,但蚕豆方面的研究缺少有报道。而某个区域内最有效的根瘤菌往往来自本地区或与本地区条件相似地区的菌株,因此在高效根瘤菌的筛选中,必须重视菌剂应用的地域适应性。
根瘤菌与豆科植物之间有很强的选择性,只有接种与豆科作物相匹配的根瘤菌,才能形成有效共生固氮体系,生物固氮作用才得以充分发挥。文献报道中尚未见针对蚕豆耐旱根瘤菌筛选及应用的研究报道。
目前在豆科作物上普遍推广接种高效根瘤菌的主要是大豆,而对种植面积相对少的杂粮豆科作物蚕豆的高效根瘤菌推广应用鲜见报道,大部分集中在蚕豆根瘤菌的生物地理分布、遗传多样性及***发育等方面,针对蚕豆根瘤菌抗逆性研究及回接提高蚕豆抗逆性的研究开展不深入,没有实质性的工作进展,特别是针对西北干旱地区蚕豆根瘤菌抗逆性研究报道更少。而某个区域内最有效的根瘤菌往往来自本地区或与本地区条件相似地区的菌株,因此在高效根瘤菌的筛选中,必须重视菌剂应用的地域适应性。因此,从西北干旱地区蚕豆根瘤中分离获得能够提高蚕豆抗逆性、促进蚕豆生长或提高蚕豆产量的根瘤菌,这对于提高西北干旱地区蚕豆根瘤菌在生产实践上的应用,发挥生物固氮的作用,减少氮肥的使用,对于保护生态环境,节约能源,降低农业生产成本,推进农业可持续发展等方面均具有重要的意义。
发明内容
本发明的目的之一是提供具有耐旱性能的蚕豆根瘤菌;
本发明的目的之二是将所述的具有耐旱性能的蚕豆根瘤菌应用于提高豆科作物抗逆性、促进豆科作物的生长或者提高豆科作物的产量等方面。
本发明的上述目的是通过以下技术方案来实现的:
本发明的一方面是提供了一株从蚕豆中分离获得的具有耐旱性能的蚕豆根瘤菌(Rhizobium),将其命名为QHCD22,其微生物保藏编号是CGMCC No.24092;其分类命名是:根瘤菌Rhizobium sp.;保藏时间是:2021年12月13日;保藏单位是:中国微生物保藏管理委员会普通微生物中心;保藏地址是: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。
本发明的第二方面是提供了该具有耐旱性能的蚕豆根瘤菌在提高豆科作物抗逆性能中的应用。
作为本发明的一种优选的具体实施方案,所述的提高豆科植物抗逆性能是提高豆科作物的耐旱性或耐盐碱性。
本发明的第三方面是提供了该具有耐旱性能的蚕豆根瘤菌在促进豆科作物生长中的应用。
作为本发明的一种优选的具体实施方案,所述的促进豆科作物生长包括提高株高、植株鲜重、根系鲜重、根瘤数或有效分枝数。
本发明的第四方面是提供了该具有耐旱性能的蚕豆根瘤菌在提高豆科作物产量中的应用。
作为本发明的一种优选的具体实施方案,所述的提高豆科作物产量包括提高单株荚数、单株粒数、单株粒重或百粒重。
本发明中所述的豆科作物包括但不限于蚕豆、大豆、豌豆、绿豆、赤豆、豇豆或菜豆中的任何一种,优选为蚕豆。
本发明所分离的具有耐旱性能的蚕豆根瘤菌(Rhizobium sp.)菌株系是从青海省西宁市湟中区李家山镇纳家村蚕豆宿主中获得,从新鲜根瘤中对根瘤菌进行分离和纯化,并对菌株进行全基因组分析,鉴定该菌株属于细菌界,变形菌门,α-变形菌纲,根瘤菌目,根瘤菌科,根瘤菌属(Rhizobium)的新菌株系,并命名为QHCD22。
本发明的根瘤菌QHCD22通过PEG模拟干旱胁迫、实验室水培回接试验以及大田回接实验证明其具有优异的耐旱性、高效结瘤、强的固氮能力等特性,接种该根瘤菌能够提高有效蚕豆抗旱能力,增加生物量以及产量,可适用于西北干旱土壤,在提高豆科作物的抗旱性、促进生长以及提高其产量等方面具有重要的应用前景。
附图说明
图1蚕豆根瘤菌QHCD22采用YMA培养基28℃培养36h的结果。
图2为蚕豆根瘤菌QHCD22的革兰氏染色镜检结果。
图3为蚕豆根瘤菌QHCD22菌株脂肪酸气相色谱图。
图4为蚕豆根瘤菌QHCD22菌株全基因组***发育树;图中的22为蚕豆根瘤菌QHCD22菌株。
图5为蚕豆根瘤菌QHCD22菌株的耐旱性检测结果。
具体实施方式
以下结合具体实施例来进一步描述本发明,本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的,并不对本发明的范围构成任何限制。本领域技术人员应该理解的是,在不偏离本发明的精神和范围下可以对本发明的细节和形式进行修改或替换,但这些修改和替换均落入本发明的保护范围内。
实施例1蚕豆根瘤菌QHCD22的分离及鉴定
(1)采集蚕豆根瘤
2020年6月中旬,在青海省西宁市湟中区李家山镇纳家村蚕豆田取样获得蚕豆根瘤,选取新鲜、粉红色的大粒根瘤放入事先准备好的干燥管(含变色硅胶)中带回实验室。
(2)根瘤菌分离纯化
将采集的干燥的根瘤在无菌水里浸泡至膨胀。用75%的乙醇溶液浸泡5min,用1%NaClO灭菌3分钟,无菌水冲洗10次,用无菌镊子挤压根瘤,用根瘤汁液进行划线法接入刚果红YMA平板培养基上(每个根瘤接三个重复平板培养基)。28℃、暗培养,长出菌落后采用划线分离法多次转至YMA培养基中获得单菌落,观察菌的形态、大小、透明度、粘稠度、颜色等,直到纯化为止(图1)。
(3)菌株形态学鉴定
革兰氏染色为阴性,菌体呈杆状。在YMA培养基,28℃培养36小时后,菌落光滑边缘整齐呈半透明乳白色(图2)。以上形态和革兰氏染色特征与根瘤菌属(Rhizobium)的形态特征类似。
(4)菌株生理生化指标测定
通过微生物脂肪酸快速鉴定***(MIDI)检测菌株的脂肪酸组成,可知待测菌株的主要脂肪酸为C16:0,C18:0,C19:0cycloω8c和C18:0 3OH,其含量分别为7.75%,5.11%,4.34%和2.67%。具体脂肪酸的结果见表1和图3;生理生化指标测定结果表明,该菌株符合根瘤菌属(Rhizobium)的主要细胞脂肪酸特征。
表1.根瘤菌QHCD22菌株脂肪酸测定数据
Matches:
Biolog GENIII检测结果:阳性反应有阳性对照、D-果糖-6-PO4、葡糖醛酰胺、四唑紫、四唑蓝、亚碲酸钾、β-Hydroxy-D,L-丁酸、氨曲南和丁酸钠;弱阳性反应有D-海藻糖、L-海藻糖和溴酸钠;阴性反应有阴性对照、葡聚糖、D-麦芽糖、D-海藻糖、D-纤维二糖、龙胆二糖、蔗糖、D-松二糖、水苏糖、pH 6、pH 5、D-蜜三糖、α-D-乳糖、D-蜜二糖、β-Methyl-D葡萄糖苷、D-水杨甙、N-Acetyl-D葡萄糖胺、N-Acetyl-β-D甘露糖胺、N-Acetyl-D半乳糖胺、N-Acetyl甘露糖胺丙酮酸、1%NaCl、4%NaCl、8%NaCl、α-D-葡萄糖、D-甘露糖、D-果糖、D-半乳糖、3-Methyl葡萄糖、L-鼠李糖、肌苷、1%乳酸钠、梭链孢酸、D-丝氨酸、D-山梨醇、D-甘露醇、D-***醇、myo-肌醇、丙三醇、D-葡萄糖-6-PO4、D-天冬氨酸、D-丝氨酸、醋竹桃霉素、利福霉素SV、二甲胺四环素、明胶、Glycyl-L-脯氨酸、L-丙氨酸、L-精氨酸、L-天(门)冬氨酸、L-谷氨酸、L-组氨酸、L-焦谷氨酸、L-丝氨酸、洁霉素、盐酸胍、硫酸四癸钠、果胶、半乳糖醛酸、L-半乳糖酸、D-葡(萄)糖酸、葡萄糖醛酸、粘液酸、奎尼酸、D-葡糖二酸、万古霉素、p-Hydroxy-苯乙酸、丙酮酸盐、D-乳酸甲酯、L-乳酸、柠檬酸、α-Keto-戊二酸、D-羟基丁二酸、L-羟基丁二酸、Bromo-琥珀酸、萘啶酮酸、氯化锂、吐温40、γ-Amino-丁酸、α-Hydroxy-丁酸、α-Keto-丁酸、乙酰乙酸、丙酸、醋酸和甲酸,详见表2。
表2根瘤菌QHCD22菌株生理生化指标
注:+,阳性;-,阴性;w,弱阳性。
(5)全基因组分析
采用A5-MiSeq和SPAdes对去除接头序列的测序数据进行从头拼装,构建contigs和scaffolds。对得到的拼装结果进行评估和比较,并使用Pilon软件进行碱基校正。使用PGCGAP软件对基因组草图序列进行Prokka注释。
(6)菌株基因组基本特性
注释完成后获得菌株的基因组大小为7,592,996bp,G+C含量为60.8%。具体见表3所示。基因组特性与根瘤菌属(Rhizobium)基本一致。
表3菌株全基因组相关信息
(7)菌株全基因组ANI值分析
使用JSpeciesWS网站(http://jspecies.ribohost.com/jspeciesws/#home)对菌株进行ANI值分析,可以得出菌株与已知种Rhizobium indicum FA23T的ANI值为95.10%。通常讲,同一物种菌株间的ANI值≥95%。因此,菌株与Rhizobium indicum应为同一种。具体见表4。
表4菌株ANI分析(%)
注:1.Rhizobium anhuiense CCBAU 23252T;2.Rhizobium leguminosarum USDA2370T;3.Rhizobium laguerreae FB206T;4.Rhizobium ruizarguesonis GB30T;5.Rhizobium hidalgonense FH14T;6.Rhizobium esperanzae CNPSo 668T;7.Rhizobiumindicum FA23T;8.Rhizobium phaseoli ATCC 14482T
(8)菌株全基因组DDH值分析
使用GGDC(Genome-to-Genome Distance Calculator 2.1,http://ggdc.dsmz.de/ggd c.php#)分析,菌株与已知模式种Rhizobium indicum MCC 3961T的基因组序列比较的DDH值为79.2%(表5)。同一个物种间的DDH(DNA-DNA hybridization)值≥70%。另外,构建基于菌株和不同物种模式菌的全基因组***进化树(图4),可以看出与模式菌株Rhizobium indicum MCC 3961T聚为同一分支。因此,进一步证实菌株属于Rhizobium indicum。
表5菌株DDH值分析(%)
菌株的全基因组***发育树见图4。
(9)确定菌株属、种
综合以上菌体和菌落形态、生理生化特征(全细胞脂肪酸和Biolog GENIII)分析以及16S rRNA基因,全基因组分析,菌株被鉴定为根瘤菌属Rhizobium indicum种。
(10)根瘤菌株甘油长期保存
2ml保菌管中加入1ml 20%甘油,将菌株加入到甘油中,-80℃长期保存。
试验例1蚕豆根瘤菌QHCD22的耐旱性检测试验
1.耐旱性检测试验
以菌株在PEG-6000渗透势下能否增殖为划分根瘤菌耐旱标准。在YMA培养液中加入不同浓度的聚乙二醇(PEG-6000)人工模拟干旱条件,设置4个不同的PEG-6000渗透势水平:
(1)仅使用YMA液体培养液,渗透势为0mPa;(2)10%PEG-6000的YMA液体培养液,渗透势为-0.2mPa;(3)15%PEG-6000的YMA液体培养液,渗透势为-0.4mPa;(4)20%PEG-6000的YMA液体培养液,渗透势为-0.6mPa,以0mPa PEG-6000为对照(CK),每水平设3次重复。在模拟干旱胁迫0~-0.6mPa下各渗透势水平下的浊度及相对值(即各菌株在各水平下的D600nm值与相应0mPa的D600nm值的比值)进行分析,评判菌株的耐旱性。
表6菌株在干旱模拟条件下的吸光值(OD600nm)
注:HD-2菌株分离自青海省海东市化隆县昂思多镇尕麻南村蚕豆植株根瘤中,为根瘤菌;HD-6菌株分离自青海省海东市循化县察汗都斯乡阿河滩村蚕豆植株根瘤中,为根瘤菌;HD-19菌株分离自青海省海东市乐都区蒲台乡侯白家村蚕豆植株根瘤中,为根瘤菌;HD-38菌株分离自青海省海东市互助县五峰乡陈家台村蚕豆植株根瘤中,为根瘤菌;保护菌株为本发明所分离的QHCD22菌株。
以0%PEG-6000培养液为对照,不同胁迫条件下供试菌株主要表现为3种生长模式;以购自中国农业微生物菌种保藏管理中心(ACCC)的根瘤菌株ACCC 15854(Rhizobiumleguminosarum)为对照,该保护菌株OD值下降率明显低于对照菌株(表6)。在不同胁迫条件下保护菌株主要表现的生长模式为:该保护菌株一方面存在一个中间致死渗透势,即菌株在-0.4mPa渗透势时存活数量减少或增殖能力减弱,而在-0.6mPa这一更低渗透势时存活数量又增高,表现出较强的耐旱性(图5)。
2.菌株回接验证
(1)温室回接鉴定
取蚕豆根瘤菌QHCD22的菌悬液与青蚕14号蚕豆回接,以不接种根瘤菌处理(NN)为对照,进行接种根瘤菌并激活处理(NA,以根瘤菌全部转变为粉色为准),选取生长均一的蚕豆植株进行人工控水模拟干旱胁迫处理考察蚕豆植株的耐旱性表现。
表7菌株回接蚕豆形态及生理指标
注:NN:不接种根瘤菌;NA:接种根瘤菌;SPAD:叶绿素含量值;RWC:叶片相对含水量;PRO:脯氨酸含量;SOD:超氧化物歧化酶活性;TCC:根系活力;CK:正常供水处理;DS:干旱胁迫处理。
结果表明,与不接种根瘤菌处理(NN)相比,蚕豆接种菌株处理(NA)在正常供水(CK)下,其蚕豆植株形态指标和生理生化指标如:株高、根瘤数、根瘤鲜/干,植株鲜/干,叶绿素含量(SPAD)、叶片相对含水量(RWC)、脯氨酸含量(PRO)、超氧化物歧化酶活性(SOD)、根系活力(TCC)均高于不接种根瘤菌处理(NN)。在干旱胁迫3天(DS)处理下比较发现,接种根瘤菌QHCD22的蚕豆植株各指标均高于不接种。可见,该根瘤菌QHCD22菌株回接到蚕豆植株上具有提高蚕豆抗旱性的能力。
(2)田间回接鉴定
取根瘤菌QHCD22菌株的菌悬液与不同蚕豆品种(青海13号、青蚕14号、马牙蚕豆)种子回接,以ACCC15854为阳性对照,不接种菌剂为阴性对照(CK),随机区组设计,5行区,行长5.0米,行距35厘米,小区面积8.75m2(1.75×5),3次重复(田间回接鉴定试验不进行人工灌溉,2021年6-8月份青海省降雨偏少,气温偏高,出现干旱气候,而这三个月为蚕豆生长发育重要时期)。
成熟期每个处理中部随机取10株进行考种,考种指标为株高、总茎枝、有效荚数、结荚节数、结荚高度、单荚粒数和百粒重。取样后整个小区实收计产(见表8)。
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Claims (6)
1.一株蚕豆根瘤菌(Rhizobium sp.),其特征在于,其微生物保藏编号是CGMCCNo.24092。
2.权利要求1所述的蚕豆根瘤菌在提高豆科作物抗逆性能中的应用;所述的豆科作物是蚕豆,所述的抗逆性能是耐旱性能。
3.权利要求1所述的蚕豆根瘤菌在干旱条件下促进豆科作物生长中的应用;所述的豆科作物是蚕豆。
4.按照权利要求3所述的应用,其特征在于,所述的促进豆科作物生长包括提高株高、植株鲜重、根系鲜重、根瘤数或有效分枝数。
5.权利要求1所述的蚕豆根瘤菌在干旱条件下提高豆科作物产量中的应用;所述的豆科作物是蚕豆。
6.按照权利要求5所述的应用,其特征在于,所述的提高豆科作物产量包括提高单株荚数、单株粒数、单株粒重或百粒重。
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