黑果枸杞根际促生菌的筛选鉴定及促生能力分析
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摘要
以新疆阿克陶县盐碱地中生长的黑果枸杞(Lycium ruthenicum)根际土壤为材料,分离兼具耐盐和良好促生能力的根际促生菌,共得到43株能够耐受1mol/L NaCl的菌株,其中13株具有溶磷和产铁载体能力,进而在13株菌中筛选出7株具有分泌生长素能力的菌株.通过溶磷、产吲哚乙酸等功能指标对比,选择出4株功能较强的菌株进行盆栽促生实验,结果发现菌株H29和X49能显著促进盐胁迫下黑果枸杞幼苗的生长.与阴性对照相比,H29处理植株的株高、叶片数、根长、地上部鲜质量和干质量分别增加32.86%,41.67%,44.76%,175.00%和114.29%,X49处理植株的根长、根表面积、地下部鲜质量和干质量分别增加51.75%,130.05%,200.00%和250.00%.分子鉴定结果显示H29为链霉菌属(Streptomyces)菌株,X49为微杆菌属(Microbacterium)菌株.上述结果可为开发和推广适合盐碱化土壤的专用生物肥料提供优良菌种.
The rhizosphere soil was exploited from Lycium ruthenicum fields in Akto County of Xinjiang to isolate rhizosphere bacteria with both salt tolerance and the desirable growth-promoting ability.We obtained 43 isolates in total,and all isolates were capable of withstanding 1 mol/L NaCl.From the obtained NaCl-tolerant bacteria,we isolated 13 strains with good tricalcium phosphate-solubilization and siderophore-secretion abilities,and among them,further isolated 7 strains with the ability to produce plant hormone indolo acetic acid(IAA).According to comparative functional analysis,we selected 4 strains with more desirable multi-functional indexes to carry out growth-promoting experiments using pot-cultivated plant materials(i.e.L.ruthenicum seedlings).Compared with the negative control(non-bacteria-treated),H29-treated plants exhibited increases in height,leaf number,root length,upground fresh and dry mass by 32.86%,41.67%,44.76%,175.00% and 114.29%,respectively;and X49-treated plants showed increases in root length,root surface area,underground fresh and dry mass by 51.75%,130.05%,200.00% and 250.00%,respectively.In addition,molecular characterization identified H29 and X49 as genus Streptomyces and Microbacterium,respectively.The above results presented bacteria resource which could be beneficial for developing salinity-soil-specialized bio-fertilizers and their application.
The rhizosphere soil was exploited from Lycium ruthenicum fields in Akto County of Xinjiang to isolate rhizosphere bacteria with both salt tolerance and the desirable growth-promoting ability.We obtained 43 isolates in total,and all isolates were capable of withstanding 1 mol/L NaCl.From the obtained NaCl-tolerant bacteria,we isolated 13 strains with good tricalcium phosphate-solubilization and siderophore-secretion abilities,and among them,further isolated 7 strains with the ability to produce plant hormone indolo acetic acid(IAA).According to comparative functional analysis,we selected 4 strains with more desirable multi-functional indexes to carry out growth-promoting experiments using pot-cultivated plant materials(i.e.L.ruthenicum seedlings).Compared with the negative control(non-bacteria-treated),H29-treated plants exhibited increases in height,leaf number,root length,upground fresh and dry mass by 32.86%,41.67%,44.76%,175.00% and 114.29%,respectively;and X49-treated plants showed increases in root length,root surface area,underground fresh and dry mass by 51.75%,130.05%,200.00% and 250.00%,respectively.In addition,molecular characterization identified H29 and X49 as genus Streptomyces and Microbacterium,respectively.The above results presented bacteria resource which could be beneficial for developing salinity-soil-specialized bio-fertilizers and their application.
引文
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[2]呼红梅,王莉.氮、磷、钾对盐胁迫谷子幼苗形态和生理指标的影响[J].江苏农业科学,2016,44(2):117-122.
[3] MAATHUI F J M,AHMAD I,PATISHTAN J.Regulation of Na+fluxes in plants[J].Frontiers in Plant Science,2014,5:00467.
[4] NABTI E,SCHMID M,HARTMANN A.Application of halotolerant bacteria to restore plant growth under salt stress[M]∥MAHESHWARI D M,SARAF M.Biodiversity and sustainable exploitation.Berlin:Springer,2015:235-259.
[5] XU G,MAGEN H,KAFKAF U.Advances in chloride nutrition in plants[J].Adv Agron,2000,68:97-150.
[6] VURUKONDA S S K P,VARDHARAJULA S,SHRIVASTAVA M,et al.Enhancement of drought stress tolerance in crops by plant growth promoting rhizobacteria[J].Microbiological Research,2016,184:13.
[7]李交昆,余黄,曾伟民,等.根际促生菌强化植物修复重金属污染土壤的研究进展[J].生命科学,2017,29(5):434-442.
[8]李华山,雷鹏,许宗奇,等.耐盐促生菌Agrobacterium sp.DF-2增强黄瓜幼苗耐盐性的研究[J].江苏农业学报,2017,33(3):654-661.
[9]刘方春,邢尚军,马海林,等.干旱胁迫下植物根际促生细菌对侧柏生长及生理生态特征的影响[J].林业科学,2014,50(6):67-73.
[10] KHAN N,BANO A.Modulation of phytoremediation and plant growth by the treatment with PGPR,Ag nanoparticle and untreated municipal wastewater[J].Int J Phytoremediat,2016,18:1258-1269.
[11]李凤霞,张德罡,姚拓.高寒地区燕麦根际高效PGPR菌培养条件研究[J].甘肃农业大学学报,2004(3):316-320.
[12]黄静,盛下放,何琳燕.具溶磷能力的植物内生促生细菌的分离筛选及其生物多样性[J].微生物学报,2010,50(6):710-716.
[13] LYRA M,SANTOS D,SILVA M,et al.Isolation and molecularcharacterizationofendophyticbacteria associated with the culture of forage cactus(Opuntia spp.)[J].Journal of Applied Biotechnology,2013,1(1):11-16.
[14]马骢毓,张英,孙广正,等.披碱草根际促生菌筛选及其接种剂的促生作用[J].植物营养与肥料学报,2016,22(4):1039-1048.
[15] SCHWYN B,NEILANDS J B.Universal chemical assay for the detection and determination of siderophores[J].Analytical Biochemistry,1987,160(1):47-56.
[16] GLICKMANN E,DESSANX Y.A critical examination of the specificity of the salkowski reagent for compounds produced by phytopathogenic bacteria[J].Applied and Environmental Microbiology,1995,61(2):793-796.
[17]张东艳,刘晔,吴越,等.花生根际产IAA菌的筛选鉴定及其效应研究[J].中国油料作物学报,2016,38(4):104-110.
[18] DHARA P,CHAITANYA K J,NEELAM T,et al.Growth enhancement of chickpea in saline soils using plant growth-promoting phizobacteria[J].Journal of Plant Growth Regulation,2012,31(1):53-62.
[19]王龙强,米永伟,蔺海明.盐胁迫对枸杞属两种植物幼苗离子吸收和分配的影响[J].草业学报,2011,20(4):129-136.
[20] TANK N D,SARAF M S.Salinity resistant PGPR ameliorates NaCl stress on tomato plants[J].Plant Interact,2010,5:51-58.
[21] MONIS P T,GIGLIO S,SAINT C P.Comparison of SYTO9and SYBR GreenⅠfor real-time polymerase chain reaction and investigation of the effect of dye concentration on amplification and DNA melting curve analysis[J].Analytical Biochemistry,2005,340(1):24-34.
[22]徐俊兵.扬州市土壤有机质和速效磷钾的分布研究[J].土壤,2004,36(1):99-103.
[23] SPERBER J I.Solution of apatite by soil microorganisms producing organic acids[J].Australia Journal of Agricultural Research,1958,9:782-789.
[24]冯欣,刁治民,曹玲珍,等.PGPR作为微生物肥料的研究进展[J].安徽农学通报,2005(6):85-87;77.
[25] PATEL D,JHA C K,TANK N,et al.Growth enhancement of chickpea in saline soils using plant growth-promoting rhizobacteria[J].Plant Growth Regul,2012,31(1):53-62.
[26] RAJANKAR P N,TAMBEKHAR D H,WATE S R.Study of phosphate solubilization efficiencies of fungi and bacteria isolated from saline belt of Purna river basin[J].Research Journal of Agriculture&Biological Sciences,2007,3(4):701-703.
[27] ELAZEEM S A,MEHANA T A,SHABAYEK A A.Some plant growth promoting traits of rhizobacteria isolated from Suez Canal region,Egypt[J].Afr Crop Sci Conf Proc,2007,8:1517-1525.
[28] TANK N D,SARAF M S.Phosphate solubilization,exopolysaccharide production and indole-3-acetic acid secretion by rhizobacteria isolated form Trigonella foenum-graenum[J].Ind J Microbiol,2003,43:37-40.
[29]张英,芦光新,谢永丽,等.溶磷菌分泌有机酸与溶磷能力相关性研究[J].草地学报,2015,23(5):1033-1038.
[30]刘永锋,陆凡,陈志谊,等.拮抗细菌T429和T392的生物活性及其对水稻白叶枯病的防治效果[J].江苏农业学报,2012,28(4):733-737.
[31] DATTA C,BASU P S.Indole acetic acid production by a Rhizobium species from root nodules of a leguminous shrub,Cajanus cajan[J].Microbiological Research,2000,155(2):123-127.
[32] MUNNS R.Comparative physiology of salt and water stress[J].Plant Cell Environ,2003,25:239-250.
[33]刘佳莉,方芳,史煦涵,等.2株盐碱地燕麦根际促生菌的筛选及其促生作用研究[J].草业学报,2013,22(2):132-139.
[34]崔晓双,王伟,张如,等.基于根际营养竞争的植物根际促生菌的筛选及促生效应研究[J].南京农业大学学报,2015,38(6):958-966.
[35]吴秉奇,梁永江,丁延芹,等.两株烟草根际拮抗菌的生防和促生效果研究[J].中国烟草科学,2013,34(1):66-71.
[36] NADEEM S M,HUSSAIN I,NAVEED M,et al.Performance of plant growth promoting rhizobacteria containing ACC-deaminase activity for improving growth of maize under salt stressed conditions[J].Pak J Agric Sci,2006,43:114-121.
[37] YONG H L,WANG H L,DO K L,et al.Factors relating to induced systemic resistance in watermelon by plant growth promoting Pseudomonas spp.[J].Plant Pathol,2001,17:174-179.
[2]呼红梅,王莉.氮、磷、钾对盐胁迫谷子幼苗形态和生理指标的影响[J].江苏农业科学,2016,44(2):117-122.
[3] MAATHUI F J M,AHMAD I,PATISHTAN J.Regulation of Na+fluxes in plants[J].Frontiers in Plant Science,2014,5:00467.
[4] NABTI E,SCHMID M,HARTMANN A.Application of halotolerant bacteria to restore plant growth under salt stress[M]∥MAHESHWARI D M,SARAF M.Biodiversity and sustainable exploitation.Berlin:Springer,2015:235-259.
[5] XU G,MAGEN H,KAFKAF U.Advances in chloride nutrition in plants[J].Adv Agron,2000,68:97-150.
[6] VURUKONDA S S K P,VARDHARAJULA S,SHRIVASTAVA M,et al.Enhancement of drought stress tolerance in crops by plant growth promoting rhizobacteria[J].Microbiological Research,2016,184:13.
[7]李交昆,余黄,曾伟民,等.根际促生菌强化植物修复重金属污染土壤的研究进展[J].生命科学,2017,29(5):434-442.
[8]李华山,雷鹏,许宗奇,等.耐盐促生菌Agrobacterium sp.DF-2增强黄瓜幼苗耐盐性的研究[J].江苏农业学报,2017,33(3):654-661.
[9]刘方春,邢尚军,马海林,等.干旱胁迫下植物根际促生细菌对侧柏生长及生理生态特征的影响[J].林业科学,2014,50(6):67-73.
[10] KHAN N,BANO A.Modulation of phytoremediation and plant growth by the treatment with PGPR,Ag nanoparticle and untreated municipal wastewater[J].Int J Phytoremediat,2016,18:1258-1269.
[11]李凤霞,张德罡,姚拓.高寒地区燕麦根际高效PGPR菌培养条件研究[J].甘肃农业大学学报,2004(3):316-320.
[12]黄静,盛下放,何琳燕.具溶磷能力的植物内生促生细菌的分离筛选及其生物多样性[J].微生物学报,2010,50(6):710-716.
[13] LYRA M,SANTOS D,SILVA M,et al.Isolation and molecularcharacterizationofendophyticbacteria associated with the culture of forage cactus(Opuntia spp.)[J].Journal of Applied Biotechnology,2013,1(1):11-16.
[14]马骢毓,张英,孙广正,等.披碱草根际促生菌筛选及其接种剂的促生作用[J].植物营养与肥料学报,2016,22(4):1039-1048.
[15] SCHWYN B,NEILANDS J B.Universal chemical assay for the detection and determination of siderophores[J].Analytical Biochemistry,1987,160(1):47-56.
[16] GLICKMANN E,DESSANX Y.A critical examination of the specificity of the salkowski reagent for compounds produced by phytopathogenic bacteria[J].Applied and Environmental Microbiology,1995,61(2):793-796.
[17]张东艳,刘晔,吴越,等.花生根际产IAA菌的筛选鉴定及其效应研究[J].中国油料作物学报,2016,38(4):104-110.
[18] DHARA P,CHAITANYA K J,NEELAM T,et al.Growth enhancement of chickpea in saline soils using plant growth-promoting phizobacteria[J].Journal of Plant Growth Regulation,2012,31(1):53-62.
[19]王龙强,米永伟,蔺海明.盐胁迫对枸杞属两种植物幼苗离子吸收和分配的影响[J].草业学报,2011,20(4):129-136.
[20] TANK N D,SARAF M S.Salinity resistant PGPR ameliorates NaCl stress on tomato plants[J].Plant Interact,2010,5:51-58.
[21] MONIS P T,GIGLIO S,SAINT C P.Comparison of SYTO9and SYBR GreenⅠfor real-time polymerase chain reaction and investigation of the effect of dye concentration on amplification and DNA melting curve analysis[J].Analytical Biochemistry,2005,340(1):24-34.
[22]徐俊兵.扬州市土壤有机质和速效磷钾的分布研究[J].土壤,2004,36(1):99-103.
[23] SPERBER J I.Solution of apatite by soil microorganisms producing organic acids[J].Australia Journal of Agricultural Research,1958,9:782-789.
[24]冯欣,刁治民,曹玲珍,等.PGPR作为微生物肥料的研究进展[J].安徽农学通报,2005(6):85-87;77.
[25] PATEL D,JHA C K,TANK N,et al.Growth enhancement of chickpea in saline soils using plant growth-promoting rhizobacteria[J].Plant Growth Regul,2012,31(1):53-62.
[26] RAJANKAR P N,TAMBEKHAR D H,WATE S R.Study of phosphate solubilization efficiencies of fungi and bacteria isolated from saline belt of Purna river basin[J].Research Journal of Agriculture&Biological Sciences,2007,3(4):701-703.
[27] ELAZEEM S A,MEHANA T A,SHABAYEK A A.Some plant growth promoting traits of rhizobacteria isolated from Suez Canal region,Egypt[J].Afr Crop Sci Conf Proc,2007,8:1517-1525.
[28] TANK N D,SARAF M S.Phosphate solubilization,exopolysaccharide production and indole-3-acetic acid secretion by rhizobacteria isolated form Trigonella foenum-graenum[J].Ind J Microbiol,2003,43:37-40.
[29]张英,芦光新,谢永丽,等.溶磷菌分泌有机酸与溶磷能力相关性研究[J].草地学报,2015,23(5):1033-1038.
[30]刘永锋,陆凡,陈志谊,等.拮抗细菌T429和T392的生物活性及其对水稻白叶枯病的防治效果[J].江苏农业学报,2012,28(4):733-737.
[31] DATTA C,BASU P S.Indole acetic acid production by a Rhizobium species from root nodules of a leguminous shrub,Cajanus cajan[J].Microbiological Research,2000,155(2):123-127.
[32] MUNNS R.Comparative physiology of salt and water stress[J].Plant Cell Environ,2003,25:239-250.
[33]刘佳莉,方芳,史煦涵,等.2株盐碱地燕麦根际促生菌的筛选及其促生作用研究[J].草业学报,2013,22(2):132-139.
[34]崔晓双,王伟,张如,等.基于根际营养竞争的植物根际促生菌的筛选及促生效应研究[J].南京农业大学学报,2015,38(6):958-966.
[35]吴秉奇,梁永江,丁延芹,等.两株烟草根际拮抗菌的生防和促生效果研究[J].中国烟草科学,2013,34(1):66-71.
[36] NADEEM S M,HUSSAIN I,NAVEED M,et al.Performance of plant growth promoting rhizobacteria containing ACC-deaminase activity for improving growth of maize under salt stressed conditions[J].Pak J Agric Sci,2006,43:114-121.
[37] YONG H L,WANG H L,DO K L,et al.Factors relating to induced systemic resistance in watermelon by plant growth promoting Pseudomonas spp.[J].Plant Pathol,2001,17:174-179.