CN113846027A - 一种耐金属贪铜菌属菌株及其应用 - Google Patents
一种耐金属贪铜菌属菌株及其应用 Download PDFInfo
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- CN113846027A CN113846027A CN202110729266.XA CN202110729266A CN113846027A CN 113846027 A CN113846027 A CN 113846027A CN 202110729266 A CN202110729266 A CN 202110729266A CN 113846027 A CN113846027 A CN 113846027A
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Abstract
本发明公开了一种耐金属贪铜菌属菌株及其应用,所述耐金属贪铜菌属菌株命名为贪铜菌(Cupriavidus metallidurans)S‑8‑2菌株,该菌株保藏号为CGMCC NO.20911。该菌株可用于制备多种重金属胁迫下植物促生剂和土壤修复剂。
Description
技术领域
本申请涉及微生物技术领域,具体涉及一种耐金属贪铜菌属菌株及其应用。
背景技术
我国的绝大多数土壤都不同程度的受到多重金属的污染,对大量受重金属污染的农用地而言,急需开发满足农业生产需要的土壤修复技术。而土壤微生物扮演着一个重要的角色,其中植物根际促生菌(Plant growth promoting rhizobacteria,PGPR)生存于植物根际和根表,可以通过解磷、固氮等来改善土壤的理化性质,降低土壤中的有毒物质毒性甚至变成无害,促使养分循环,与植物共生,为植物生长提供营养,也可通过分泌吲哚乙酸(IAA)、细胞分裂素、赤霉素等植物激素、抑制植物病原菌等来促进植物生长,提高植物抗性的同时修复污染土壤,维持土壤质量健康等功能。从废弃矿山周边抗重金属锑的优势植物金荞麦的根际土壤筛选出一株促生根际菌贪铜菌,具有溶磷作用,将植物难以吸收利用的磷转化为可吸收利用的形态,供植物生长发育,同时该菌株能产生长激素IAA直接促进植物生长,该筛选具有产IAA能力的微生物可以为促生生物肥料的研发等提供出发菌株。
因此,分离筛选出既抗重金属又能促进农作物种子萌发的PGPR,研究其生物学特性及在重金属污染区对农作物的促生作用,无疑对重金属污染区的农业生产有巨大的帮助,保证农业绿色可持续发展,改善土壤生态环境。
发明内容
本发明旨在克服现有技术的不足,提供一种耐金属贪铜菌属菌株及其应用。
为了达到上述目的,本发明提供的技术方案为:
所述耐金属贪铜菌属菌株命名为耐金属贪铜菌(Cupriavidus metallidurans)S-8-2菌株,该菌株于2020年10月19日保藏于中国典型培养物保藏中心(地址:中国武汉武汉大学),保藏号为CGMCC NO.20911。该菌株的16S rDNA序列如SEQ ID NO.1所示。
该耐金属贪铜菌属菌株可用于制备多种重金属胁迫下植物促生剂。
优选地,所述多种重金属包括镉、砷、铅、锑(As、Sb、Pb、Cd)中的至少一种,即,该耐金属贪铜菌属菌株能耐多种重金属,抗镉、砷、铅、锑等重金属。
优选地,所述促生剂为具有溶磷作用的制剂,即,该耐金属贪铜菌属菌株在重金属胁迫下具有溶磷作用,能将植物难以吸收利用的磷转化为速效磷等可用于土壤磷肥的磷素。
优选地,所述促生剂为能够利用色氨酸合成生长素IAA的制剂。
优选地,所述促生剂为能分泌ACC脱氨酶的制剂,即,该耐金属贪铜菌属菌株可以通过降解乙烯前体物质1-氨基环丙烷-1-羧酸(ACC),调节植物内源乙烯的水平,减轻重金属胁迫下对农作物的影响,促进农作物的生长发育。
优选地,所述促生剂为广谱抗生素制剂。
优选地,所述促生剂为促农作物油菜种子萌发剂。
优选地,所述促生剂为抗氧化体系活力促进剂,即,该耐金属贪铜菌属菌株对重金属胁迫下油菜种子萌发过程中对种子的抗氧化体系活力的促进作用。
该耐金属贪铜菌属菌株还能用于制备土壤修复剂,即,该耐金属贪铜菌属菌株可结合农作物种植用于重金属农田污染区的土壤修复。
下面对本发明作进一步说明:
本发明提供的耐金属贪铜菌(Cupriavidus metallidurans)S-8-2为首次从湖南锡矿山废弃矿山优势植物金荞麦的根际土壤中筛选分离得到的,该菌株具有高浓度重金属锑砷抗性,检测结果表明该菌是一种能够抗多重金属的新的贪铜菌。
本发明提供的耐金属贪铜菌(Cupriavidus metallidurans)S-8-2,能够溶解并利用培养基中的磷酸钙,分泌小分子有机酸,同时具有产生长激素IAA、产ACC脱氨酶、广谱抗生素抗性等特性,能抗高浓度重金属,一方面转化土壤中的重金属,解除农作物的重金属胁迫,使多价态As、Sb的价态和形态发生改变,降低其在农作物根部的积累,从而改良土壤营养成份和吸收状况,促进植物吸收更多的营养物质,促进植物生长。另一方面具有促生特性,能促进农作物种子萌发及生长。
本发明提供的耐金属贪铜菌(Cupriavidus metallidurans)S-8-2可以色氨酸为前体合成 IAA,由于其吸附在农作物种子和根系的表面,可直接被农作物利用,同时也可能与农作物本身内生的IAA共同作用刺激农作物体内细胞的生长和增殖,从而促进重金属污染农田土壤中的农作物根系的生长发育及有效吸收土壤中的水分和养分。同时可能参与农作物体内其他生命活动的调节。
本发明提供的耐金属贪铜菌(Cupriavidus metallidurans)S-8-2,对高浓度重金属锑胁迫下油菜种子萌发具有显著的促进作用,并对油菜种子抗氧化体系活力有明显增强作用。
附图说明
图1为本发明的耐金属贪铜菌(Cupriavidus metallidurans)S-8-2在CDM培养基中的菌落形态;
图2为耐金属贪铜菌(Cupriavidus metallidurans)S-8-2发酵液产有机酸的高效液相分析图,菌株在含10mM Sb3+的液体NBRIP液体培养基,28℃、120rmp/min条件下,持续发酵2天后进行液相分析,进样量为0.02ml;
图3为测定溶磷能力的溶磷标准曲线;
图4为采用Sackowski显色法测定产IA A能力的IAA标准曲线;
图5为二硝基苯肼法测定α-酮丁酸浓度的标准曲线;
图6为重金属锑胁迫下耐金属贪铜菌(Cupriavidus metallidurans)S-8-2菌株对油菜种子根系发育的影响。
具体实施方式
下面结合具体的实施例对本发明做进一步的详细说明,所述是对本发明的解释而不是限定。
下述实施例中所用的材料和试剂,如无特殊说明,均可从商业途径得到。下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
实施案例1菌株的分离和鉴定
1.1耐金属贪铜菌(Cupriavidus metallidurans)S-8-2的分离
耐金属贪铜菌(Cupriavidus metallidurans)S-8-2的分离包括取样,富集和初筛,复筛 3个步骤,具体如下:
1.1.1取样
样品取自湖南锡矿山废弃矿山的根际土壤中,在优势植物金荞麦植株周围间隔120°角度沿着根系生长方向挖出植物的根系,轻轻抖落根部将其带有的大量大块的非根际土去除,再用无菌刷将附着在植物根表面的土壤刷于无菌袋中,做好标记,于常温下运回实验室,于4℃冰箱中保存备用。
1.1.2富集和初筛
称取土样100g于灭菌的玻璃瓶中,加入1ml浓度为100mM的酒石酸锑钾C8H4K2O12Sb2.3H2O,轻轻搅匀置28℃恒温箱中富集培养7d,富集过程中注意补加无菌水,确保样品保持原始湿度。
称取上述富集的土样10g于装有90ml无菌生理盐水的三角瓶中,于28℃摇床以160r/min振荡培养20min,再按梯度稀释到10-3,10-4,10-5,分别取各个梯度的稀释液 0.1ml涂布含100μM C8H4K2O12Sb2.3H2O的CDM选择性平板上,每个稀释度涂布3个平板,置于28℃恒温培养箱中培养7d后,观察有无单菌落出现,挑取单菌落多次划线确保得到纯培养。
1.1.3复筛
将1.1.2得到的菌株在LB培养基中,30℃,120rpm,培养24h。而后对其发酵液进行IAA检查,取发酵液按梯度稀释涂于含5mM Sb3+的CDM固体培养基中,30℃培养箱中倒置培养3天,挑选较大的菌株多次分离传代,分离得到了一株抗重金属能力较高的菌株。 1.2耐金属贪铜菌(Cupriavidus metallidurans)S-8-2的鉴定
对上述分离纯化得到的纯培养菌株进行一系列生理生化鉴定,并进行DNA提取,16S rDNA的扩增和测序。
1.2.1如图1所示,该菌株在CDM固态培养基上形成淡黄色、凸起、表面光滑、边缘整齐的椭圆形或圆形菌落。
镜检显示短杆状、无鞭毛、无芽孢的革兰氏阴性细菌,其他检测项目如表1所示。
表1 S-8-2菌株的生理生化特征
注:上表中+表示阳性反应,存在或有,–表示阴性反应,不存在或没有
1.2.2利用引物27F和1492R进行扩增16S rDNA,引物序列如下:
27F:5′-AGAGTTTGATCATGGCTCAG-3′(SEQ ID NO.2);
1492R:5′-TAGGGTTACCTTGTTACGACTT-3′(SEQ ID NO.3)。
PCR扩增条件为95℃5min,95℃30s、58℃30s、72℃90s、72℃7min,35个循环。
对PCR扩增产物进行测序,测序结果如SEQ ID NO.1所示,S-8-2菌株的16SrDNA 序列的扩增长度为2481kb。
经同源性比对,其与Cupriavidus metallidurans CH34 (NCBI accessionno.NR_074704.1)具有98.71%的相似性,结合S-8-2菌株形态学和生理生化特性,鉴定该菌株为Cupriavidus metallidurans菌属的一个菌株,命名为贪铜菌 (Cupriavidusmetallidurans)S-8-2,将其保藏于中国典型培养物保藏中心(CCTCC)(地址:中国武汉武汉大学),保藏号为CCTCC NO:20911。
实施例2:耐金属贪铜菌(Cupriavidus metallidurans)S-8-2在重金属锑胁迫下产有机酸种类及量的测定
S-8-2菌株在含10mM Sb3+的液体NBRIP液体培养基,28℃、120rmp/min条件下,持续发酵3天,然后进行高效液相色谱(HPLC)分析发酵液中的有机酸。分析条件为色谱柱: C18(250mm×4.6mm,5μm);紫外检测波长215nm;流速0.4ml/min,进样量20μl;流动相为pH 2.5的磷酸氢二钾(分析纯,SIGMA)。分析结果表明,S-8-2能有效利用培养液中的葡萄糖转化为丙酸,进而溶解培养液内的磷酸钙,将金属钙螯合物溶解,释放速效磷。
如图2所示,本发明的耐金属贪铜菌(Cupriavidus metallidurans)S-8-2产丙酸最高,柠檬酸、葡萄糖醛酸次之。
实施例3:耐金属贪铜菌(Cupriavidus metallidurans)S-8-2在重金属锑胁迫下溶磷能力的测定
于含10mM Sb3+的液体NBRIP液体培养基中,设置不加菌、加菌、不加Ca3(PO4)2对照,每个处理3个重复。处理组接种1%处于对数期S-8-2菌液,接种后28℃、120rmp/min条件下培养72小时,培养液经8000rpm,离心l5min,取上清液,上清液过0.22pm孔径滤膜后, 用钼蓝法测定速效磷浓度。结果表明:接种S-8-2菌株的有效磷浓度为501.63±22.47mg/l,而无菌对照组仅为3.05±0.11mg/l,处理组对Ca--P的活化能力是无菌对照的164倍。
如图3所示,本发明的耐金属贪铜菌(Cupriavidus sp)S-8-2在重金属锑胁迫下溶磷能力体现为有效磷浓度为501.63士22.47mg/l。
实施例4:耐金属贪铜菌(Cupriavidus metallidurans)S-8-2合成生长激素IAA含量的测定
通过使用Salkowski法对S-8-2菌株进行产IAA定量检测,在含有0.5g/l色氨酸的LB 液体培养基中,28℃、150rmp条件下培养48h,用分光光度计测定OD535,利用IAA标准曲线计算各处理的IAA。实验发现,2mM Sb3+和10mM Sb3胁迫对菌株产IAA的能力并没有很大影响,IAA浓度与对照组相差不大,基本稳定在94.75μg/ml左右,培养36h左右,产IAA浓度已经趋于饱和,往后继续培养未见其含量上升。
如图4所示,本发明的耐金属贪铜菌(Cupriavidus sp)S-8-2合成IAA的量 94.75μg/ml。
实施例5:耐金属贪铜菌(Cupriavidus metallidurans)S-8-2的ACC脱氨酶活性检测
将菌株S-8-2在TSB培养液中28℃、160rmp黑暗培养12h后,离心收集菌体沉淀。用Tris-HCl缓冲液(0.1mol/l,pH 7.6)洗涤离心2次,然后,将离心收集的菌体沉淀用 600μlTris-HCl缓冲液(0.1mol/l,pH 8.5)重悬,并加入30μl甲苯后迅速振荡30秒,以破碎细胞来获取ACC脱氨酶,使用2,4-二硝基苯肼法测定菌株的ACC脱氨酶活性,于 540nm下测定其吸光度值,利用α-丁酮酸标准曲线来计算菌株ACC脱氨酶活性。
如图5所示,S-8-2菌株具有ACC脱氨酶活性,其酶活达到389.06μmol/(mg·h)。
实施例6:耐金属贪铜菌(Cupriavidus metallidurans)S-8-2对不同重金属的最低耐受浓度 (MIC)
最低抑菌浓度(MIC)指具有抑菌的物质对目的菌株的产生抑制的最低浓度。本实验通过测定菌株S-8-2在7种重金属的最高浓度得以实现。
将活化的耐金属贪铜菌(Cupriavidus metallidurans)S-8-2划线按接种于含有不同浓度重金属的LB培养基平板,于28℃生化培养箱中培养3天后,观察菌体是否生长或生长情况。结果如表2所示,菌株S-8-2能耐受多种重金属。最小抑制浓度分别为Sb+3(10mM)、As+3 (40mM)、Pb+2(4mM)、Cd+2(5mM)、Cr+6(6mM)、Cu+2(8mM)、Zn+2 (25mM)。
表2 S-8-2菌株在含不同浓度重金属的LB培养基上的生长情况
注:上表中一代表不生长;+代表生长
实施例7:耐金属贪铜菌(Cupriavidus metallidurans)S-8-2对广谱抗生素的抗敏性能力
取200uL培养至对数期的菌液涂布于TSA固体培养基上,将10种带有不同浓度的抗生素的药敏纸片放在平板上,28℃培养3d,观察其生长情况,并转接2次,验证实验结果,结果如表3所示。S-8-2具有广谱抗生素敏感性,有利于进一步实现其用于农田土壤修复中。
表3 S-8-2菌株对抗生素的抗敏性测定
注上表中+:代表敏感及程度—:代表不敏感
实施例8:耐金属贪铜菌(Cupriavidus metallidurans)S-8-2锑胁迫下的促生作用
促生作用通过10mM Sb3+离子胁迫下耐金属贪铜菌(Cupriavidus metallidurans)S-8-2 对植株种子萌发促生实验体现。具体过程如下:
①将滤纸垫于培养皿中,尽量铺满皿底,121℃湿热灭菌20min,70℃烘干,待用。
②菌悬液的准备:将菌株接种于LB培养基中,37℃,200rpm,培养18h,离心收集菌体,用蒸馏水悬浮,加蒸馏水至OD600=0.8,此菌悬液约为108CFU/ml。
③种子表面消毒并浸种:挑出饱满的油菜种子,无菌蒸馏水清洗三次,75%酒精洗两次,75%酒精浸泡2min,无菌蒸馏水洗三次,备用。用上述制备的菌悬液对已经消毒的种子进行浸种,对照组使用蒸馏水;浸种4小时后进行下一步实验。
④在培养皿中加入3ml 1/4Hoagland培养液(完全浸湿滤纸);重金属胁迫组在上述条件下加入C8H4K2O12Sb2溶液,使Sb3+浓度保持在10mM;对照组加入同等的蒸馏水。每个培养皿置入20粒浸种的种子,置于人工气候箱,培养条件为温度25℃,光照/黑暗14h/10h,培养7d。每天需要补充适量蒸馏水以弥补蒸发掉的水分,每天同一时间去观察发芽情况,记录发芽指标。7天后计算种子萌发率,测量根长、茎长、鲜重干重等生物量指标,初步断定菌株S-8-2对锑胁迫下油菜的促生作用。
如图6所示,本发明的供试菌株耐金属贪铜菌(Cupriavidus metallidurans)S-8-2在锑胁迫下的对油菜种子有促生作用。
如表4所示,对照组和实验组分别随机采取3株萌发种子作为一个生物学重复,对照组和实验组分别随机采取3株萌发种子作为一个生物学重复,结合根系扫描仪进行生物量测定,同时分别采用丙酮提取法、TTC法、考马斯亮蓝G-250法和蒽酮法测定叶绿素含量、根系活力、可溶性蛋白和可溶性糖含量。结果表明,处理组比对照组的生物量显著增加(P<0.01),萌发率提高31.9%,鲜重茎部和根部分别提高50.00%146.67%、干重茎部和根部分别提高44.44%和150.00%;根系根长增加254.76%、根表面积增加 220.81%、根系总根尖数提高200.00%。此外,接种菌株萌发后的种子根系活力、总叶绿素、根系可溶性蛋白、茎部可溶性蛋白和可溶性糖和与对照组相比也显著增加,增加量分别达70.3%、53.3%、208.33%、133.33%、45.7%和61.2%。而两组处理间根系可溶性糖差异不明显。表明接种耐金属贪铜菌(Cupriavidus metallidurans)S-8-2能促进高浓度重金属锑胁迫下种子的萌发和生长,提高其部分生理活性。
表4 S-8-2在锑胁迫下的对油菜种子萌发生理指标的影响
*处理组与对照组有差异显著(P<0.05)
对照组和实验组分别随机采取5株萌发种子作为一个生物学重复,采用愈创木酚法、NBT还原法和高锰酸钾滴定容量法测定萌发种子的过氧化物酶(POD)、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性,每个指标设定3个重复,分茎部和根系2个部位进行。萌发后的种子抗氧化酶活性结果显示(表5),接种后,处理组种子根系部分的SOD 酶活性较对照组有显著增加,增加量达93.0%和30.4%,地上部仅有CAT酶活性有显著增加,增加量为47.0%。表明菌株S-8-2能显著提高高浓度重金属锑胁迫下种子萌发的 3种抗氧化酶活性中的1~2种,尤其对植株地下根系部分的抗氧化酶活性影响更大。
表5 S-8-2在锑胁迫下的对油菜种子萌发抗氧化酶活性的影响
*处理组与对照组有差异显著(P<0.05)
综上所述,本发明的耐金属贪铜菌(Cupriavidus metallidurans)S-8-2由于其能够抗重金属(Sb、As、Cr、Cd、Pb、Zn、Cu),能通过吸附重金属和转化重金属价态,降低土壤中重金属的毒性,促进植物的生长。其能够在重金属胁迫下溶解并利用磷酸钙,生成有机酸小分子,高效溶解难溶磷,释放可溶磷,增加土壤中速效磷含量,可以解除植物缺磷胁迫,促进植物生长。同时对土壤中的抗生素具有广谱抗敏性,具有ACC脱氨酶活性,能减轻重金属对植物细胞的毒性,还能够合成吲哚乙酸,促进植物生长,同时其对土壤重金属的转化消解和促进速效磷含量的提高,可协助改良农田土壤。因此,由于上述作用所具有的促植物生长的共性,所以可作为广泛的促生菌应用于植物促生长当中。
本发明的耐金属贪铜菌(Cupriavidus metallidurans)S-8-2能够从多个方面促进重金属胁迫下种子萌发,本发明的耐金属贪铜菌(Cupriavidus metallidurans)S-8-2处理重金属锑胁迫下油菜种子萌发率,茎部及根部生物量均具有显著的促进作用,也能显著提高抗氧化能力,对土壤速效磷含量也有显著提高作用,可应用于油菜促生制剂或微生物肥料的制备。
前述对本发明的具体示例性实施方案的描述是为了说明和例证的目的。这些描述并非想将本发明限定为所公开的精确形式,根据上述教导,可以进行很多改变和变化。对示例性实施例进行选择和描述的目的在于解释本发明的特定原理及其实际应用,从而使得本领域的技术人员能够实现并利用本发明的各种不同的示例性实施方案以及各种不同的选择和改变。本发明的范围意在由权利要求书及其等同形式所限定。
序列表
<110> 湖南人文科技学院
<120> 一种耐金属贪铜菌属菌株及其应用
<141> 2021-06-29
<160> 3
<170> SIPOSequenceListing 1.0
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atcgccctcc ttgcggttag gctaactact tctggcaaaa cccactccca tggtgtgacg 60
ggcggtgtgt acaagacccg ggaacgtatt caccgcggca tgctgatccg cgattactag 120
cgattccagc ttcacgtagt cgagttgcag actacgatcc ggactacgat gcattttctg 180
ggattagctc cccctcgcgg gttggcaacc ctctgtatgc accattgtat gacgtgtgaa 240
gccctaccca taagggccat gaggacttga cgtcatcccc accttcctcc ggtttgtcac 300
cggcagtctc tctagagtgc cctttcgtag caactagaga caagggttgc gctcgttgcg 360
ggacttaacc caacatctca cgacacgagc tgacgacagc catgcagcac ctgtgtccac 420
tttctctttc gagcacctaa tgcatctctg cttcgttagt ggcatgtcaa gggtaggtaa 480
ggtttttcgc gttgcatcga attaatccac atcatccacc gcttgtgcgg gtccccgtca 540
attcctttga gttttaatct tgcgaccgta ctccccaggc ggtcaacttc acgcgttagc 600
tacgttactg agaaaatgaa tccccaacaa ctagttgaca tcgtttaggg cgtggactac 660
cagggtatct aatcctgttt gctccccacg ctttcgtgca tgagcgtcag tgacgtccca 720
gggggctgcc ttcgccatcg gtattcctcc acatctctac gcatttcact gctacacgtg 780
gaattctacc ccccctctga catactctag ccttgcagtc acaagcgcaa ttcccaagtt 840
aagctcgggg atttcacgcc tgtcttacaa aaccgcctgc gcacgcttta cgcccagtaa 900
ttccgattaa cgctcgcacc ctacgtatta ccgcggctgc tggcacgtag ttagccggtg 960
cttattcttc cggtaccgtc atccgcccca ggtattaacc agtgcgtttt ctttccggac 1020
aaaagtgctt tacaacccga aggccttctt cacacacgcg gcattgctgg atcagggttg 1080
cccccattgt ccaaaattcc ccactgctgc ctcccgtagg agtctgggcc gtgtctcagt 1140
cccagtgtgg ctgatcgtcc tctcagacca gctactgatc gtcgccttgg tgggccttta 1200
ccccaccaac tagctaatca gacatcggcc gctcctgctg cgtgaggcct tgcggtcccc 1260
cactttcacc ctcaggtcgt atgcggtatt agctaatctt tcgactagtt atcccccaca 1320
acagggtacg ttccgatgta ttactcaccc gttcgccact cgccgccaga ccgaagtccg 1380
cgctgccgtc gactgca 1397
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Claims (10)
1.一种耐金属贪铜菌属菌株,其特征在于,所述耐金属贪铜菌属菌株命名为耐金属贪铜菌 (Cupriavidus metallidurans) S-8-2菌株,该菌株保藏号为CGMCC NO.20911。
2.如权利要求1所述的耐金属贪铜菌属菌株在制备多种重金属胁迫下植物促生剂中的应用。
3.如权利要求2所述的应用,其特征在于,所述多种重金属包括镉、砷、铅、锑中的至少一种。
4.如权利要求2所述的应用,其特征在于,所述促生剂为具有溶磷作用的制剂。
5.如权利要求2所述的应用,其特征在于,所述促生剂为能够利用色氨酸合成生长素IAA的制剂。
6.如权利要求2所述的应用,其特征在于,所述促生剂为能分泌ACC脱氨酶的制剂。
7.如权利要求2所述的应用,其特征在于,所述促生剂为广谱抗生素制剂。
8.如权利要求2所述的应用,其特征在于,所述促生剂为促农作物油菜种子萌发剂。
9.如权利要求2所述的应用,其特征在于,所述促生剂为抗氧化体系活力促进剂。
10.如权利要求1所述的耐金属贪铜菌属菌株在制备土壤修复剂中的应用。
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