一株野生大豆内生真菌YD-25的筛选鉴定及其抗重金属活性研究
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摘要
为了丰富野生大豆内生真菌资源,探究野生大豆内生真菌的生物学活性。从野生大豆植株内分离筛选出1株内生真菌YD-25,通过形态学和分子生物学相结合的方式鉴定菌株;采用控制单因素变量试验和正交试验确定菌株的最适生长条件;通过对比胞内、胞外产物和对照组培养大豆幼苗的株高测定菌株的促生效果;测定菌株抗重金属铜、铅、镉的能力和重金属胁迫下菌体的抗氧化酶活性变化。结果表明:YD-25菌落初期为白色,后期变为橘黄色,菌丝分支且产孢;经鉴定该菌株为黑附球菌(Epicoccum nigrum)。该菌株生长的最适pH为3.0,优化培养条件为可溶性淀粉4%、酵母浸粉0.5%、KH_2PO_4 0.15%、MgSO_4 0.05%,培养8 d。YD-25菌株的胞外产物对大豆幼苗有较好的促生长作用,第4天和第6天的促生率分别达到了15.4%和18.1%。同时具有良好的耐重金属镉、铅、铜的能力,除受Cd~(2+)胁迫的YD-25菌株的SOD活性之外,受其它重金属胁迫的YD-25菌株各项酶活性均随着重金属浓度提高有不同程度的提高。
In order to enrich the endophytic fungi resources of wild soybeans and explore the biological activities of endophytic fungi of wild soybeans. An endophytic fungus strain YD-25 was isolated from Glycine soja plants and identified by combining morphology with molecular biology. The optimum growth conditions of the strain were determined by controlling single factor variable experiment and orthogonal experiment. The growth promoting effect of the strain was determined by comparing the plant height of the soybean seedlings in the intracellular, extracellular, and control groups. The ability of the strain to resist heavy metals such as copper, lead and cadmium and the enzyme activity of the cells under heavy metal stress were determined. The results showed that the YD-25 colony was white at the beginning, and later turned into orange, the mycelium branched and sporulated. The strain was identified as Epicoccum nigrum. The optimal pH of the strain was 3.0, and the optimized culture conditions were soluble starch 4%, yeast dip powder 0.5%, KH_2PO_4 0.15%, MgSO_4 0.05%, and cultured for 8 d. The extracellular product of YD-25 strain had a good growth promoting effect on soybean seedlings, and the growth promoting rates on 4 th and 6 th day reached 15.4% and 18.1%, respectively. At the same time, it had good resistance to heavy metal cadmium, lead and copper. Except for the SOD activity of Cd~(2+) stressed YD-25 strain, the enzyme activities of YD-25 strains under other heavy metal stresses were increased in different degree with the increase of heavy metal concentration.
In order to enrich the endophytic fungi resources of wild soybeans and explore the biological activities of endophytic fungi of wild soybeans. An endophytic fungus strain YD-25 was isolated from Glycine soja plants and identified by combining morphology with molecular biology. The optimum growth conditions of the strain were determined by controlling single factor variable experiment and orthogonal experiment. The growth promoting effect of the strain was determined by comparing the plant height of the soybean seedlings in the intracellular, extracellular, and control groups. The ability of the strain to resist heavy metals such as copper, lead and cadmium and the enzyme activity of the cells under heavy metal stress were determined. The results showed that the YD-25 colony was white at the beginning, and later turned into orange, the mycelium branched and sporulated. The strain was identified as Epicoccum nigrum. The optimal pH of the strain was 3.0, and the optimized culture conditions were soluble starch 4%, yeast dip powder 0.5%, KH_2PO_4 0.15%, MgSO_4 0.05%, and cultured for 8 d. The extracellular product of YD-25 strain had a good growth promoting effect on soybean seedlings, and the growth promoting rates on 4 th and 6 th day reached 15.4% and 18.1%, respectively. At the same time, it had good resistance to heavy metal cadmium, lead and copper. Except for the SOD activity of Cd~(2+) stressed YD-25 strain, the enzyme activities of YD-25 strains under other heavy metal stresses were increased in different degree with the increase of heavy metal concentration.
引文
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[17]陈建勋,王晓峰.植物生理学实验指导[M].广州:华南理工大学出版社,2015.(Chen J X,Wang X F.Instruction for plant physiology experiments[M].Guangzhou:South China University of Technology Press,2015.)
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[19]詹寿发,卢丹妮,毛花英,等.2株溶磷、解钾与产IAA的内生真菌菌株的筛选、鉴定及促生作用研究[J].中国土壤与肥料,2017(3):142-151.(Zhan S F,Lu D N,Mao H Y,et al.Screening,identification and growth promotion of two endophytic fungi strains solving phosphorus,potassium and producing IAA[J].Soil and Fertilizer in China,2017(3):142-151.)
[20]刘劲松,张健君,杨淑芳,等.内生菌参与植物/微生物联合修复重金属污染土壤的研究进展[J].中国植保导刊,2014,34(2):27-30.(Liu J S,Zhang J J,Yang S F,et al.Advances in the study of endophytic bacteria participating in phyto-microbial remediation of heavy metal contaminated soils[J].China Plant Protection Guide,2014,34(2):27-30.)
[21]赵颖,于飞,卜宁.碱蓬内生真菌JP3的分离、鉴定及促生作用研究[J].沈阳师范大学学报(自然科学版),2015(1):116-120.(Zhao Y,Yu F,Bu N.Isolation,identification and growth promotion of endophytic fungus JP3 from Suaeda salsa[J].Journal of Shenyang Normal University(Natural Science Edition),2015(1):116-120.)
[22]姜敏,曹理想,张仁铎.重金属抗性内生真菌与其宿主植物重金属抗性关系初探[J].农业环境科学学报,2007(6):2038-2042.(Jiang M,Cao L X,Zhang R D.Preliminary study on the relationship between heavy metal resistant endophytic fungi and heavy metal resistance in host plants[J].Journal of Agro-Environment Science,2007(6):2038-2042.)
[23]贾彤,王瑞宏,曹苗文.白羊草Epichloё属内生真菌的分离鉴定及其重金属耐受性[J].生态学报,2018,38(18):6623-6631.(Jia T,Wang R H,Cao M W.Isolation and identification of endophytic fungi from epichlo genus and its heavy metal tolerance[J].Journal of Ecology,2018,38(18):6623-6631.)
[24]Abril A,Zurdo-Pineiro J L,Peix A.Solubilization of phosphate by a strain of Rhizobium leguminosarum bv.trifolii isolated from Phaseolus vulgaris in El Chaco Arido soil(Argentina)[J].Developments in Plant and Soil Sciences,2007(102):135-138.
[25]陈芳芳,姜清华,朱新开,等.氮肥运筹对生选6号籽粒产量及花后叶片衰老特性的影响[J].大麦与谷类科学,2013(2):1-6.(Chen F F,Jiang Q H,Zhu X K,et al.Effects of nitrogen fertilizer application on grain yield and senescence characteristics of post-floral leaves[J].Barley and Cereal Science,2013(2):1-6.)
[26]惠俊爱,党志.土壤不同镉浓度对玉米CT38生长及抗氧化酶活性的影响[J].生态环境学报,2014,23(5):884-889.(Hui H A,Dang Z.Effects of different cadmium concentrations in soil on CT38 growth and antioxidant enzyme activities in maize[J].Journal of Eco-Environment,2014,23(5):884-889.)
[2]Kogel K H,Franken P,Hückelhoven R.Endophyte or parasitewhat decides?[J].Current Opinion in Plant Biology,2006,9(4):358-363.
[3]官珊,钟国华,孙之潭,等.植物内生真菌的研究进展[J].仲恺农业技术学院学报,2005(1):61-66.(Guan S,Zhong GH,Sun Z T.Advances in endophytic fungi in plant[J].Journal of Zhongkai Agrotechnical College,2005(1):61-66.)
[4]黎万奎,胡之璧.内生菌与天然药物[J].中国天然药物,2005(4):193-199.(Li W K,Hu Z B.Endophytic bacteria and natural medicines[J].Chinese Natural Medicines,2005(4):193-199.)
[5]Tan R X,Zou W X.Endophytes:A rich source of functional metabolites[J].Cheminform,2001,32(44):448-459.
[6]Petrini O.Microbial ecology of leaves[M].New York:SpringerVerlag,1991:179-197.
[7]王果.河南省野生大豆资源遗传多样性分析[D].陕西:西北农林科技大学,2006:1-2.(Wang G.Genetic diversity dnalysis of wild soybean resources in Henan province[D].Shanxi:Northwest University of Agriculture and Forestry Science and Technology,2006:1-2.)
[8]徐豹.中国野生大豆(G.soja)研究十年[J].吉林农业科学,1989(1):5-13.(Xu B.Ten years of research on wild soybean(G.soja)in China[J].Jilin Agricultural Sciences,1989(1):5-13.)
[9]庄炳昌.中国野生大豆研究二十年[J].吉林农业科学,1999(5):3-10.(Zhuang B C.Twenty years of research on wild soybeans in China[J].Jilin Agricultural Sciences,1999(5):3-10.)
[10]李润芳,王栋,李湛,等.我国野生大豆遗传多样性研究进展(英文)[J].农业科学与技术,2017,18(12):2326-2330,2334.(Li R F,Wang D,Li Z,et al.Advances in research on genetic diversity of wild soybean in China[J].Agricultural Science&Technology,2017,18(12):2326-2330,2334.)
[11]李娜娜,孔维国,张煜,等.野生大豆耐盐性研究进展[J].西北植物学报,2012,32(5):1067-1072.(Li N N,Kong WG,Zhang Y,et al.Advances in salt tolerance of wild soybean[J].Journal of Northwest Botany,2012,32(5):1067-1072.)
[12]王志友,王昌陵,董丽杰,等.辽宁省野生大豆种质资源及利用现状[J].杂粮作物,2008(4):241-243.(Wang Z Y,Wang C L,Dong L J,et al.Wild soybean germplasm resources and utilization status in Liaoning province[J].Miscellaneous Crops,2008(4):241-243.)
[13]高小宽,刘国杰,白丽荣.聚乙二醇(PEG)模拟干旱胁迫对野生大豆与栽培大豆萌发的影响[J].大豆科学,2012,31(6):1027-1029.(Gao X K,Liu G J,Bai L R.Effects of polyethylene glycol(PEG)simulated drought stress on germination of wild soybean and cultivated soybean[J].Soybean Science,2012,31(6):1027-1029.)
[14]纪展波,蒲伟凤,李桂兰,等.野生大豆、半野生大豆和栽培大豆对苗期干旱胁迫的生理反应[J].大豆科学,2012,31(4):598-604.(Ji Z B,Pu W F,Li G L,et al.Physiological responses of wild soybean,semi-wild soybean and cultivated soybean to drought stress at seedling stage[J].Soybean Science,2012,31(4):598-604.)
[15]魏景超.真菌鉴定手册[M].上海:上海科学技术出版社出版,1979.(Wei J C.Fungal identification manual[M].Shanghai:Shanghai Scientific&Technical Publishers,1979.)
[16]Rao K V M,Sresty T V S.Antioxidative parameters in the seedlings of pigeonpea[Cajanus cajan(L.)Millspaugh]in response to Zn and Ni stresses[J].Plant Science,2000,157(1):1-128.
[17]陈建勋,王晓峰.植物生理学实验指导[M].广州:华南理工大学出版社,2015.(Chen J X,Wang X F.Instruction for plant physiology experiments[M].Guangzhou:South China University of Technology Press,2015.)
[18]Sheng X F,Xia J J,Jiang C Y,et,al.Characterization of heavy metal-resistant endophytic bacteria from rape(Brassica napus)roots and their potential in promoting the growth and lead accumulation of rape[J].Environmental Pollution,2008,156(3):1164-1170.
[19]詹寿发,卢丹妮,毛花英,等.2株溶磷、解钾与产IAA的内生真菌菌株的筛选、鉴定及促生作用研究[J].中国土壤与肥料,2017(3):142-151.(Zhan S F,Lu D N,Mao H Y,et al.Screening,identification and growth promotion of two endophytic fungi strains solving phosphorus,potassium and producing IAA[J].Soil and Fertilizer in China,2017(3):142-151.)
[20]刘劲松,张健君,杨淑芳,等.内生菌参与植物/微生物联合修复重金属污染土壤的研究进展[J].中国植保导刊,2014,34(2):27-30.(Liu J S,Zhang J J,Yang S F,et al.Advances in the study of endophytic bacteria participating in phyto-microbial remediation of heavy metal contaminated soils[J].China Plant Protection Guide,2014,34(2):27-30.)
[21]赵颖,于飞,卜宁.碱蓬内生真菌JP3的分离、鉴定及促生作用研究[J].沈阳师范大学学报(自然科学版),2015(1):116-120.(Zhao Y,Yu F,Bu N.Isolation,identification and growth promotion of endophytic fungus JP3 from Suaeda salsa[J].Journal of Shenyang Normal University(Natural Science Edition),2015(1):116-120.)
[22]姜敏,曹理想,张仁铎.重金属抗性内生真菌与其宿主植物重金属抗性关系初探[J].农业环境科学学报,2007(6):2038-2042.(Jiang M,Cao L X,Zhang R D.Preliminary study on the relationship between heavy metal resistant endophytic fungi and heavy metal resistance in host plants[J].Journal of Agro-Environment Science,2007(6):2038-2042.)
[23]贾彤,王瑞宏,曹苗文.白羊草Epichloё属内生真菌的分离鉴定及其重金属耐受性[J].生态学报,2018,38(18):6623-6631.(Jia T,Wang R H,Cao M W.Isolation and identification of endophytic fungi from epichlo genus and its heavy metal tolerance[J].Journal of Ecology,2018,38(18):6623-6631.)
[24]Abril A,Zurdo-Pineiro J L,Peix A.Solubilization of phosphate by a strain of Rhizobium leguminosarum bv.trifolii isolated from Phaseolus vulgaris in El Chaco Arido soil(Argentina)[J].Developments in Plant and Soil Sciences,2007(102):135-138.
[25]陈芳芳,姜清华,朱新开,等.氮肥运筹对生选6号籽粒产量及花后叶片衰老特性的影响[J].大麦与谷类科学,2013(2):1-6.(Chen F F,Jiang Q H,Zhu X K,et al.Effects of nitrogen fertilizer application on grain yield and senescence characteristics of post-floral leaves[J].Barley and Cereal Science,2013(2):1-6.)
[26]惠俊爱,党志.土壤不同镉浓度对玉米CT38生长及抗氧化酶活性的影响[J].生态环境学报,2014,23(5):884-889.(Hui H A,Dang Z.Effects of different cadmium concentrations in soil on CT38 growth and antioxidant enzyme activities in maize[J].Journal of Eco-Environment,2014,23(5):884-889.)