目的:旨在分离筛选有利于水稻种植的抗镉菌种资源。方法:从水稻根际土壤分离纯化抗镉细菌,采用火焰原子吸收分光光度法测定菌株对镉的吸附去除率,利用水培法检测抗镉细菌对镉胁迫水稻幼苗生长的影响,运用16SrRNA基因序列分析来鉴定菌株的种属。结果:分离得到4株抗镉细菌Cdr-1~Cdr-4,其中菌株Cdr-2在含镉400 mg/L的培养液生长后,对镉离子吸附去除率为61.27%。菌株Cdr-2对无镉水稻营养液中培养的水稻幼苗具有促生长作用,但它对受10 mg/L镉离胁迫的水稻幼苗生长有加强毒害作用。经16SrRNA基因测序鉴定菌株Cdr-2为产气克雷伯氏菌。结论:从水稻根际土壤分离出一株兼有抗镉性和促生长性的产气克雷伯氏菌Cdr-2。意义:研究结果可为产气克雷伯氏菌Cdr-2在水稻种植上应用提供参考。 Aim: Isolating and screening of the cadmium-resistant bacteria (Cd-r-BA) that were benefited to rice cultivation. Methods: The Cd-r-BA was isolated from rice rhizospheric soil. The adsorption and removal rate of cadmium when the Cd-r-BA grows in cadmium-containing broth was detected by the flame atomic absorption spectrometric method. The effects of Cd-r-BA to cadmium-stressed rice seedlings were detected by using water culture method. The identification of the object bacterium was depended on its 16SrRNA gene sequence. Results: The results showed that four Cd-r-BA strains of Cdr-1~Cdr-4 were screened out. Especially, when the strain Cdr-2 grows in broth containing 400 mg/L of cadmium, the adsorption and removal rate of 61.27% cadmium was detected. The growth of the rice seedlings was prompted by the strain Cdr-2 under cadmium-free rice culture nutrients in water culture box, while the toxic effects of the cadmium to the rice seedlings was enhanced by the strain Cdr-2 under 10 mg/L of cadmium condition. The strain Cdr-2 was identified as species Klebsiella aerogenes by 16SrRNA gene sequencing. Conclusion: The conclusion was drawn that aKlebsiellaaerogenesstrain Cdr-2 that characterized by cadmium-resistance and growth-promoting was isolated and screened from rice rhizosphericsoil. Significance: The results could provide reference data for the application of strain Cdr-2 to the rice cultivation.
目的:旨在分离筛选有利于水稻种植的抗镉菌种资源。方法:从水稻根际土壤分离纯化抗镉细菌,采用火焰原子吸收分光光度法测定菌株对镉的吸附去除率,利用水培法检测抗镉细菌对镉胁迫水稻幼苗生长的影响,运用16S rRNA基因序列分析来鉴定菌株的种属。结果:分离得到4株抗镉细菌Cdr-1~Cdr-4,其中菌株Cdr-2在含镉400 mg/L的培养液生长后,对镉离子吸附去除率为61.27%。菌株Cdr-2对无镉水稻营养液中培养的水稻幼苗具有促生长作用,但它对受10 mg/L镉离胁迫的水稻幼苗生长有加强毒害作用。经16S rRNA基因测序鉴定菌株Cdr-2为产气克雷伯氏菌。结论:从水稻根际土壤分离出一株兼有抗镉性和促生长性的产气克雷伯氏菌Cdr-2。意义:研究结果可为产气克雷伯氏菌Cdr-2在水稻种植上应用提供参考。
镉,水稻,抗镉细菌,产气克雷伯氏菌
Xinyu Zhang1, Ninghong Yin1, Dandan Chen1, Yue Xu1, Jing Luo1, Aiqing Xu1,2*
1School of Life and Health Science, Hunan University of Science and Technology, Xiangtan Hunan
2Ecological Remediation and Safe Utilization of Heavy Metal-Polluted Soils, College of Hunan Province, Xiangtan Hunan
Received: Mar. 28th, 2023; accepted: Jun. 1st, 2023; published: Jun. 9th, 2023
Aim: Isolating and screening of the cadmium-resistant bacteria (Cd-r-BA) that were benefited to rice cultivation. Methods: The Cd-r-BA was isolated from rice rhizospheric soil. The adsorption and removal rate of cadmium when the Cd-r-BA grows in cadmium-containing broth was detected by the flame atomic absorption spectrometric method. The effects of Cd-r-BA to cadmium-stressed rice seedlings were detected by using water culture method. The identification of the object bacterium was depended on its 16S rRNA gene sequence. Results: The results showed that four Cd-r-BA strains of Cdr-1~Cdr-4 were screened out. Especially, when the strain Cdr-2 grows in broth containing 400 mg/L of cadmium, the adsorption and removal rate of 61.27% cadmium was detected. The growth of the rice seedlings was prompted by the strain Cdr-2 under cadmium-free rice culture nutrients in water culture box, while the toxic effects of the cadmium to the rice seedlings was enhanced by the strain Cdr-2 under 10 mg/L of cadmium condition. The strain Cdr-2 was identified as species Klebsiella aerogenes by 16S rRNA gene sequencing. Conclusion: The conclusion was drawn that a Klebsiella aerogenes strain Cdr-2 that characterized by cadmium-resistance and growth-promoting was isolated and screened from rice rhizosphericsoil. Significance: The results could provide reference data for the application of strain Cdr-2 to the rice cultivation.
Keywords:Cadmium, Rice Plant, Cadmium-Resistant Bacteria, Klebsiella aerogenes
Copyright © 2023 by author(s) and
This work is licensed under the Creative Commons Attribution International License (CC BY 4.0).
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镉(Cadmium, Cd)是元素周期表第五周期IIB族元素。镉是生物体非必需元素,对于动植物都是一种毒性非常强的重金属,被国际癌症机构IARC列为致癌类物质。Cd由于其在环境中具有很强的迁移转化特性及对人体的高度危害性而被列为《国家重金属污染综合防治“十二五”规划》重点关注的5大重金属污染元素之一 [
水稻(Oryza sativa L.)是世界上最重要、消耗最大的粮食作物之一,对其产量的需求随着世界人口的不断增长正不断增加 [
水稻从稻田吸收Cd并在稻米中积累,人们在食用含Cd的大米后,镉从消化道进入人体,则会出现呕吐、胃肠痉挛、腹疼、腹泻等症状,甚至可因肝肾综合症死亡。大部分Cd会被吸收并积累在体内产生毒性,危害人体健康。Cd在人体器官中积累,其中肾脏和肝脏的积累量占体内Cd总量的约60%。长期的Cd毒性易导致脏器功能受损,严重的甚至诱发癌变 [
微生物修复即利用微生物将环境中的污染物降解或转化为其它无害物质的过程。微生物修复可应用于水体或土壤的重金属污染修复。土壤微生物群落与土壤重金属污染之间的关系是当今国内外环境科学领域的一个研究热点。一方面,重金属污染会对土壤微生物群落产生影响,如降低土壤微生物量、可培养细菌菌落数量,或明显改变土壤微生物群落的结构 [
本研究在分离筛选抗(耐)镉细菌资源过程中,从水稻根际土壤中分离纯化得到一株兼有抗镉性和促生长性的产气克雷伯氏菌Cdr-2。研究结果为水稻栽培积累了有应用潜力菌种资源的参考性实验数据。
采样土壤:采自湘潭市某有色金属冶炼厂附近的水稻根际土壤样品;水稻种子:Y两优1928 (长沙利诚种业有限公司);牛肉膏,蛋白胨,氯化钠,琼脂粉,Cd(NO3)2∙4H2O (AR,天津市科密欧化学试剂有限公司),镉离子标准溶液(1000 μg/mL,国家有色金属及电子材料分析测试中心);Yoshida水稻营养液(800×母液)。
全自动高压蒸汽灭菌器(登冠),电热恒温鼓风干燥箱,立式恒温振荡器,洁净工作台,人工气候培养箱(上海一恒),高速冷冻离心机(卢湘仪),原子吸收分光光度计(AA-7000,岛津),植物水培盒(6孔,容积1 L)。
在湘潭市某有色金属冶炼厂附近的稻田中随机采取4份水稻根际土样。制作含Cd2+100 mg/L的牛肉膏蛋白胨固体培养基平板,各取1 g土样分别加入50 mL灭菌生理盐水制成土样悬液,移取0.2 mL土样悬液涂布平板后置于28℃恒温培养箱中培养48 hr。每个土样挑取2个菌落用于划线分离纯化得到纯菌落,转接到斜面培养基,28℃培养48 hr后,于4℃冰箱保藏备用。
配制含Cd2+浓度为0,200 mg/L、400 mg/L和600 mg/L的牛肉膏蛋白胨培养液,分装成20 mL/瓶。无菌操作移取抗镉菌Cdr-1、Cdr-2、Cdr-3或Cdr-4种子液200 μL,分别接种于灭菌培养液中,28℃恒温震荡培养48 hr,观察培养液的浊度。移取1.5 ml 含Cd2+为200 mg/L,400 mg/L,600 mg/L的48 hr培养液,转速10,000 r/min离心10 min收集培养液上清。用去离子水稀释上清液使其中Cd2+浓度为1 mg/L以下。将镉离子标准溶液(1000 mg/L)用去离子水稀释,配制Cd2+浓度分别为0.1 mg/L、0.2 mg/L、0.4 mg/L、0.6 mg/L、0.8 mg/L和1.0 mg/L的稀释液,用于制作火焰原子吸收分光光度法测镉浓度的标准曲线。利用火焰原子吸收分光光度法测量各上清液吸光值(Abs 228.8 nm),根据标准曲线拟合公式计算上清液中的镉残留量以及镉离子的吸附去除率。
称取水稻种子100 g放入洁净保鲜盒中,先用无菌水浮选去除瘪谷等杂质,用0.2%高锰酸钾溶液浸泡种子消毒0.5 hr,无菌水清洗后用28℃无菌水浸泡种子,置于恒温培养箱中28℃温育至种子破口露白。把破口露白的萌发种子随机分成5组,标记为CK (空白对照)、1#、2#、3#和4#组,每组平行重复3小组。处理方式是:CK (空白对照):不接触任何细菌处理;1#组~4#组:将萌发种子分别放入用无菌生理盐水稀释100倍的Cdr-1、Cdr-2、Cdr-3或Cdr-4菌株的48 hr培养液,使种子沾上相应菌液。每小组随机挑取50颗沾上菌液的种子摆放在带湿润纱布的平板上,放入28℃恒温培养箱中培养,观察菌株对水稻种子发芽的影响情况。
按说明书用无菌去离子水稀释水稻营养液母液制成工作液。取6个植物水培盒,分别盛装按表1处理的水稻培养液1000 mL。从2.3中随机选取各处理中长势状态基本一致的水稻芽苗,用定植海绵固定在植物水培盒定植孔中,其中5孔各定植2株水稻芽苗,留下1个空孔用于通气和观察水情,放入人工气候培养箱中培养。培养条件是28℃、湿度95%、60%光照培养12 hr;28℃、湿度95%、黑暗培养12 hr。交替培养共15天,观察记录水稻幼苗的长势。培养结束后测量水稻幼苗生理指标,包括株高、根长、叶片数、干重等,比较分析菌株对镉胁迫水稻幼苗生长的影响。
编号 | 组名 | Yoshida营养液/ml | 菌液体积(菌株) | 硝酸镉母液(3000 mg/L)/ml |
---|---|---|---|---|
1# | 正常对照组 | 1000 | 0 | 0 |
2# | 10 mg/L镉胁迫组 | 998.3 | 0 | 1.7 |
3# | Cd-2菌株处理组 | 990 | 10 ml (Cd-2) | 0 |
4# | 10 mg/L镉胁迫 + Cd-2菌株处理组 | 988.3 | 10 ml (Cd-2) | 1.7 |
5# | Cd-3菌株处理组 | 990 | 10 ml (Cd-3) | 0 |
6# | 10 mg/L镉胁迫 + Cd-3菌株处理组 | 988.3 | 10 ml (Cd-3) | 1.7 |
表1. 植物水培盒中的水稻培养液处理方式
用牛肉膏蛋白胨培养液培养Cdr-2菌株48 hr后,取1.5 mL含菌培养液10,000 r/min离心10 min,收集菌体细胞,委托上海生工生物工程有限公司(武汉)进行菌样的16S rRNA基因测序,测序引物为细菌16S rRNA基因的通用引物27F/1492R。所测序列提交到GenBank数据库进行BLAST比对搜索相似序列,鉴定所测菌株Cdr-2的种属类别。
在构建菌株Cdr-2与克雷伯氏菌属典型菌株的系统发育树时,首先在LPSN数据库中检索克雷伯氏菌属(Klebsiella)下列出的菌种名,找到部分典型菌株的16S rRNA基因序列在Genbank中的登录号;其次运用ClustalW1.8.3程序将所选典型菌株与待测菌株Cdr-2的16S rRNA基因序列进行多序列比对;最后运用分子进化遗传分析软件MEGA7.0.26构建N-J系统发育树。系统发育树的可靠性检测的自举值设定为1000次,以大肠杆菌(E. coli)典型菌株做外群。
从水稻根际土壤样品中分离筛选到4株抗镉细菌,Cdr-1#~Cdr-4#。其菌落呈圆形,光滑湿润,淡黄色,边缘较平整。
根据观察各菌株在培养液中的菌体生长浑浊程度判断,菌株Cdr-1在Cd2+浓度400 mg/L和600 mg/L的培养液中不生长,其它三个菌株都能耐受Cd2+浓度600 mg/L。
实验测得镉浓度的标准曲线拟合公式:Abs = 0.61510 Conc + 0.028249 (R = 0.9987)。各抗镉菌株对镉离子吸附去除能力分析结果见表2。结果表明在实验条件下,各菌株在含镉培养基中生长繁殖后,镉离子吸附去除率在50%左右。在初始镉400 mg/L培养液生长时,菌株Cdr-4的吸附去除率最高,为65.30%,其次是菌株Cdr-2,为61.27%。
菌株编号 | 初始镉浓度(mg/L) | 稀释倍数 | 吸光值 (228 nm) | 测定镉浓度 (mg/L) | 残留镉浓度 (mg/L) | 镉去除率 (%) |
---|---|---|---|---|---|---|
Cdr-1 | 200 | 200 | 0.3195 | 0.4735 | 94.70 | 52.65 |
Cdr-1 | 400 | 400 | 0.2897 | 0.4215 | 168.60 | 57.85 |
Cdr-1 | 600 | 600 | 0.3279 | 0.4872 | 292.32 | 51.28 |
Cdr-2 | 200 | 200 | 0.3320 | 0.4938 | 98.76 | 50.62 |
Cdr-2 | 400 | 400 | 0.2665 | 0.3873 | 154.92 | 61.27 |
Cdr-2 | 600 | 600 | 0.3581 | 0.5363 | 321.78 | 46.37 |
Cdr-3 | 200 | 200 | 0.3322 | 0.4941 | 98.82 | 50.59 |
Cdr-3 | 400 | 400 | 0.3322 | 0.4941 | 197.64 | 50.59 |
Cdr-3 | 600 | 600 | 0.3340 | 0.4971 | 298.26 | 50.29 |
Cdr-4 | 200 | 200 | 0.3239 | 0.4907 | 98.14 | 50.93 |
Cdr-4 | 400 | 400 | 0.2417 | 0.3470 | 138.80 | 65.30 |
Cdr-4 | 600 | 600 | 0.3286 | 0.4883 | 292.98 | 51.17 |
表2. 抗镉菌株对镉离子吸附去除能力检测分析结果
种子催芽48 hr后,所有水稻种子已破口露白,都能正常萌发,说明水稻种子的活力正常。培养48 hr 后观察,2#组、3#组水稻芽苗的长势与空白对照基本一致,1#组显得稍矮,4#组较差。结果表明Cdr-2菌株和Cdr-3菌株对水稻芽苗没有病害感染作用。
培养15天后,各水培盒中水稻幼苗的长势情况见图1。直观显示1#和2#长势相当;3#与1#相比株高增加非常明显;4#、5#、6#的幼苗有营养不良、黄化萎蔫迹象。结果表明,10 mg/L的镉离子对水稻幼苗茎秆部分生长的胁迫作用不明显;Cdr-2菌株对无镉胁迫水稻幼苗的正常生长具有促进作用;Cdr-2菌株与10 mg/L的镉离子处理组(4#)、Cdr-3菌株处理组(5#)、Cdr-3菌株与10 mg/L的镉离子处理组(6#)中水稻幼苗的生长遭受胁迫抑制。
图1. 各水培盒中水稻幼苗的长势情况照片(左→右处理编号1#~6#)
水稻幼苗的生理指标测量及其分析结果见表3。数据显示,10 mg/L镉胁迫组(编号2#)的根长比正常对照组(编号1#)显著降低,表明镉离子能够引起对根细胞的生长发育直接毒害作用。菌株Cdr-2处理组(编号3#)的株高值、干重值都最大,表明菌株Cdr-2对水稻幼苗生长有显著的促进作用。菌株Cdr-3处理组(编号5#)、含10 mg/L镉离子处理组(编号2#、4#、6#)株高、根长、干重的指标值都较正常对照组(编号1#)低,表明在含10 mg/L的镉离子水培环境中,菌株Cdr-2更加重了对水稻幼苗的胁迫作用,在不含镉或者含10 mg/L的镉离子水培环境中,Cdr-3菌株都抑制水稻幼苗的生长。
编号 | 组名 | 叶片众数 | 株高/cm | 根长/cm | 干重均值/g | ||
---|---|---|---|---|---|---|---|
均值 | 标准差 | 均值 | 标准差 | ||||
1# | 正常对照组 | 4 | 16.1 | 2.132 | 17.4 | 3.017 | 0.7163 |
2# | 10 mg/L镉胁迫组 | 4 | 15.5 | 3.697 | 9.3 | 2.139 | 0.5324 |
3# | Cd-2菌株处理组 | 4 | 28.7 | 4.138 | 12.3 | 1.231 | 0.9203 |
4# | 10 mg/L镉胁迫 + Cd-2菌株处理组 | 3 | 10.5 | 4.137 | 6.5 | 2.082 | 0.3176 |
5# | Cd-3菌株处理组 | 3 | 7.7 | 1.824 | 6.5 | 1.743 | 0.3019 |
6# | 10 mg/L镉胁迫 + Cd-3菌株处理组 | 3 | 6.5 | 2.227 | 5.3 | 1.419 | 0.2003 |
表3. 水稻幼苗生理指标测量与分析结果
测得Cdr-2菌株16S rRNA基因序列在GenBank中进行BLAST搜索比对分析,显示所测序列仅与Klebsiella aerogenes的一些菌株的16S rRNA基因具有99%以上的同源性一致性。结果表明菌株Cdr-2的亲缘种是Klebsiella aerogenes (产气克雷伯氏菌)。菌株Cdr-2与克雷伯氏菌属的一些典型菌株构建的系统发育树见图2,表明菌株Cdr-2与Klebsiella aerogenes G3_AM (登录号MT373520)聚类在一个分枝,两者的亲缘关系最近。
图2. 基于16S rRNA基因构建菌株Cdr-2与克雷伯氏菌属典型菌株的N-J系统发育树
从水稻根际土壤中分离纯化得到4株抗镉细菌,菌株Cdr-1~Cdr-4。在实验条件下,各菌株在含镉培养液中生长繁殖后,镉离子吸附去除率在50%左右。其中在含镉400 mg/L培养液中,菌株Cdr-4的镉离子吸附去除率为65.30%,菌株Cdr-2为61.27%。水培结果表明,10 mg/L的镉离子对水稻幼苗茎秆生长的胁迫作用不明显,但对根的生长发育有明显的毒害作用;Cdr-2菌株对无镉胁迫水稻幼苗的正常生长具有促进作用;在含10 mg/L的镉离子水培环境中,菌株Cdr-2更加重了对水稻幼苗的胁迫作用;在不含镉或者含10 mg/L的镉离子水培环境中,Cdr-3菌株都抑制水稻幼苗的生长。经16S rRNA基因测序,鉴定Cdr-2菌株为产气克雷伯氏菌(Klebsiella aerogenes)。研究发现了产气克雷伯氏菌Cdr-2菌株的强抗镉性以及促水稻幼苗生长性能,为该菌株的进一步开发利用奠定了基础。
湖南省教育厅资助科研项目(19K030);湖南省自然科学基金项目(2020JJ6026)。
张芯瑜,印宁鸿,陈丹丹,徐 樾,罗 婧,许爱清. 抗镉细菌分离鉴定及其对镉胁迫水稻幼苗生长的影响Isolation and Identification of Cadmium-Resistant Bacteria and Their Influence on Growth of Cadmium-Stressed Rice Seedlings[J]. 微生物前沿, 2023, 12(02): 63-70. https://doi.org/10.12677/AMB.2023.122008
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