攀枝花矿区糙野青茅根际耐铬放线菌筛选及促生能力评价
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摘要
从攀枝花矿区糙野青茅根际土中分离放线菌,筛选获得对重金属铬具有强耐受性、抗菌促生和产胞外酶能力的放线菌,为微生物-植物联合修复重金属污染提供菌种资源.采用稀释涂布法进行放线菌的分离,通过放线菌在不同铬浓度高氏一号培养基上的生长状况判断其对重金属铬的耐受性,进一步采用原子吸收分光光度法分析菌株对铬耐受性强的菌株耐铬及去铬能力;通过对菌株产铁载体、溶磷、产吲哚乙酸(IAA)、产纤维素酶、产淀粉酶、产木糖酶、产酪蛋白酶能力及抗菌活性的筛选,综合评价菌株抗菌促生能力.结果显示,分离获得的27株根际菌株,光学显微形态及扫描电镜结果均显示为放线菌,其中有14株能在含铬的培养基上生长,具有耐铬能力.16S rRNA基因测序结果显示,14株放线菌分属于链霉菌属(streptomyces)、小单孢菌属(Micromonospora)和冢村氏菌属(Tsukamurella).菌株SCAU9002和SCAU9008在铬浓度为300mg·L~(-1)的处理下培养6 d,去铬率分别达到81.17%和78.41%;菌株SCAU9002、SCAU9006、SCAU9008、SCAU9009、SCAU9013同时具有3种促生功能,盆栽实验结果进一步表明,在重金属胁迫下,经菌株处理后的玉米植株更高,生物量更大,根系更发达,促生效果较好.此外,菌株SCAU9006对6种病原菌具有拮抗作用,5株菌同时具有3种及以上产胞外水解酶的能力,4株菌能在7%的NaCl浓度下生长,13株菌在pH5.0~8.0的范围内均可生长.结果表明,根际放线菌在促进矿区植物生长和铬尾矿污染土壤的生物修复研究中具有很大潜力.
In order to provide strains possessed both heavy metal resistant and plant growth promoting ability,the rhizospheric actinobacteria of Deyeuxia scabrescens was isolated and screened. The chromium resistant strains were selected from GAUZE's medium NO.1 with different chromium concentration.Atomic absorption spectrophotometry( AAS) was employed to analyze the chromium stress resistance and removal ability of isolates. The antimicrobial activity of isolates was tested by antagonistic experiment. The plant growth promoting activity including phosphate solubilizing ability,siderophore secreted,production of indole-3-acetic acid( IAA),chitinase,amylase,xylase and casease activity,as well as antimicrobial activity of isolates were also analyzed. The results showed a total of 27 strains were isolated from rhizosphere of Deyeuxia scabrescens. These strains were identified as actinobacteria by microscopic morphology analysis. Out of the 14 isolates could grow on medium with chromium and showed chromium resistant ability. The 16 S rRNA genesequences analysis showed that the isolates were belonged to 3 genus,including Streptomyces,Micromonospora and Tsukamurella. Among of 14 isolates,SCAU9002 and SCAU9008 possessed the high chromium removal rate with 81. 17% and 78. 41% in medium( 300 mg Cr6+/L,6 d incubation),respectively. Furthermore,strains SCAU9002,SCAU9006,SCAU9008,SCAU9009 and SCAU9013 harbored multiple plant growth promoting properties.Subsequently,pot experiment demonstrated that actinobacteria can improve both corn biomass and root growth under heavy metal stress. In additon,strains SCAU9006 have antimicrobial activities to six pathogens,five strains possessed capacity for producing at least three exoenzyme,four strains were able to tolerate 7% NaCl concentrations,13 out of 14 strains can grow under pH between 5. 0 to 8. 0. The results revealed that the rhizosphere actinobacteria isolated from Deyeuxia scabrescens possessed potential application prospect in bioremediation for chromium contaminated soil.
In order to provide strains possessed both heavy metal resistant and plant growth promoting ability,the rhizospheric actinobacteria of Deyeuxia scabrescens was isolated and screened. The chromium resistant strains were selected from GAUZE's medium NO.1 with different chromium concentration.Atomic absorption spectrophotometry( AAS) was employed to analyze the chromium stress resistance and removal ability of isolates. The antimicrobial activity of isolates was tested by antagonistic experiment. The plant growth promoting activity including phosphate solubilizing ability,siderophore secreted,production of indole-3-acetic acid( IAA),chitinase,amylase,xylase and casease activity,as well as antimicrobial activity of isolates were also analyzed. The results showed a total of 27 strains were isolated from rhizosphere of Deyeuxia scabrescens. These strains were identified as actinobacteria by microscopic morphology analysis. Out of the 14 isolates could grow on medium with chromium and showed chromium resistant ability. The 16 S rRNA genesequences analysis showed that the isolates were belonged to 3 genus,including Streptomyces,Micromonospora and Tsukamurella. Among of 14 isolates,SCAU9002 and SCAU9008 possessed the high chromium removal rate with 81. 17% and 78. 41% in medium( 300 mg Cr6+/L,6 d incubation),respectively. Furthermore,strains SCAU9002,SCAU9006,SCAU9008,SCAU9009 and SCAU9013 harbored multiple plant growth promoting properties.Subsequently,pot experiment demonstrated that actinobacteria can improve both corn biomass and root growth under heavy metal stress. In additon,strains SCAU9006 have antimicrobial activities to six pathogens,five strains possessed capacity for producing at least three exoenzyme,four strains were able to tolerate 7% NaCl concentrations,13 out of 14 strains can grow under pH between 5. 0 to 8. 0. The results revealed that the rhizosphere actinobacteria isolated from Deyeuxia scabrescens possessed potential application prospect in bioremediation for chromium contaminated soil.
引文
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Basil A J,Strap J L,Knotek-Smith H M,et al.2004.Studies on the microbial populations of the rhizosphere of big sagebrush(Artemisia tridentata)[J].Journal of Industrial Microbiology&Biotechnology,31(6):278-288
Benimeli C S,Polti M A,Albarracín V H,et al.2011.Bioremediation Potential of Heavy Metal-Resistant Actinobacteria and Maize Plants in Polluted Soil[M]//Biomanagement of Metal-Contaminated Soils.Springer Netherlands.459-477
白艳丽,王利佳,刘秀芝.2008.微波消解---原子吸收分光光度法测定土壤中的铜锌镍铬锰铅镉[J].黑龙江环境通报,32(4):58-60
曹书苗.2016.放线菌强化植物修复土壤铅镉污染的效应及机理[D].西安:长安大学.10-15
Chen L,Luo S,Li X,et al.2014.Interaction of Cd-hyperaccumulator Solanum nigrum L.and functional endophyte Pseudomonas sp.Lk9on soil heavy metals uptake[J].Soil Biology&Biochemistry,68(1):300-308
Clegg S,Gobran G R.1997.Rhizospheric P and K in forest soil manipulated with ammonium sulfate and water[J].Canadian Journal of Soil Science,77(4):525-533
邓振山,党军龙,张海州,等.2012.植物根际促生菌的筛选及其对玉米的促生效应[J].微生物学通报,39(7):980-988.
D9nmez G,K9cberber N.2005.Bioaccumulation of hexavalent chromium by enriched microbial cultures obtained from molasses and Na Cl containing media[J].Process Biochemistry,40(7):2493-2498
Gordon S A,Weber R P.1951.Colorimetric estimation of indoleacetic acid[J].Plant Physiology,26(1):192-195
侯伶龙,黄荣,周丽蓉,等.2010.鱼腥草对土壤中镉的富集及根系微生物的促进作用[J].生态环境学报,19(4):817-821
纪春艳,王新荣,程路明,等.2008.新疆棉花立枯病根际拮抗放线菌的筛选[J].植物保护,34(1):53-56
Khan A G.2005.Role of soil microbes in the rhizospheres of plants growing on trace metal contaminated soils in phytoremediation[J].Journal of Trace Elements in Medicine&Biology,18(4):355-364
Kumar V,Bharti A,Gusain O,et al.2011.Scanning electron microscopy of Streptomyces without use of any chemical fixatives[J].Scanning,33(6):446-449
Langella F,Grawunder A,Stark R,et al.2014.Microbially assisted phytoremediation approaches for two multi-element contaminated sites[J].Environmental Science and Pollution Research,21(11):6845-6858
Li X,Huang P,Wang Q,et al.2014.Staurosporine from the endophytic Streptomyces sp.strain CNS-42 acts as a potential biocontrol agent and growth elicitor in cucumber[J].Antonie Van Leeuwenhoek,106(3):515-525
李静,张瀚能,赵翀,等.2016.高效纤维素降解菌分离筛选、复合菌系构建及秸秆降解效果分析[J].应用与环境生物学报,22(4):689-696
廖萍,刘茂柯,张瀚能,等.2015.铬污染区糙野青茅内生耐铬细菌筛选及其促生能力[J].应用与环境生物学报,21(6):1025-1031
刘世全,曹红霞,张建青,等.2014.不同水氮供应对小南瓜根系生长、产量和水氮利用效率的影响[J].中国农业科学,47(7):1362-1371
Ming N,Zhang X D,Wang J Q,et al.2009.Rhizosphere effects on soil bacterial abundance and diversity in the Yellow River Deltaic ecosystem as influenced by petroleum contamination and soil salinization[J].Soil Biology&Biochemistry,41(12):2535-2542
Morgan J A W,Bending G D,White P J.2005.Biological costs and benefits to plant-microbe interactions in the rhizosphere[J].Journal of Experimental Botany,56(417):1729-1739
Phieler R,Merten D,Roth M,et al.2015.Phytoremediation using microbially mediated metal accumulation in Sorghum bicolor[J].Environmental Science&Pollution Research,22(24):19408-19416
Prakash D,Nawani N N.2014.A rapid and improved technique for scanning electron microscopy of actinomycetes[J].Journal of Microbiological Methods,99(4):54-57
Puschenreiter M,Stoger G,Lombi E,et al.2001.Phytoextraction of heavy metal contaminated soils with Thlaspi goesingense and Amaranthus hybridus:Rhizosphere manipulation using EDTA and ammonium sulfate[J].Journal of Plant Nutrition and Soil Science,164(6):615-621
Ribeiro C M,Cardoso E J.2012.Isolation,selection and characterization of root-associated growth promoting bacteria in Brazil Pine(Araucaria angustifolia)[J].Microbiological Research,167(2):69-78
Rojas-Tapias D F,Bonilla R R,Dussan J.2012.Effect of inoculation with plant growth-promoting bacteria on growth and copper uptake by sunflowers[J].Water Air&Soil Pollution,223(2):643-654
荣良燕,姚拓,赵桂琴,等.2011.产铁载体PGPR菌筛选及其对病原菌的拮抗作用[J].植物保护,37(1):59-64
Shin S H,Lim Y,Lee S E,et al.2001.CAS agar diffusion assay for the measurement of siderophores in biological fluids[J].Journal of Microbiological Methods,44(1):89-95
Tamura K,Stecher G,Peterson D,et al.2013.MEGA6:Molecular Evolutionary Genetics Analysis Version 6.0[J].Molecular Biology&Evolution,30(12):2725
孙三杰,李建明,宗建伟,等.2012.亚低温与干旱胁迫对番茄幼苗根系形态及叶片结构的影响[J].应用生态学报,23(11):3027-3032
田宏,姚拓,张德罡.2004.兰州地区草坪草根际溶磷菌分离及溶磷能力初步测定[J].草原与草坪,(4):39-41
田婧,李邵,马宁,等.2016.植物根际促生菌作用机理研究进展[J].安徽农业科学,21(10):1-2
徐丽华,李文均,刘志恒.2007.放线菌系统学---原理、方法及实践[M].北京:科学出版社
Whipps J M.2001.Microbial interactions and biocontrol in the rhizosphere[J].Journal of Experimental Botany,52(52):487-511
杨文玲,王继雯,慕琦,等.2013.耐Cr(VI)菌株的筛选及条件优化[J].河南科学,31(8):1175-1179
袁红莉,王贺祥.2009.农业微生物学及实验教程[M].北京:中国农业大学出版社.441-443
钟雄明,蔡俊鹏.2007.大闸蟹肠道中产酶菌株及其胞外酶的研究[J].水利渔业,27(1):91-93
周赓,杨辉,潘虎,等.2017.一株耐镉链霉菌的筛选、鉴定与基本特性分析[J].环境科学学报,37(6):12-16
Bacon C W,Yates I E,Hinton D M,et al.2001.Biological control of Fusarium moniliforme in maize[J].Environmental Health Perspectives,109(Suppl 2):325
Basil A J,Strap J L,Knotek-Smith H M,et al.2004.Studies on the microbial populations of the rhizosphere of big sagebrush(Artemisia tridentata)[J].Journal of Industrial Microbiology&Biotechnology,31(6):278-288
Benimeli C S,Polti M A,Albarracín V H,et al.2011.Bioremediation Potential of Heavy Metal-Resistant Actinobacteria and Maize Plants in Polluted Soil[M]//Biomanagement of Metal-Contaminated Soils.Springer Netherlands.459-477
白艳丽,王利佳,刘秀芝.2008.微波消解---原子吸收分光光度法测定土壤中的铜锌镍铬锰铅镉[J].黑龙江环境通报,32(4):58-60
曹书苗.2016.放线菌强化植物修复土壤铅镉污染的效应及机理[D].西安:长安大学.10-15
Chen L,Luo S,Li X,et al.2014.Interaction of Cd-hyperaccumulator Solanum nigrum L.and functional endophyte Pseudomonas sp.Lk9on soil heavy metals uptake[J].Soil Biology&Biochemistry,68(1):300-308
Clegg S,Gobran G R.1997.Rhizospheric P and K in forest soil manipulated with ammonium sulfate and water[J].Canadian Journal of Soil Science,77(4):525-533
邓振山,党军龙,张海州,等.2012.植物根际促生菌的筛选及其对玉米的促生效应[J].微生物学通报,39(7):980-988.
D9nmez G,K9cberber N.2005.Bioaccumulation of hexavalent chromium by enriched microbial cultures obtained from molasses and Na Cl containing media[J].Process Biochemistry,40(7):2493-2498
Gordon S A,Weber R P.1951.Colorimetric estimation of indoleacetic acid[J].Plant Physiology,26(1):192-195
侯伶龙,黄荣,周丽蓉,等.2010.鱼腥草对土壤中镉的富集及根系微生物的促进作用[J].生态环境学报,19(4):817-821
纪春艳,王新荣,程路明,等.2008.新疆棉花立枯病根际拮抗放线菌的筛选[J].植物保护,34(1):53-56
Khan A G.2005.Role of soil microbes in the rhizospheres of plants growing on trace metal contaminated soils in phytoremediation[J].Journal of Trace Elements in Medicine&Biology,18(4):355-364
Kumar V,Bharti A,Gusain O,et al.2011.Scanning electron microscopy of Streptomyces without use of any chemical fixatives[J].Scanning,33(6):446-449
Langella F,Grawunder A,Stark R,et al.2014.Microbially assisted phytoremediation approaches for two multi-element contaminated sites[J].Environmental Science and Pollution Research,21(11):6845-6858
Li X,Huang P,Wang Q,et al.2014.Staurosporine from the endophytic Streptomyces sp.strain CNS-42 acts as a potential biocontrol agent and growth elicitor in cucumber[J].Antonie Van Leeuwenhoek,106(3):515-525
李静,张瀚能,赵翀,等.2016.高效纤维素降解菌分离筛选、复合菌系构建及秸秆降解效果分析[J].应用与环境生物学报,22(4):689-696
廖萍,刘茂柯,张瀚能,等.2015.铬污染区糙野青茅内生耐铬细菌筛选及其促生能力[J].应用与环境生物学报,21(6):1025-1031
刘世全,曹红霞,张建青,等.2014.不同水氮供应对小南瓜根系生长、产量和水氮利用效率的影响[J].中国农业科学,47(7):1362-1371
Ming N,Zhang X D,Wang J Q,et al.2009.Rhizosphere effects on soil bacterial abundance and diversity in the Yellow River Deltaic ecosystem as influenced by petroleum contamination and soil salinization[J].Soil Biology&Biochemistry,41(12):2535-2542
Morgan J A W,Bending G D,White P J.2005.Biological costs and benefits to plant-microbe interactions in the rhizosphere[J].Journal of Experimental Botany,56(417):1729-1739
Phieler R,Merten D,Roth M,et al.2015.Phytoremediation using microbially mediated metal accumulation in Sorghum bicolor[J].Environmental Science&Pollution Research,22(24):19408-19416
Prakash D,Nawani N N.2014.A rapid and improved technique for scanning electron microscopy of actinomycetes[J].Journal of Microbiological Methods,99(4):54-57
Puschenreiter M,Stoger G,Lombi E,et al.2001.Phytoextraction of heavy metal contaminated soils with Thlaspi goesingense and Amaranthus hybridus:Rhizosphere manipulation using EDTA and ammonium sulfate[J].Journal of Plant Nutrition and Soil Science,164(6):615-621
Ribeiro C M,Cardoso E J.2012.Isolation,selection and characterization of root-associated growth promoting bacteria in Brazil Pine(Araucaria angustifolia)[J].Microbiological Research,167(2):69-78
Rojas-Tapias D F,Bonilla R R,Dussan J.2012.Effect of inoculation with plant growth-promoting bacteria on growth and copper uptake by sunflowers[J].Water Air&Soil Pollution,223(2):643-654
荣良燕,姚拓,赵桂琴,等.2011.产铁载体PGPR菌筛选及其对病原菌的拮抗作用[J].植物保护,37(1):59-64
Shin S H,Lim Y,Lee S E,et al.2001.CAS agar diffusion assay for the measurement of siderophores in biological fluids[J].Journal of Microbiological Methods,44(1):89-95
Tamura K,Stecher G,Peterson D,et al.2013.MEGA6:Molecular Evolutionary Genetics Analysis Version 6.0[J].Molecular Biology&Evolution,30(12):2725
孙三杰,李建明,宗建伟,等.2012.亚低温与干旱胁迫对番茄幼苗根系形态及叶片结构的影响[J].应用生态学报,23(11):3027-3032
田宏,姚拓,张德罡.2004.兰州地区草坪草根际溶磷菌分离及溶磷能力初步测定[J].草原与草坪,(4):39-41
田婧,李邵,马宁,等.2016.植物根际促生菌作用机理研究进展[J].安徽农业科学,21(10):1-2
徐丽华,李文均,刘志恒.2007.放线菌系统学---原理、方法及实践[M].北京:科学出版社
Whipps J M.2001.Microbial interactions and biocontrol in the rhizosphere[J].Journal of Experimental Botany,52(52):487-511
杨文玲,王继雯,慕琦,等.2013.耐Cr(VI)菌株的筛选及条件优化[J].河南科学,31(8):1175-1179
袁红莉,王贺祥.2009.农业微生物学及实验教程[M].北京:中国农业大学出版社.441-443
钟雄明,蔡俊鹏.2007.大闸蟹肠道中产酶菌株及其胞外酶的研究[J].水利渔业,27(1):91-93
周赓,杨辉,潘虎,等.2017.一株耐镉链霉菌的筛选、鉴定与基本特性分析[J].环境科学学报,37(6):12-16