CN111100812A - 一株拉塔伯克霍尔德菌pn1及其应用 - Google Patents
一株拉塔伯克霍尔德菌pn1及其应用 Download PDFInfo
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Abstract
本发明公开了一株拉塔伯克霍尔德菌PN1及其应用。该菌株的分类命名为Burkholderialata,菌株编号为PN1,保藏在中国典型培养物保藏中心,保藏编号为:CCTCC NO:M 2019880。本发明从毛竹根系内筛选的菌株PN1对毛竹枯梢病病原菌竹喙球菌有显著的抑菌作用,其发酵液抑菌率可达至58.82%,且具有高效的解磷和分泌IAA的能力;本发明的毛竹内生细菌PN1能显著促进毛竹实生苗生长,试验数据显示菌株PN1制成的液体菌剂对毛竹地径、苗高和叶绿素等生长指标具有显著的提高。因此,本发明为开发环境友好型毛竹生物专用肥料提供了优良的菌株资源。
Description
技术领域
本发明涉及微生物和生物肥料技术领域,具体涉及一株拉塔伯克霍尔德菌PN1及其应用。
背景技术
毛竹(Phyllostachys edulis)是我国南方农林产业结构调整、林业增效、林农增收的主要经济林种。自毛竹枯梢病在浙江黄岩首次发现后,相继在南方省份爆发成灾,造成了严重经济损失,严重制约了毛竹资源的培育和竹产业的发展。该病害病原菌为竹喙球菌(Ceratosphaeria phyllostachydis),给竹林培育带来了极大的危害,已被列为国家森林植物检疫对象。此外,随着近些年对毛竹需求量的增加,毛竹林地养分耗竭的现象也与日剧增。如,研究显示磷素已成为限制毛竹生产力提高的首要因子,而磷在生态系统能量代谢、核酸和蛋白质合成以及激酶调控等方面发挥着不可替代的作用。由于土壤中仅有约0.1%的磷能直接被植物吸收利用,其余主要以难溶性磷酸盐存在。因此,人们通过施用大量磷肥来满足植物对于磷素的需求,然而磷肥中的磷酸根离子极易与土壤Ca2+、Fe3+和Al3+等离子发生鳌合作用形成难溶性磷酸盐,因此施用磷肥中仅有5-25%可被植物吸收利用,残余部分导致土壤磷素富集和土壤肥力丧失,引发了一系列的环境问题。这些亟待解决的问题使得提高毛竹抗病能力和竹林土壤养分有效性成为一种迫切需要。
植物体内分布着丰富的内生细菌,它们通过分泌IAA、利用解磷和固氮等功能促进植物生长,在长期协同进化的过程中与植物形成互惠互利的关系。研究表明,从土壤中筛选出的促生细菌在定殖过程中易受土著微生物的干扰,定殖成功率不高,导致应用效果欠佳。因此,利用筛选内生细菌进行植物病害防治和促生方面的应用已成为近年来研究热点。然而目前关于毛竹内生细菌的相关研究较少,对毛竹内生细菌抗病促生方面的研究也尚未见报道。因此,挖掘毛竹内生菌的功能和实用特性,对于保护竹林健康,提高竹林生产力,有巨大的潜力和价值。
发明内容
本发明的第一个目的是提供一种促进毛竹生长的拉塔伯克霍尔德菌(Burkholderia lata)PN1。
本发明从江西省宜春市大港林场、官山林场和井冈山大井林场毛竹人工林中选取长势良好的3-4年生毛竹根系中,筛选获得了一株促进毛竹生长的拉塔伯克霍尔德菌(Burkholderia lata),菌株号为PN1。已保藏于中国典型培养物保藏中心(CCTCC),地址:中国湖北省武汉市武汉大学,邮编:430072,保藏编号为:CCTCC NO:M 2019880,保藏日期为2019年10月31日。
Burkholderia lata PN1菌株的主要生物学特征:无芽孢革兰氏阴性(G-)短杆菌,菌落圆形、边缘整齐,表面湿润粘稠。菌株淡乳白色,接触酶阳性,氧化酶阳性,3%氢氧化钾试验阴性,淀粉水解试验阴性,明胶水解阴性,硝酸还原试验阴性,甲基红阴性、VP阴性,吲哚阳性。Burkholderia lata PN1菌株的16S rDNA序列如SEQ ID NO:1所示。将所测的16SrDNA序列与GeneBank数据库中的序列进行比对分析。结果显示,菌株PN1与Burkholderialata同源性很高,相识度达到100%。结合形态、生理生化特征(如表1所示)及16S rDNA序列分析,鉴定为Burkholderia lata。
本发明的第二个目的是提供上述的拉塔伯克霍尔德菌(Burkholderia lata)PN1在促进毛竹生长中的应用,特别是在促进毛竹实生苗生长中的应用。
本发明的第三个目的是提供上述的拉塔伯克霍尔德菌(Burkholderia lata)PN1在抑制毛竹枯梢病病原菌竹喙球菌中的应用。
本发明的第四个目的是提供上述的拉塔伯克霍尔德菌(Burkholderia lata)PN1在溶解难溶性磷酸盐中的应用。
优选,所述的难溶性磷酸盐包括磷酸三钙、磷酸铁、磷酸氢钙、磷酸铝或植酸钙。
本发明的第五个目的是提供上述的拉塔伯克霍尔德菌(Burkholderia lata)PN1制备植物生长素IAA中的应用。
本发明的第六个目的是提供一种生物肥料,所述的生物肥料含有上述的拉塔伯克霍尔德菌(Burkholderia lata)PN1。优选,所述的生物肥料为细菌肥料。
本发明的第七个目的是提供一种抑制毛竹枯梢病病原菌竹喙球菌的制剂,所述的制剂含有上述的拉塔伯克霍尔德菌(Burkholderia lata)PN1。
与现有技术相比,本发明的有益效果是:
本发明的拉塔伯克霍尔德菌(Burkholderia lata)PN1对毛竹枯梢病病原菌竹喙球菌有显著的抑菌活性。在液体振荡培养条件下,具有较强的解磷能力和分泌IAA能力。将拉塔伯克霍尔德菌(Burkholderia lata)PN1制成菌剂接种毛竹实生苗,结果表明,该菌剂显著地促进毛竹实生苗生长。因此,本发明为开发促进毛竹生长的细菌肥料提供了优良的菌株资源。
本发明的拉塔伯克霍尔德菌(Burkholderia lata)PN1,于2019年10月31日保藏于中国典型培养物保藏中心(CCTCC),地址为中国湖北省武汉市武汉大学,邮编:430072,保藏编号为:CCTCC NO:M 2019880。
附图说明
图1为拉塔伯克霍尔德菌PN1的菌落形态图。
图2为拉塔伯克霍尔德菌PN1的16S rDNA基因序列系统发育树,其中PN1代表本发明的拉塔伯克霍尔德菌PN1。
图3为拉塔伯克霍尔德菌PN1对病原菌(竹喙球菌)平板对抗。
图4为拉塔伯克霍尔德菌PN1在NBRIP培养基产生的解磷圈。
图5为拉塔伯克霍尔德菌PN1在第3d和6d对NBRIP培养基中磷酸钙的溶解能力检测结果。
图6为拉塔伯克霍尔德菌PN1分泌IAA能力检测结果。
图7为拉塔伯克霍尔德菌PN1接种180d对一年生毛竹实生苗生长的影响。
具体实施方式
根据下述实施例,可以使本领域的技术人员更好地理解本发明。实施例所描述的仅用于说明本发明,而不应当也不会限制权利要求书中所详细描述的本发明。
实施例1:毛竹内生细菌PN1的分离鉴定
分别在江西省宜春市大港林场、官山林场和井冈山大井林场毛竹人工林中选取长势良好的3-4年生毛竹,铲去表土,用铁锹将毛竹根系挖出,收集毛竹细根。毛竹根系用蒸馏水冲洗3遍,按下述步骤进行表面消毒:75%v/v酒精浸泡1min,3.25%v/v次氯酸钠浸泡3min,75%v/v酒精浸泡30s,然后无菌水冲洗3遍。将处理过的根系组织进行研磨,收集研磨液。用最后一次冲洗后的无菌水涂布NA平板(NA培养基的配方:牛肉膏3g,蛋白胨10g,NaCl5g,琼脂16g,蒸馏水1000mL,pH 7.2,混合均匀灭菌即得),检验是否表面灭菌彻底。采用稀释平板法,将平板放置在30℃下培养。
培养36h后在NA平板上出现细菌菌落,菌落大部分成乳白色,外观十分相似。本发明人对该菌落进行进一步的纯化,通过形态学和分子鉴定发现,该菌株菌落呈圆形,颜色呈淡乳白色,不透明(图1)。对该株细菌显微观察,结果显示为杆菌,生理生化特征分析显示均属于好氧性革兰氏阴性杆菌(表1)。该菌株的16S rDNA序列见SEQ ID NO:1所示,利用16SrDNA序列构建系统发育树,结果显示与该毛竹内生细菌最接近的是Burkholderia lata,菌株号为PN1,命名为拉塔伯克霍尔德菌(Burkholderia lata)PN1(图2)。PN1的生理生化特征也与B.lata的标准菌株描述一致。因此,PN1是Burkholderia lata的分离株。
表1拉塔伯克霍尔德菌PN1生理生化特征
备注:“-”表示反应阴性;“+”表示反应阳性
拉塔伯克霍尔德菌(Burkholderia lata)PN1已保藏于中国典型培养物保藏中心(CCTCC),地址:中国湖北省武汉市武汉大学,邮编:430072,保藏编号为:CCTCC NO:M2019880,保藏日期为2019年10月31日。
实施例2:菌株PN1对毛竹枯梢病病原菌拮抗作用
用打孔器(直径=5mm)在培养5d的毛竹枯稍病病原菌竹喙球菌(C.phyllostachydis)菌落边缘打取菌饼,接于PDA平板中央,将用NA斜面活化的待测细菌菌株划线接种于PDA平板两侧,以只接病原菌为对照(CK),每个处理3个重复。28℃培养4d观察抑菌带的有无,并测量抑菌带宽度。
将平板对峙试验中抑菌较好的拉塔伯克霍尔德菌PN1接种于NB培养基(NB培养基的配方:牛肉膏3g,蛋白胨10g,NaCl 5g,蒸馏水1000mL,pH 7.2,混合均匀后灭菌即得)中。发酵用150mL摇瓶,每瓶装液量为50mL,于30℃(200r/min)培养2d。发酵液经10000r/m离心20min后,取上清液用0.22μm微孔滤膜过滤即得无菌滤液。取无菌滤液5mL与20mL冷却到50℃的PDA培养基(配方为:马铃薯200g、葡萄糖20g、琼脂15g,蒸馏水1000mL、自然pH;配制方法为:先将马铃薯洗净去皮,再称取200g马铃薯切成小块,加水煮烂,纱布过滤,收取滤液,再和葡萄糖、琼脂,混合,溶于水,搅拌溶解,于121℃灭菌20min,冷却即得)混合倒平板;以无菌水代替无菌滤液为对照。再将病原菌菌块点接于平板中间。每个处理3个重复,28℃培养4d后,测定滤液对竹喙球菌的生长抑菌率。计算公式如下:生长抑菌率(%)=(对照平板菌落直径-带滤液平板菌落直径)/(对照平板菌落直径-菌块直径)×100%。结果显示拉塔伯克霍尔德菌PN1对竹喙球菌具有较好抑制作用(图3),抑菌率能达至58.82%(表2)。
表2拉塔伯克霍尔德菌PN1对竹喙球菌的抑菌活性
注:同列不同小写字母代表在0.05水平差异显著。
实施例3:菌株PN1解磷能力测定试验
将拉塔伯克霍尔德菌PN1接种于NBRIP固体培养基(葡萄糖10g,Ca3(PO4)2 5g,MgCl25g,KCl 0.2g,MgSO4.7H2O 0.25g,(NH4)2SO4 0.1g,蒸馏水1000mL,琼脂15g,混合均匀灭菌即得)中,培养4d后观察是否产生溶磷圈。采用Pikovskaya等人的方法对拉塔伯克霍尔德菌PN1进行解磷能力的定量测定。将拉塔伯克霍尔德菌PN1在NB培养基震荡培养24h后,按1%v/v的接种量接种于装有50mL NBRIP培养液的100mL三角瓶中,设接相同体积空白种子液的NBRIP培养液(没有接入菌种)为对照。各接种处理五个重复,30℃,180r/min下振荡培养6d,分别在培养第3d和第6d将发酵液离心10min(4℃,10000r/min),采用钼锑抗比色法测定不同培养时间段下上清液中可溶性磷的含量。结果显示拉塔伯克霍尔德菌PN1在NBRIP固体培养基均有解磷圈,显示具有较好的溶磷能力(图4)。拉塔伯克霍尔德菌PN1在第3d和6d对NBRIP培养液中磷酸钙均具有较强的溶解能力,解磷量分别为179mg/L和236.33mg/L(图5)。
实施例4:菌株PN1产IAA能力试验
采用Salkowski比色法测定拉塔伯克霍尔德菌PN1产IAA能力。取IAA标品,配置成0、0.5、2.5、5.0、7.5、10、12.5、15.0、17.5、25.0、50.0mg/L的浓度梯度,取2mL各浓度IAA加入等量的氯化铁比色液(PC比色液),30℃中暗保温30min,波长530nm下利用分光光度计测定吸光度,绘制标准曲线,得方程y=31.868x(R2=0.9947)。将活化后的拉塔伯克霍尔德菌PN1菌株接种至King培养基(King培养基配方为:蛋白胨2g、甘油1g、K2SO4 0.15g、MgSO44.7g、H2O 0.15g、琼脂1.5g、H2O 1000mL、pH 7.2,混合均匀后灭菌即得)摇床培养15d,按标准曲线制作方法测定发酵液中IAA含量。试验结果如图6所示,拉塔伯克霍尔德菌PN1具有较强的分泌IAA能力,其IAA分泌量为15.17mg/L。
实施例5:菌株PN1温室盆栽试验
将活化2~3次后的拉塔伯克霍尔德菌PN1接种至含有50mL NB培养基(NB培养基的配方牛肉膏3g,蛋白胨10g,NaCl 5g,蒸馏水1000mL,pH 7.2,混合均匀后灭菌即得)的100mL三角瓶中,30℃,180r/min振荡培养48h得到发酵液。取发酵液于4℃,6000r/min离心5min,收集菌体,菌体沉淀用无菌生理盐水洗涤3次后得到菌悬液,用无菌生理盐水调节菌悬液至108cfu/mL制成菌剂。将上述菌剂接种至1年生毛竹实生苗根际,以等量无菌生理盐水为对照(CK),接种量为5mL/株。每处理10个重复,置于温室统一管理,光照为12h/d,适时浇水。拉塔伯克霍尔德菌PN1接种180d对一年生毛竹实生苗生长的影响见图7。从图7可看出,施用PN1菌剂显著促进毛竹实生苗的生长。施菌处理对毛竹实生苗的苗高、地径和叶绿素含量有着显著提高。施菌后180d后毛竹实生苗苗高和地径分别为35.13cm和2.26cm,和对照相比分别增长了54.42%和39.51%(表3);施菌后180d后毛竹叶绿素a、叶绿素b和叶绿素总量分别为4.12、1.02和5.14,分别比对照提高了63.49%、52.24%和61.13%(表4)。
表3在接种拉塔伯克霍尔德菌PN1 180d后对毛竹实生苗生长的影响
注:同列不同小写字母代表在0.05水平差异显著。
表4在接种菌株PN1 180d后对毛竹实生苗生长的影响
注:同列不同小写字母代表在0.05水平差异显著。
以上仅是本发明的优选实施方式,应当指出的是,上述优选实施方式不应视为对本发明的限制,本发明的保护范围应当以权利要求所限定的范围为准。对于本技术领域的普通技术人员来说,在不脱离本发明的精神和范围内,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
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<110> 江西农业大学
<120> 一株拉塔伯克霍尔德菌PN1及其应用
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<170> SIPOSequenceListing 1.0
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<212> DNA
<213> 拉塔伯克霍尔德菌PN1(Burkholderia lata PN1)
<400> 1
taatacatcg gaacatgtcc tgtagtgggg gatagcccgg cgaaagccgg attaataccg 60
catacgatct acggatgaaa gcgggggacc ttcgggcctc gcgctatagg gttggccgat 120
ggctgattag ctagttggtg gggtaaaggc ctaccaaggc gacgatcagt agctggtctg 180
agaggacgac cagccacact gggactgaga cacggcccag actcctacgg gaggcagcag 240
tggggaattt tggacaatgg gcgaaagcct gatccagcaa tgccgcgtgt gtgaagaagg 300
ccttcgggtt gtaaagcact tttgtccgga aagaaatcct tggctctaat acagtcgggg 360
gatgacggta ccggaagaat aagcaccggc taactacgtg ccagcagccg cggtaatacg 420
tagggtgcga gcgttaatcg gaattactgg gcgtaaagcg tgcgcaggcg gtttgctaag 480
accgatgtga aatccccggg ctcaacctgg gaactgcatt ggtgactggc aggctagagt 540
atggcagagg ggggtagaat tccacgtgta gcagtgaaat gcgtagagat gtggaggaat 600
accgatggcg aaggcagccc cctgggccaa tactgacgct catgcacgaa agcgtgggga 660
gcaaacagga ttagataccc tggtagtcca cgccctaaac gatgtcaact agttgttggg 720
gattcatttc cttagtaacg tagctaacgc gtgaagttga ccgcctgggg agtacggtcg 780
caagattaaa actcaaagga attgacgggg acccgcacaa gcggtggatg atgtggatta 840
attcgatgca acgcgaaaaa ccttacctac ccttgacatg gtcggaatcc cgctgagagg 900
tgggagtgct cgaaagagaa ccgatacaca ggtgctgcat ggctgtcgtc agctcgtgtc 960
gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt gtccttagtt gctacgcaag 1020
agcactctaa ggagactgcc ggtgacaaac cggaggaagg tggggatgac gtcaagtcct 1080
catggccctt atgggtaggg cttcacacgt catacaatgg tcggaacaga gggttgccaa 1140
cccgcgaggg ggagctaatc ccagaaaacc gatcgtagtc cggattgcac tctgcaactc 1200
gagtgcatga agctggaatc gctagtaatc gcggatcagc atgccgcggt gaatacgttc 1260
ccgggtcttg tacacaccgc ccgtcacgcc atgggagtgg gttttaccag aagtggctag 1320
tctaaccgca aggaggac 1338
Claims (9)
1.一种拉塔伯克霍尔德菌(Burkholderia lata)PN1,保藏编号为:CCTCC NO:M2019880。
2.权利要求1所述的拉塔伯克霍尔德菌(Burkholderia lata)PN1在促进毛竹生长中的应用。
3.根据权利要求2所述的应用,其特征在于,所述的毛竹为毛竹实生苗。
4.权利要求1所述的拉塔伯克霍尔德菌(Burkholderia lata)PN1在抑制毛竹枯梢病病原菌竹喙球菌中的应用。
5.权利要求1所述的拉塔伯克霍尔德菌(Burkholderia lata)PN1在溶解难溶性磷酸盐中的应用。
6.根据权利要求5所述的应用,其特征在于,所述的难溶性磷酸盐包括磷酸三钙、磷酸铁、磷酸氢钙、磷酸铝或植酸钙。
7.权利要求1所述的拉塔伯克霍尔德菌(Burkholderia lata)PN1在制备植物生长素IAA中的应用。
8.一种生物肥料,其特征在于,含有权利要求1所述的拉塔伯克霍尔德菌(Burkholderia lata)PN1。
9.一种抑制毛竹枯梢病病原菌竹喙球菌的制剂,其特征在于,含有权利要求1所述的拉塔伯克霍尔德菌(Burkholderia lata)PN1。
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CN109280628A (zh) * | 2018-08-08 | 2019-01-29 | 江西农业大学 | 一种肠杆菌菌种及其在促进毛竹生长中的应用 |
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US20110269177A1 (en) * | 2008-08-13 | 2011-11-03 | University College Cardiff Consultants Ltd. Cardiff School of Biosciences Cardiff University | Antimicrobial agent and method for the production thereof |
CN102604860A (zh) * | 2012-02-07 | 2012-07-25 | 南京林业大学 | 一种多噬伯克霍尔德氏菌及其在促进松树生长中的应用 |
CN105483065A (zh) * | 2016-02-24 | 2016-04-13 | 长治学院 | 一种咯伯克霍尔德氏菌及其在促进连香树生长中的应用 |
CN109280628A (zh) * | 2018-08-08 | 2019-01-29 | 江西农业大学 | 一种肠杆菌菌种及其在促进毛竹生长中的应用 |
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CN112279710A (zh) * | 2020-07-10 | 2021-01-29 | 江西农业大学 | 一种溶磷菌肥及其应用 |
CN112279710B (zh) * | 2020-07-10 | 2022-05-24 | 江西农业大学 | 一种溶磷菌肥及其应用 |
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