1株镉耐受真菌C12的鉴定及其镉耐受性研究
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摘要
【目的】对1株镉耐受性较强的弯孢属真菌C12进行分类鉴定,评估其对重金属镉的耐受能力,并初步分析和探讨其相关的镉耐性机制,为揭示真菌的重金属抗性机制及其在重金属污染治理中的应用提供依据。【方法】利用形态和分子生物学相结合的方法对菌株C12进行分类鉴定,采用固体和液体2种培养方法研究不同Cd~(2+)质量浓度(0,20,40,80,160,320mg/L)下菌株C12的生长状况,比较不同Cd~(2+)质量浓度下菌株C12培养液中pH值和有机酸含量以及菌丝内谷胱甘肽(GSH)和丙二醛(MDA)含量的变化。【结果】菌株C12被鉴定为津田弯孢(Curvularia tsudae);该菌在Cd~(2+)质量浓度高达320mg/L时仍有菌丝生长,菌丝体干质量在固体培养时随Cd~(2+)质量浓度的升高先增加后降低,在Cd~(2+)质量浓度为20mg/L时达到最大(P<0.05),而液体培养时却随Cd~(2+)质量浓度的升高逐渐降低(P<0.05);培养液中柠檬酸和苹果酸的含量较高,琥珀酸仅在高Cd~(2+)质量浓度(80~320mg/L)时才会产生,未检测到草酸和酒石酸;随Cd~(2+)质量浓度的升高,柠檬酸、苹果酸、乙酸和琥珀酸质量浓度以及菌丝内的GSH含量均先增加后降低,且在160mg/L时达到最大(P<0.05),而培养液的pH值并无显著变化(P>0.05),但均显著低于不加镉的对照(P<0.05);菌丝内的MDA含量则随Cd~(2+)质量浓度的升高先增加再降低后又升高,但差异并不显著(P>0.05)。【结论】津田弯孢C12具有较高的镉耐受性,且液体培养条件下菌丝生长和镉耐受性明显优于固体培养,GSH和有机酸(柠檬酸、苹果酸和琥珀酸)的大量积累可能是其减轻镉毒害、提高镉耐受性的潜在机制之一。
【Objective】In this study,one isolate of Curvulariasp.C12 which showed high tolerance ability to heavy metal cadmium was identified as Curvulariatsudae,and then the related tolerance mechanism was preliminarily analyzed and discussed.The results could provide the foundation for revealing the mechanism of fungal resistance to heavy metal,and for the application of fungi to control heavy metal pollution.【Method】The taxonomic status of isolate C12 was identified based on its morphological and molecular characteristics.Its tolerance ability to cadmium was assessed both in solid and liquid culture condition with different mass concentrations of Cd~(2+)(0,20,40,80,160,320mg/L).pH value and organic acids contents,as well as glutathione(GSH)and malondialdehyde(MDA)contents in the fungal mycelia weremeasured and compared under different mass concentrations of Cd~(2+)in liquid culture condition.【Result】Isolate C12 was identified as Curvularia tsudae,and it could grow under 320mg/L Cd~(2+).Along with the increase of Cd~(2+)mass concentrations,the mycelia dry weight of C.tsudae C12 was increased firstly and then decreased on solid culture condition,and reached the maximum at 20mg/L(P<0.05),but was gradually reduced in liquid culture condition(P<0.05).In liquid culture condition,oxalic acid and tartaric acid did not be detected,while citric acid and malic acid content were relatively high,and succinic acid was only produced under high concentrations of Cd~(2+)(80-320mg/L).With the increase of Cd~(2+) mass concentrations,the content of citric acid,malic acid,acetic acid and succinic acid,as well as GSH content in mycelia were increased firstly and then decreased,and reached the maximum at 160mg/L(P<0.05);while MDA content in mycelia was increased firstly,then decreased and later rose again,but did not show significant difference(P>0.05).pH values were significant lower than that of control(without cadmium)(P<0.05),however,did not change significantly(P>0.05)along with the increase of Cd~(2+)mass concentrations.【Conclusion】Curvularia tsudae C12 showed high cadmium tolerance,and its growth and cadmium tolerance were obviously better under liquid culture condition.The accumulation of GSH and organic acids(especially citric acid,malic acid and succinic acid)maybe one of the underlying mechanisms for C.tsudae C12 to reduce the damage of cadmium and to improve its cadmium tolerance.
【Objective】In this study,one isolate of Curvulariasp.C12 which showed high tolerance ability to heavy metal cadmium was identified as Curvulariatsudae,and then the related tolerance mechanism was preliminarily analyzed and discussed.The results could provide the foundation for revealing the mechanism of fungal resistance to heavy metal,and for the application of fungi to control heavy metal pollution.【Method】The taxonomic status of isolate C12 was identified based on its morphological and molecular characteristics.Its tolerance ability to cadmium was assessed both in solid and liquid culture condition with different mass concentrations of Cd~(2+)(0,20,40,80,160,320mg/L).pH value and organic acids contents,as well as glutathione(GSH)and malondialdehyde(MDA)contents in the fungal mycelia weremeasured and compared under different mass concentrations of Cd~(2+)in liquid culture condition.【Result】Isolate C12 was identified as Curvularia tsudae,and it could grow under 320mg/L Cd~(2+).Along with the increase of Cd~(2+)mass concentrations,the mycelia dry weight of C.tsudae C12 was increased firstly and then decreased on solid culture condition,and reached the maximum at 20mg/L(P<0.05),but was gradually reduced in liquid culture condition(P<0.05).In liquid culture condition,oxalic acid and tartaric acid did not be detected,while citric acid and malic acid content were relatively high,and succinic acid was only produced under high concentrations of Cd~(2+)(80-320mg/L).With the increase of Cd~(2+) mass concentrations,the content of citric acid,malic acid,acetic acid and succinic acid,as well as GSH content in mycelia were increased firstly and then decreased,and reached the maximum at 160mg/L(P<0.05);while MDA content in mycelia was increased firstly,then decreased and later rose again,but did not show significant difference(P>0.05).pH values were significant lower than that of control(without cadmium)(P<0.05),however,did not change significantly(P>0.05)along with the increase of Cd~(2+)mass concentrations.【Conclusion】Curvularia tsudae C12 showed high cadmium tolerance,and its growth and cadmium tolerance were obviously better under liquid culture condition.The accumulation of GSH and organic acids(especially citric acid,malic acid and succinic acid)maybe one of the underlying mechanisms for C.tsudae C12 to reduce the damage of cadmium and to improve its cadmium tolerance.
引文
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[17]Mcginnis M R,Rinaldi M G,Winn R E.Emerging agents of phaeohyphomycosis:pathogenic species of Bipolaris and Exserohilum[J].Journal of Clinical Microbiology,1986,24(2):250-259.
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[19]Newell C K,Steinmetz R L,Brooks H L.Chronic postoperative endophthalmitis caused by Bipolaris australiensis[J].Retina,2006,26:109-110.
[20]张玉洁,李洪超,赵之伟.矿区植物根内嗜鱼外瓶霉对重金属的耐性和超积累作用[J].土壤,2012,44(3):467-473.Zhang Y J,Li H C,Zhao Z W.Tolerance and accumulation of heavy metals by Exophiala pisciphila strain isolated from plant roots growing in metal polluted soils[J].Soils,2012,44(3):467-473.
[21]Ban Y,Tang M,Chen H,et al.The response of dark septateendophytes(DSE)to heavy metals in pure culture[J].PLoS One,2012,7(10):e47968.
[22]刘红玲,贺亚玲,蒋存凯,等.新疆橙黄疣柄牛肝菌菌丝对Cu2+、Zn2+和Pb2+的耐受性研究[J].北方园艺,2010(8):197-200.Liu H L,He Y L,Jiang C K,et al.Study on the toleration of hypha of Leccinum aurantiacum(Bull.)Gray to heavy metals Cu2+,Zn2+and Pb2+[J].Northern Horticulture,2010(8):197-200.
[23]李芳,张俊伶,冯固,等.两种外生菌根真菌对重金属Zn、Cd和Pb耐性的研究[J].环境科学学报,2003,23(6):807-812.Li F,Zhang J L,Feng G,et al.The tolerance of ectomycorrhizal fungi Suillus granulates and Paxillus involutus to heavy metals Zn,Cd,Pb[J].Acta Scientiae Circumstatiae,2003,23(6):807-812.
[24]Xu P,Zeng G,Huang D,et al.Metal bioaccumulation,oxidative stress and antioxidant defenses in Phanerochaete chrysosporium response to Cd exposure[J].Ecological Engineering,2016,87:150-156.
[25]Green I I I F,Clausen C A.Copper tolerance of brown-rot fungi:time course of oxalic acid production[J].International Biodeterioration&Biodegradation,2003,51(2):145-149.
[26]López-Climent M F,Arbona V,Pérez-Clemente R M,et al.Effect of cadmium and calcium treatments on phytochelatin and glutathione levels in citrus plants[J].Plant Biology,2014,16:79-87.
[27]Wójcik M,Skórzyńska-Polit E,Tukiendorf A.Organic acids accumulation and antioxidant enzyme activities in Thlaspicarulescens under Zn and Cd stress[J].Plant Growth Regulation,2006,48(2):145-155.
[28]Garg N,Chandel S.Role of arbuscularmycorrhiza in arresting reactive oxygen species(ROS)and strengthening antioxidant defense in Cajanuscajan(L.)Millsp.nodules under salinity(NaCl)and cadmium(Cd)stress[J].Plant Growth Regulation,2015,75(2):521-534.
[29]Clausen C A,Green I I I F.Oxalic acid overproduction by copper-tolerant brown-rot basidiomycetes on southern yellow pine treated with copper-based preservatives[J].International Biodeterioration&Biodegradation,2003,51(2):139-144.
[2]钱春香,王明明,许燕波.土壤重金属污染现状及微生物修复技术研究进展[J].东南大学学报(自然科学版),2013,43(3):669-674.Qian C X,Wang M M,Xu Y B.Current situation of soil contamination by heavy metal and research progress in bio-remediation technique[J].Journal of Southeast University(Natural Science Edition),2013,43(3):669-674.
[3]冷阳.黄孢原毛平革菌对重金属Cd的富集及其交互作用机理研究[D].长沙:湖南大学,2014.Leng Y.Research on Cd(Ⅱ)enrichment and its mechanism of interaction with Phanerochaete chrysosporium[D].Changsha:Hunan University,2014.
[4]薛高尚,胡丽娟,田云,等.微生物修复技术在重金属污染治理中的研究进展[J].中国农学通报,2012,28(11):266-271.Xue G S,Hu L J,Tian Y,et al.Research progress on microbial remediation of controlling heavy metal pollution[J].Chinese Agriculture Science Bulletin,2012,28(11):266-271.
[5]Iskandar N L,Zainudin N A,Tan S G.Tolerance and biosorption of copper(Cu)and lead(Pb)by filamentous fungi isolated from a freshwater ecosystem[J].Journal of Environmental Sciences,2011,23(5):824-830.
[6]Tahir A,Iram H.Development of a fungal consortium for the biosorption of cadmium from paddy rice field water in a bioreactor[J].Annals of Microbiology,2012,62(3):1243-1246.
[7]Fomina M A,Hillier S,Charnock J M,et al.Role of oxalic acid overexcretion in transformations of toxic metal minerals by Beauveria caledonica[J].Applied and Environmental Microbiology,2005,71(1):371-381.
[8]Chaoui A,Mazhoudi S,Ghorbal M H,et al.Cadmium and zinc induction of lipid peroxidation and effects on antioxidant enzyme activities in bean(Phaseolus vulgaris L.)[J].Plant Science,1997,127(2):139-147.
[9]Zhan F,He Y,Li T,et al.Tolerance and antioxidant response of a dark septateendophyte(DSE),Exophiala pisciphila,to cadmium stress[J].Bulletin of Environmental Contamination and Toxicology,2015,94(1):96-102.
[10]Doyle J J.A rapid DNA isolation procedure for small quantities of fresh leaftissue[J].Phytochemical Bulletin,1987,19:11-15.
[11]White T J,Bruns T,Lee S J,et al.Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics[J].PCR Protocols:A Guide to Methods and Applications,1990,18(1):315-322.
[12]Altschul S F,Madden T L,Schaffer A A,et al.Gapped Blast and Psiblast:a new generation of protein database search programs[J].Nucleic Acids,1997,25:3389-3402.
[13]湛方栋.嗜鱼外瓶霉(Exophiala pisciphila ACCC32496)镉耐性机制研究[D].昆明:云南大学,2012.Zhan F D.Study on the tolerance mechanisms of Exophiala pisciphila ACCC32496for cadmium[D].Kunming:Yunnan University,2012.
[14]Gallego S M,Benavides M P,Tomaro M L.Effect of heavy metal ion excess on sunflower leaves:evidence for involvement of oxidative stress[J].Plant Science,1996,121(2):151-159.
[15]黄健.废水处理中黄孢原毛平革菌对Cd胁迫的适应性及抗性反应研究[D].长沙:湖南大学,2014.Huang J.Adaptability and resistance of Phanerochaete chrysosporium with Cd(II)in wastewater treatment[D].Changsha:Hunan University,2014.
[16]Deng H,Tan Y P,Shivas R G.Curvularia tsudae comb.nov.et nom.nov.,formerly Pseudocochliobolus australiensis,and a revised synonymy for Curvularia australiensis[J].Mycoscience,2015,56(1):24-28.
[17]Mcginnis M R,Rinaldi M G,Winn R E.Emerging agents of phaeohyphomycosis:pathogenic species of Bipolaris and Exserohilum[J].Journal of Clinical Microbiology,1986,24(2):250-259.
[18]Flanagan K L,Bryceson A D.Disseminate dinfection due to Bipolaris australiensis in a young immunocompetent man:case report and review[J].Clinical Infectious Diseases,1997,25:311-313.
[19]Newell C K,Steinmetz R L,Brooks H L.Chronic postoperative endophthalmitis caused by Bipolaris australiensis[J].Retina,2006,26:109-110.
[20]张玉洁,李洪超,赵之伟.矿区植物根内嗜鱼外瓶霉对重金属的耐性和超积累作用[J].土壤,2012,44(3):467-473.Zhang Y J,Li H C,Zhao Z W.Tolerance and accumulation of heavy metals by Exophiala pisciphila strain isolated from plant roots growing in metal polluted soils[J].Soils,2012,44(3):467-473.
[21]Ban Y,Tang M,Chen H,et al.The response of dark septateendophytes(DSE)to heavy metals in pure culture[J].PLoS One,2012,7(10):e47968.
[22]刘红玲,贺亚玲,蒋存凯,等.新疆橙黄疣柄牛肝菌菌丝对Cu2+、Zn2+和Pb2+的耐受性研究[J].北方园艺,2010(8):197-200.Liu H L,He Y L,Jiang C K,et al.Study on the toleration of hypha of Leccinum aurantiacum(Bull.)Gray to heavy metals Cu2+,Zn2+and Pb2+[J].Northern Horticulture,2010(8):197-200.
[23]李芳,张俊伶,冯固,等.两种外生菌根真菌对重金属Zn、Cd和Pb耐性的研究[J].环境科学学报,2003,23(6):807-812.Li F,Zhang J L,Feng G,et al.The tolerance of ectomycorrhizal fungi Suillus granulates and Paxillus involutus to heavy metals Zn,Cd,Pb[J].Acta Scientiae Circumstatiae,2003,23(6):807-812.
[24]Xu P,Zeng G,Huang D,et al.Metal bioaccumulation,oxidative stress and antioxidant defenses in Phanerochaete chrysosporium response to Cd exposure[J].Ecological Engineering,2016,87:150-156.
[25]Green I I I F,Clausen C A.Copper tolerance of brown-rot fungi:time course of oxalic acid production[J].International Biodeterioration&Biodegradation,2003,51(2):145-149.
[26]López-Climent M F,Arbona V,Pérez-Clemente R M,et al.Effect of cadmium and calcium treatments on phytochelatin and glutathione levels in citrus plants[J].Plant Biology,2014,16:79-87.
[27]Wójcik M,Skórzyńska-Polit E,Tukiendorf A.Organic acids accumulation and antioxidant enzyme activities in Thlaspicarulescens under Zn and Cd stress[J].Plant Growth Regulation,2006,48(2):145-155.
[28]Garg N,Chandel S.Role of arbuscularmycorrhiza in arresting reactive oxygen species(ROS)and strengthening antioxidant defense in Cajanuscajan(L.)Millsp.nodules under salinity(NaCl)and cadmium(Cd)stress[J].Plant Growth Regulation,2015,75(2):521-534.
[29]Clausen C A,Green I I I F.Oxalic acid overproduction by copper-tolerant brown-rot basidiomycetes on southern yellow pine treated with copper-based preservatives[J].International Biodeterioration&Biodegradation,2003,51(2):139-144.
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