拮抗性木霉菌株抗逆性筛选评价标准与方法
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摘要
本研究旨在为高效筛选耐受高温胁迫、低温胁迫、盐胁迫及干燥(干旱)胁迫拮抗性木霉菌株提供筛选阈值。根据5种、20株拮抗尖孢镰孢菌效果较好的木霉菌对上述逆境因子的反应差异,建立每种逆境因子在不同水平下的木霉菌株生长和酶活变化检测阈值和标准化评价方式;分析木霉菌在不同胁迫条件下发育相关生化因子与菌株生长变化之间的关系,及相应胁迫下的孢子萌发率与菌落直径之间的相关性。通过正态性检验获得供试木霉菌株生长响应胁迫的阈值为:1mol/L NaCl(盐胁迫阈值),36℃(高温胁迫阈值),14℃(低温胁迫阈值),400 g/L PEG-6000(干旱胁迫阈值)。相应胁迫下的菌落直径与孢子萌发率明显正相关。菌株在大部分胁迫下的抗坏血酸过氧化物酶(APX)及过氧化氢酶(CAT)酶活与菌丝干重有明显的相关性。本研究为科学评价生物防治木霉菌资源的抗逆性提供了依据。
Efficient screening of Trichoderma strains with biocontrol potential and tolerance to various environment stress factors including extreme temperature, salt and drought etc was extensively required. Hence in this work, we examined a total of 20 Trichoderma isolates from five species in the tolerance performance to different stress conditions, including high and/or low temperature, salt and drought, and then established the threshold and standardized evaluation method based on colony growth and selected biochemical factor s activity/contents of Trichoderma strains at different levels of each stress factor. Eventually the correlation of colony growth changes of Trichoderma spp. with the selected enzymes activity or MDA contents was analyzed under different stress conditions, and the correlation between spore germination rate and colony diameter under the corresponding stress was also analyzed. These results showed that the thresholds for Trichoderma growth response to stress factors were 1 mol/L of NaCl(salt stress), 36 ℃(high temperature), 14 ℃(low temperature) and 400 g/L PEG-6000(drought stress). The diameter of colony was positively correlated with the spore germination rate under the corresponding stress. Most of the selected biochemical factors activity indexes under different stresses factors were significantly correlated with the dry weight of mycelium. In conclusion, the establishment of threshold of Trichoderma strains resistant to different stresses factors provided the essential basis for scientific evaluation of Trichoderma, which is important for evaluation and utilization of tolerant Trichoderma resources as biocontrol agents.
Efficient screening of Trichoderma strains with biocontrol potential and tolerance to various environment stress factors including extreme temperature, salt and drought etc was extensively required. Hence in this work, we examined a total of 20 Trichoderma isolates from five species in the tolerance performance to different stress conditions, including high and/or low temperature, salt and drought, and then established the threshold and standardized evaluation method based on colony growth and selected biochemical factor s activity/contents of Trichoderma strains at different levels of each stress factor. Eventually the correlation of colony growth changes of Trichoderma spp. with the selected enzymes activity or MDA contents was analyzed under different stress conditions, and the correlation between spore germination rate and colony diameter under the corresponding stress was also analyzed. These results showed that the thresholds for Trichoderma growth response to stress factors were 1 mol/L of NaCl(salt stress), 36 ℃(high temperature), 14 ℃(low temperature) and 400 g/L PEG-6000(drought stress). The diameter of colony was positively correlated with the spore germination rate under the corresponding stress. Most of the selected biochemical factors activity indexes under different stresses factors were significantly correlated with the dry weight of mycelium. In conclusion, the establishment of threshold of Trichoderma strains resistant to different stresses factors provided the essential basis for scientific evaluation of Trichoderma, which is important for evaluation and utilization of tolerant Trichoderma resources as biocontrol agents.
引文
[1]陈捷.木霉菌生物学与应用研究--回顾与展望[J].菌物学报,2014,33(6):1129-1135.
[2]AndréS,Monika S.Biology and biotechnology of Trichoderma[J].Applied Microbiology and Biotechnology,2010,87(3):787-799.
[3]Harman G E.Overview of mechanisms and uses of Trichoderma spp.[J].Phytopathology,2006,96(2):190-194.
[4]陈勇,朱廷恒,汪琨,等.提高木霉逆境适应性与生物防治效果的基因工程研究进展[J].中国生物工程杂志,2012,32(6):120-124.
[5]Monte E.Understanding Trichoderma:between biotechnology and microbial ecology[J].International Microbiology the Official Journal of the Spanish Society for Microbiology,2001,4(1):1.
[6]Benítez T,Rincón A M,Limón M C,et al.Biocontrol mechanism of Trichoderma strains[J].International Microbiology,2005,7(4):249-260.
[7]高永东,王兵,刘力行,等.木霉菌及其REMI突变株与油菜联合吸附镉污染土壤研究[J].现代农业科技,2008(20):161-162.
[8]马文亭,滕应,凌婉婷,等.里氏木霉FS10-C对伴矿景天吸取修复镉污染土壤的强化作用[J].土壤,2012,44(6):991-995.
[9]田晔,滕应,赵静,等.一株抗铜木霉对含铜废水的修复潜力研究[J].中国矿业大学学报,2012,41(4):657-662.
[10]陈捷.木霉菌诱导植物抗病性研究新进展[J].中国生物防治学报,2015,31(5):733-741.
[11]Vinalea F,Sivasithamparam K,Ghisalberti E L.Trichoderma plant pathogen interactions[J].Soil Biology and Biochemistry,2008,40(1):1-10.
[12]Kredics L,Antal Z,Manczinger L,et al.Influence of environmental parameters on Trichoderma strains with biocontrol potential[J].Food Technology and Biotechnology,2003,41(7/8):37-42.
[13]张晶晶,黄亚丽,马宏,等.木霉厚垣孢子可湿性粉剂的研制[J].植物保护,2016,42(5):103-109.
[14]杨力明,杨谦,李森,等.哈茨木霉Cu-ZnSOD的克隆表达及活性鉴定[J].哈尔滨工业大学学报,2009(3):102-107..
[15]Zhang S,Gan Y,Xu B.Application of plant growth promoting fungi Trichoderma longibrachiatum T6 enhances tolerance of wheat to salt stress through improvement of antioxidative defense system and gene expression:[J].Frontiers in Plant Science,2016,7(868):1405.
[16]Parvaiz A,Abeer H,Elsayed Fathi A A,et al.Role of Trichoderma harzianum in mitigating NaCl stress in Indian mustard(Brassica juncea L.)through antioxidative defense system[J].Frontiers in Plant Science,2015,6:868.
[17]Bj?rkman T,Blanchard L M,Harman G E.Growth enhancement of shrunken-2(sh2)sweet corn by Trichoderma harzianum 1295-22:effect of environmental stress[J].Journal of the American Society for Horticultural Science American Society for Horticultural Science,1998,123(1):35-40.
[18]Li S Y,Li F L,Wang Y J,et al.Effect of Trichoderma harzianum on cucumber seed germination with NaCl stress[J].Science Technology&Engineering,2016,16(9):164-166.
[19]曲乐,郭凯,李纪顺,等.外源一氧化氮供体硝普钠对盐胁迫下椒样薄荷不定根和根毛发育的影响[J].山东科学,2013,26(5):33-38.
[20]尹大川,邓勋,Ilan Chet,等.干旱和Na Cl胁迫下哈茨木霉的响应[J].安徽农业科学,2013(30):11997-12000.
[21]崔西苓,李世贵,杨佳,等.耐盐碱抗烟草黑胫病木霉菌株的筛选与鉴定[J].中国农业科技导报,2014,16(3):81-89.
[22]Zhang Y,Tang H R,Luo Y.Variation in antioxidant enzyme activities of two strawberry cultivars with short-term low temperature stress[J].2014,4(4):458-462.
[23]Loh K P,Qi J,Tan B K,et al.Leonurine protects middle cerebral artery occluded rats through antioxidant effect and regulation of mitochondrial function[J].Stroke,2010,41(11):2661.
[24]Tijerino A,Cardoza R E,Moraga J,et al.Overexpression of the trichodiene synthase gene tri5,increases trichodermin production and antimicrobial activity in Trichoderma brevicompactum[J].Fungal Genetics and Biology Fg&B,2011,48(3):285.
[25]Druzhinina I S,Seidl-Seiboth V,Herrera-Estrella A,et al.Trichoderma:the genomics of opportunistic success.Nat Rev Microbiol 9:749-759[J].Nature Reviews Microbiology,2011,9(10):749-759.
[26]王丽荣,蒋细良,Estifanos,等.铜胁迫下的哈茨木霉Th-33转录组分析[J].中国生物防治学报,2017,33(1):103-113.
[27]唐俊,陆娟,许惠芬,等.康氏木霉HEX1蛋白的原核表达及纯化[J].西北农林科技大学学报(自然科学版),2014,42(10):147-151.
[28]宋瑛瑛,黄丽,唐明.不同碳源和氮源对4种深色有隔内生真菌生长的影响[J].西北农林科技大学学报(自然科学版),2016,44(3):181-187.
[29]王美琴,陈俊美,李新凤.不同碳、氮源对番茄两种内生真菌菌丝生长的影响研究[J].山西农业科学,2008,36(11):75-77.
[2]AndréS,Monika S.Biology and biotechnology of Trichoderma[J].Applied Microbiology and Biotechnology,2010,87(3):787-799.
[3]Harman G E.Overview of mechanisms and uses of Trichoderma spp.[J].Phytopathology,2006,96(2):190-194.
[4]陈勇,朱廷恒,汪琨,等.提高木霉逆境适应性与生物防治效果的基因工程研究进展[J].中国生物工程杂志,2012,32(6):120-124.
[5]Monte E.Understanding Trichoderma:between biotechnology and microbial ecology[J].International Microbiology the Official Journal of the Spanish Society for Microbiology,2001,4(1):1.
[6]Benítez T,Rincón A M,Limón M C,et al.Biocontrol mechanism of Trichoderma strains[J].International Microbiology,2005,7(4):249-260.
[7]高永东,王兵,刘力行,等.木霉菌及其REMI突变株与油菜联合吸附镉污染土壤研究[J].现代农业科技,2008(20):161-162.
[8]马文亭,滕应,凌婉婷,等.里氏木霉FS10-C对伴矿景天吸取修复镉污染土壤的强化作用[J].土壤,2012,44(6):991-995.
[9]田晔,滕应,赵静,等.一株抗铜木霉对含铜废水的修复潜力研究[J].中国矿业大学学报,2012,41(4):657-662.
[10]陈捷.木霉菌诱导植物抗病性研究新进展[J].中国生物防治学报,2015,31(5):733-741.
[11]Vinalea F,Sivasithamparam K,Ghisalberti E L.Trichoderma plant pathogen interactions[J].Soil Biology and Biochemistry,2008,40(1):1-10.
[12]Kredics L,Antal Z,Manczinger L,et al.Influence of environmental parameters on Trichoderma strains with biocontrol potential[J].Food Technology and Biotechnology,2003,41(7/8):37-42.
[13]张晶晶,黄亚丽,马宏,等.木霉厚垣孢子可湿性粉剂的研制[J].植物保护,2016,42(5):103-109.
[14]杨力明,杨谦,李森,等.哈茨木霉Cu-ZnSOD的克隆表达及活性鉴定[J].哈尔滨工业大学学报,2009(3):102-107..
[15]Zhang S,Gan Y,Xu B.Application of plant growth promoting fungi Trichoderma longibrachiatum T6 enhances tolerance of wheat to salt stress through improvement of antioxidative defense system and gene expression:[J].Frontiers in Plant Science,2016,7(868):1405.
[16]Parvaiz A,Abeer H,Elsayed Fathi A A,et al.Role of Trichoderma harzianum in mitigating NaCl stress in Indian mustard(Brassica juncea L.)through antioxidative defense system[J].Frontiers in Plant Science,2015,6:868.
[17]Bj?rkman T,Blanchard L M,Harman G E.Growth enhancement of shrunken-2(sh2)sweet corn by Trichoderma harzianum 1295-22:effect of environmental stress[J].Journal of the American Society for Horticultural Science American Society for Horticultural Science,1998,123(1):35-40.
[18]Li S Y,Li F L,Wang Y J,et al.Effect of Trichoderma harzianum on cucumber seed germination with NaCl stress[J].Science Technology&Engineering,2016,16(9):164-166.
[19]曲乐,郭凯,李纪顺,等.外源一氧化氮供体硝普钠对盐胁迫下椒样薄荷不定根和根毛发育的影响[J].山东科学,2013,26(5):33-38.
[20]尹大川,邓勋,Ilan Chet,等.干旱和Na Cl胁迫下哈茨木霉的响应[J].安徽农业科学,2013(30):11997-12000.
[21]崔西苓,李世贵,杨佳,等.耐盐碱抗烟草黑胫病木霉菌株的筛选与鉴定[J].中国农业科技导报,2014,16(3):81-89.
[22]Zhang Y,Tang H R,Luo Y.Variation in antioxidant enzyme activities of two strawberry cultivars with short-term low temperature stress[J].2014,4(4):458-462.
[23]Loh K P,Qi J,Tan B K,et al.Leonurine protects middle cerebral artery occluded rats through antioxidant effect and regulation of mitochondrial function[J].Stroke,2010,41(11):2661.
[24]Tijerino A,Cardoza R E,Moraga J,et al.Overexpression of the trichodiene synthase gene tri5,increases trichodermin production and antimicrobial activity in Trichoderma brevicompactum[J].Fungal Genetics and Biology Fg&B,2011,48(3):285.
[25]Druzhinina I S,Seidl-Seiboth V,Herrera-Estrella A,et al.Trichoderma:the genomics of opportunistic success.Nat Rev Microbiol 9:749-759[J].Nature Reviews Microbiology,2011,9(10):749-759.
[26]王丽荣,蒋细良,Estifanos,等.铜胁迫下的哈茨木霉Th-33转录组分析[J].中国生物防治学报,2017,33(1):103-113.
[27]唐俊,陆娟,许惠芬,等.康氏木霉HEX1蛋白的原核表达及纯化[J].西北农林科技大学学报(自然科学版),2014,42(10):147-151.
[28]宋瑛瑛,黄丽,唐明.不同碳源和氮源对4种深色有隔内生真菌生长的影响[J].西北农林科技大学学报(自然科学版),2016,44(3):181-187.
[29]王美琴,陈俊美,李新凤.不同碳、氮源对番茄两种内生真菌菌丝生长的影响研究[J].山西农业科学,2008,36(11):75-77.