抗镉植物内生真菌的筛选及其促生能力研究
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摘要
筛选具有抗镉促生能力的内生真菌,为植物-微生物联合修复农田镉污染提供优势菌株。分离秦岭铅锌尾矿区内优势植物杠柳(Periploca sepium Bunge)的内生真菌,利用加镉培养基筛选抗镉能力较高的内生真菌,分析其形态学和内转录间隔区基因序列(Internal transcribed sequence, ITS)特征,鉴定其所属菌属,研究其Cd~(2+)去除率及吲哚-3-乙酸(IAA)活性、铁载体活性、胞外过氧化氢酶(CAT)活性和溶磷解钾活性等促生潜力,验证内生真菌对小麦幼苗抗镉能力的提升作用。结果表明:筛选到5株可耐受30 000 mg/L Cd~(2+)的内生真菌,经鉴定,菌株GR1为Talaromyces sp.,菌株GR4为Colletotrichum sp.,菌株GR9为Purpureocillium sp.,而菌株GR7和菌株GR10均为Fusarium sp.。Cd~(2+)含量为1 000 mg/L时,菌株GR7的Cd~(2+)去除率最高。4株菌株有IAA活性,其中菌株GR4的IAA活性最高(99.417 mg/L)。菌株GR7的铁载体活性最高(84.64%)。菌株GR9的胞外CAT活性最高(10.01 U/mL)。菌株GR1发酵液中的有效磷含量最高(201.67 mg/L)。菌株GR4发酵液中的有效钾含量最高(105.11 mg/L)。5株抗镉内生真菌均能显著提高小麦幼苗的耐镉能力,与空白相比,添加GR1发酵液的小麦幼苗株高增加了80.22%,根长增加了61.26%,鲜质量增加了27.66%,干质量增加了60.87%,叶绿素含量增加了28.42%,可溶性糖含量增加了7.83%,可溶性蛋白含量增加了13.28%,整体促进效果较为显著(P<0.05)。5株抗镉内生真菌均有抗镉促生潜力,可为农作物-微生物联合修复农田镉污染提供优势菌株,提高农田镉污染修复效率。
To provide a dominant strain for remediation of cadmium contaminated farmland by plants-microorganism combined remediation technology,the cadmium resistant endophytic fungi with growth-promoting ability were screened.Periploca sepium Bunge,a dominant plant in the lead-zinc tailings area of Qinling mountains, was used to isolate endophytic fungi.The endophytic fungi with high cadmium resistance were screened and its bacteria genus were indentified by adding cadmium medium and analysed their morphology and internal transcribed sequence(ITS).Their Cd~(2+) removal rates were detected and their growth-promoting potentials such as 3-indoleacetic acid(IAA) activity,siderophore activity,extracellular catalase(CAT) activity,phosphorus and potassium dissolution activity were measured to verify the promotion of cadmium resistant ability of endophytic fungi of wheat seedling.The results showed that,5 strains of endophytic fungi which could tolerate 30 000 mg/L Cd~(2+) were screened.Strain GR1 was belonging to Talaromyces sp.,strain GR4 was belonging to Colletotrichum sp.,strain GR9 was belonging to Purpureocillium sp.,both strain GR7 and GR10 were belonging to Fusarium sp..When the content of Cd~(2+) was 1 000 mg/L,strain GR7 had the highest removal rate of Cd~(2+).4 strains had the IAA activity,strain GR4 was the highest(99.417 mg/L).Strain GR7 had the highest siderophore activity(84.64%).Strain GR9 had the highest extracellular CAT activity(10.01 U/mL).Strain GR1 fermentation broth had the highest effective phosphorus content(201.67 mg/L).Strain GR4 fermentation broth had the highest effective potassium content(105.11 mg/L).All of 5 cadmium-resistant endophytic fungi could significantly improve the cadmium tolerance of wheat seedlings.Compared with the blank,the plant height,root length,fresh weight,dry weight,chlorophyll content,soluble sugar content,soluble protein content of wheat seedlings with strain GR1 fermentation broth increased by 80.22%,61.26%,27.66%,60.87%,28.42%,7.83%,13.28% respectively.Overall promotion effect is significant(P<0.05).5 strains of cadmium resistant endophytic fungi had cadmium resistant and growth-promoting potential,which could be used as the dominant strains for remediation of cadmium contaminated farmland by crops-microorganism combined remediation technology and to promote remediating efficiency of cadmium contaminated farmland.
To provide a dominant strain for remediation of cadmium contaminated farmland by plants-microorganism combined remediation technology,the cadmium resistant endophytic fungi with growth-promoting ability were screened.Periploca sepium Bunge,a dominant plant in the lead-zinc tailings area of Qinling mountains, was used to isolate endophytic fungi.The endophytic fungi with high cadmium resistance were screened and its bacteria genus were indentified by adding cadmium medium and analysed their morphology and internal transcribed sequence(ITS).Their Cd~(2+) removal rates were detected and their growth-promoting potentials such as 3-indoleacetic acid(IAA) activity,siderophore activity,extracellular catalase(CAT) activity,phosphorus and potassium dissolution activity were measured to verify the promotion of cadmium resistant ability of endophytic fungi of wheat seedling.The results showed that,5 strains of endophytic fungi which could tolerate 30 000 mg/L Cd~(2+) were screened.Strain GR1 was belonging to Talaromyces sp.,strain GR4 was belonging to Colletotrichum sp.,strain GR9 was belonging to Purpureocillium sp.,both strain GR7 and GR10 were belonging to Fusarium sp..When the content of Cd~(2+) was 1 000 mg/L,strain GR7 had the highest removal rate of Cd~(2+).4 strains had the IAA activity,strain GR4 was the highest(99.417 mg/L).Strain GR7 had the highest siderophore activity(84.64%).Strain GR9 had the highest extracellular CAT activity(10.01 U/mL).Strain GR1 fermentation broth had the highest effective phosphorus content(201.67 mg/L).Strain GR4 fermentation broth had the highest effective potassium content(105.11 mg/L).All of 5 cadmium-resistant endophytic fungi could significantly improve the cadmium tolerance of wheat seedlings.Compared with the blank,the plant height,root length,fresh weight,dry weight,chlorophyll content,soluble sugar content,soluble protein content of wheat seedlings with strain GR1 fermentation broth increased by 80.22%,61.26%,27.66%,60.87%,28.42%,7.83%,13.28% respectively.Overall promotion effect is significant(P<0.05).5 strains of cadmium resistant endophytic fungi had cadmium resistant and growth-promoting potential,which could be used as the dominant strains for remediation of cadmium contaminated farmland by crops-microorganism combined remediation technology and to promote remediating efficiency of cadmium contaminated farmland.
引文
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[2] 刘菊梅,栗利曼,沈渭寿,等.黄河包头段灌区玉米重金属污染及人群健康风险评价[J].南方农业学报,2015,46(9):1591-1595.
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[6] 丁锐,李萌凯,任光宇,等.羊耳蒜内生真菌的分离鉴定与抑菌活性筛选[J].北方园艺,2017(1):126-130.
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[9] BABU A G,REDDY M S.Dual inoculation of arbuscular mycorrhizal and phosphate solubilizing fungi contributes in sustainable maintenance of plant health in fly ash ponds[J].Water,Air and Soil Pollution,2011,219(1/4):3-10.
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[12] RAJKUMAR M,SANDHYA S,PRASAD M N,et al.Perspectives of plant-associated microbes in heavy metal phytoremediation[J].Biotechnology Advances,2012,30(6):1562-1574.
[13] 张兴旭.醉马草-内生真菌共生体对胁迫的响应及其次生代谢产物活性的研究[D].兰州:兰州大学,2012.
[14] 魏玮.植物内生真菌对镉和盐胁迫下水稻幼苗缓解作用的研究[D].沈阳:沈阳师范大学,2017.
[15] 刘军生,解修超,罗阳兰,等.Aspergillus sp.EA-LJS80的分离及其发酵液提取物七叶皂苷C的生物活性[J].菌物学报,2018,37(1):79-87.
[16] 王祥红,汤志宏,李静.曲霉菌的分离及其形态观察[J].生物学通报,2013,48(7):54-55.
[17] 魏景超.真菌鉴定手册[M].上海:上海科学技术出版社,1979.
[18] 金忠民,郝宇,刘丽杰,等.一株铅镉抗性菌株的分离鉴定及其生物学特性[J].环境工程学报,2015,9(7):3551-3557.
[19] 代金霞,周波,田平雅.荒漠植物柠条产ACC脱氨酶根际促生菌的筛选及其促生特性研究[J].生态环境学报,2017,26(3):386-391.
[20] 肖艳红,李菁,刘祝祥,等.1株蛇足石杉根际铁载体细菌的分离、筛选与鉴定[J].微生物学杂志,2013,33(2):63-67.
[21] 彭建,王丹英,徐春梅,等.钼酸铵法测定水稻过氧化氢酶活性[J].中国农学通报,2009,25(16):61-64.
[22] 李海蛟,孟歌,郑飞,等.液体培养条件下槐生多年卧孔菌、微酸多年卧孔菌和白蜡多年卧孔菌的抗氧化活性研究[J].菌物学报,2017,36(2):229-241.
[23] 王金昌,郑国华,傅筱冲.一株解钾解磷菌株的筛选[J].江西科学,2014,32(1):51-53,103.
[24] 王誉瑶,韦中,徐阳春,等.溶磷菌株组合的溶磷效应及对玉米生长的影响[J].植物营养与肥料学报,2017,23(1):262-268.
[25] 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.
[26] 边子星,颜彩燕,姚肖健,等.濒危华石斛种子活力测定方法研究[J].热带作物学报,2017,38(3):403-407.
[27] 袁进.BS-12两性修饰对Cd2+污染土壤中小麦生长的影响[D].杨凌:西北农林科技大学,2011.
[28] 张志良.植物生理学实验指导[M].北京:高等教育出版社,2000:160-161.
[29] MIETHKE M,MARAHIEL M A.Siderophore-based iron acquisition and pathogen control[J].Microbiology and Molecular Biology Reviews,2007,71(3):413-451.
[30] 孙萌,李韵雅,吴祎,等.解淀粉芽孢杆菌SYBC H47铁载体合成酶基因dhbA、dhbB、dhbC的克隆表达与序列分析[J].基因组学与应用生物学,2017,36(10):4175-4183.
[31] 杨慧,范丙全,龚明波,等.一株新的溶磷草生欧文氏菌的分离、鉴定及其溶磷效果的初步研究[J].微生物学报,2008,48(1):51-56.
[32] 谷艳.氧化尾矿与白茅根际尾矿中可培养溶磷菌比较研究[J].土壤通报,2018,49(1):119-125.
[33] 周小梅,赵运林,董萌,等.成团泛菌(Pantoea agglomerans)对镉胁迫下蒌蒿耐受性的影响[J].生态毒理学报,2016,11(4):176-183.
[34] 尹艺,赵颖,马莲菊,等.碱蓬内生真菌对镉胁迫水稻幼苗生长及生理生化指标的影响[J].贵州农业科学,2014,42(3):23-26.
[35] GUO J P,TAN J L,WANG Y L,et al.Isolation of talathermophilins from the thermophilic fungus Talaromyces thermophilus YM3-4[J].Journal of natural products,2011,74(10):2278-2281.
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