深绿木霉T2发酵液蛋白分离物诱导抗病作用研究
|
摘要
采用生长速率法和产孢量测定法,研究了不同培养基和温度、pH、接菌量、培养基量对深绿木霉T2生长和产孢量的影响。结果表明,PDA培养基有利于深绿木霉T2的生长;深绿木霉T2发酵产孢最优条件为温度25℃、pH 7、接菌量为0.05mL/L、培养基量为100mL/250mL,此条件下发酵72h时产孢量最大,为1.5×109个/mL;76h时产孢量下降;发酵液通过盐析及凝胶层析获得具有3个洗脱峰的蛋白质液,分别为Ⅰ,Ⅱ和Ⅲ;采用生长速率法和诱导活体接种法分别测定不同浓度的3个洗脱液对百合灰霉菌的抑菌率及其诱导抗病效果,结果表明洗脱峰Ⅰ蛋白质的100倍液对百合灰霉菌的抑菌率最小,仅为1.16%,而其诱导抗病效果最好,为84.77%。
The effects of medium,temperature,pH value,inoculation amount on the growth and sporulation of Trichoderma atroviride T2 were studied and the result indicated that PDA medium performed better and its sporulation optimum fermentation conditions were 25℃,pH 7,inoculation amount of 0.5 mL/L,medium content of 100mL/250 mL,sporulation reached the maximum at 72 hunder these conditions,it was 1.5×109/mL sporulation began to decline at 76 hby growth rate method and spore production measurement method.Three proteins eluted peaks from fermentation broth Trichoderma atroviride T2 were obtained by(NH4)2SO4precipitation and gel filtration chromatography,they wereⅠ,Ⅱ and Ⅲ,respectively and their inhibition rates and induced resistance effects on their different concentrations to lily Botrytis cinerea were measured by growth rate method and the induced in vivo inoculation method.The inhibition rate of protein elution peakⅠ100times was the minimum(1.16%),while its induced resistance was the best(84.77%).
The effects of medium,temperature,pH value,inoculation amount on the growth and sporulation of Trichoderma atroviride T2 were studied and the result indicated that PDA medium performed better and its sporulation optimum fermentation conditions were 25℃,pH 7,inoculation amount of 0.5 mL/L,medium content of 100mL/250 mL,sporulation reached the maximum at 72 hunder these conditions,it was 1.5×109/mL sporulation began to decline at 76 hby growth rate method and spore production measurement method.Three proteins eluted peaks from fermentation broth Trichoderma atroviride T2 were obtained by(NH4)2SO4precipitation and gel filtration chromatography,they wereⅠ,Ⅱ and Ⅲ,respectively and their inhibition rates and induced resistance effects on their different concentrations to lily Botrytis cinerea were measured by growth rate method and the induced in vivo inoculation method.The inhibition rate of protein elution peakⅠ100times was the minimum(1.16%),while its induced resistance was the best(84.77%).
引文
[1]杨合同.木霉分类与鉴定[M].北京:中国大地出版社,2009.
[2]Yohei Yoshioka,Haruki Ichikawa,Hushna Ara Naznin,et al.Systemic resistance induced in Arabidopsis thaliana by Trichoderma asperellum SKT-1,amicrobial pesticide of seedborne diseases of rice[J].Pest Manag Sci,2012,68:60-66.
[3]胡琼.木霉对植物促生作用的研究进展[J].北方园艺,2010(7):197-200
[4]赵蕾,滕安娜.木霉对植物的促生及诱导抗性研究进展[J].植物保护,2010,36(3):43-46
[5]Harman G E,Petzoldt R,Comis A,et al.Interactions between Trichoderma harzianum strain T22and maize inbred line Mo17and effects of this interaction on diseases caused by Pythium ultimum and Colletotrichum graminicola[J].Phytopathology,2004,94:147-153.
[6]Harman G,Howell C R,Viterbo A,et al.Trichoderma species opportunistic,avirulent plant symbionts[J].Nature,2004(2):3-56.
[7]杨合同,唐文华.木霉菌与植物病害的生物防治[J].山东科学,1999,12(4):7-14.
[8]方中达.植病研究方法[M].北京:中国农业出版社,1998.
[9]庄敬华,高增贵,刘限,等.不同发酵条件对木霉产孢类型的影响[J].中国生物防治,2005,21(1):37-40.
[10]郝林华,牛德庆,陈靠山,等.拟康氏木霉液态发酵条件的研究[J].菌物学报,2005,24(2):235-244.
[11]武永红,黄敏,曹励民,等.香菇C91-3菌丝发酵液中蛋白的分离纯化及其抗肿瘤机理研究[J].时珍国医国药,2012(3):538-542.
[12]梁巧兰,魏列新,徐秉良,等.三种化学物质诱导观赏百合对黑斑病抗性的研究[J].植物保护,2011,37(2):36-40.
[13]朱丽梅,侯建文,罗凤霞,等.百合灰霉病的诊断及其病原物的分离纯化[J].金陵科技学院学报,2009(3):59-63.
[14]刘梅,徐同.木霉的营养生长及发酵条件[J].云南农业大学学报,2000,15(3):263-278.
[15]Papavizas G C,Dunn M T,Lewis J A.Liquid fermentation technology for experimental production of biocontrol fungi[J].Phytopathology,1984,74:1171-1175.
[16]Jackson A M,Whipps J M,Lynch J M.Effects of temperature,pH and water potential on grow th of four fungi with disease biocontrol potential[J].World J.Microbiol.Bio technol,1991,7:494-501.
[17]Lewis J A,Papavizas G C.Production of chlamydospores and co nidia by Trichoderma spp.in liquid and solid growth media[J].Soil Biol Biochem,1983,15:351-357.
[18]王慧中,赵培洁,陈卫辉.哈茨木霉浅层液体培养适宜条件的研究[J].浙江农业学报,1995,7(1):61-62.
[19]王慧中,赵培洁,陈卫辉.哈茨木霉浅层液体培养最适pH的研究[J].江西农业大学学报,1995,17(1):77-79.
[20]陈卫辉,赵培洁,王慧中,等.哈茨木霉液体培养技术研究[J].江西农业大学学报,1998,20(2):170-174.
[21]赵培洁,王慧中,陈卫辉.利用虫草头孢菌发酵废液培养哈茨木霉[J].中国生物防治,1995,11(1):48.
[22]赵培洁,王慧中,陈卫辉.哈茨木霉浅层液体培养最适采收时间的研究[J].植物病理学报,1995,25(3):220.
[23]魏林,梁志怀,罗赫荣,等.木霉代谢产物对豇豆种子活力及幼苗生理特性的影响[J].湖南农业学,2004(6):14-18.
[24]张旭东,刘云龙.木霉生防菌对植物生长的影响[J].云南农业大学学报,2001,16(4):299-303.
[25]程玲娟,徐秉良,梁巧兰.深绿木霉对几种牧草草种发芽率的影响[J].草原与草坪,2008,6:28-31.
[26]Hanson L E,Howell C R.Elicitors of plant defense reponse from biocontrol strains of Trichodermaviren[J].Phytopathology,2004,94(8):171-176.
[27]Sriram S,Manasa S B,Savitha M J.Potential use of elicitors from Trichoderma in induced systemic resistance for the management of Phytophthora capsici in red pepper[J].Journal Biology Control,2009,23(4):449-456.
[28]Woo S L,Scala F,Ruocco M,et al.The molecular biology of the interactions between Trichoderma spp,phytopathogenic fungi,and plants[J].Phytopathology,2006,96:181-185.
[2]Yohei Yoshioka,Haruki Ichikawa,Hushna Ara Naznin,et al.Systemic resistance induced in Arabidopsis thaliana by Trichoderma asperellum SKT-1,amicrobial pesticide of seedborne diseases of rice[J].Pest Manag Sci,2012,68:60-66.
[3]胡琼.木霉对植物促生作用的研究进展[J].北方园艺,2010(7):197-200
[4]赵蕾,滕安娜.木霉对植物的促生及诱导抗性研究进展[J].植物保护,2010,36(3):43-46
[5]Harman G E,Petzoldt R,Comis A,et al.Interactions between Trichoderma harzianum strain T22and maize inbred line Mo17and effects of this interaction on diseases caused by Pythium ultimum and Colletotrichum graminicola[J].Phytopathology,2004,94:147-153.
[6]Harman G,Howell C R,Viterbo A,et al.Trichoderma species opportunistic,avirulent plant symbionts[J].Nature,2004(2):3-56.
[7]杨合同,唐文华.木霉菌与植物病害的生物防治[J].山东科学,1999,12(4):7-14.
[8]方中达.植病研究方法[M].北京:中国农业出版社,1998.
[9]庄敬华,高增贵,刘限,等.不同发酵条件对木霉产孢类型的影响[J].中国生物防治,2005,21(1):37-40.
[10]郝林华,牛德庆,陈靠山,等.拟康氏木霉液态发酵条件的研究[J].菌物学报,2005,24(2):235-244.
[11]武永红,黄敏,曹励民,等.香菇C91-3菌丝发酵液中蛋白的分离纯化及其抗肿瘤机理研究[J].时珍国医国药,2012(3):538-542.
[12]梁巧兰,魏列新,徐秉良,等.三种化学物质诱导观赏百合对黑斑病抗性的研究[J].植物保护,2011,37(2):36-40.
[13]朱丽梅,侯建文,罗凤霞,等.百合灰霉病的诊断及其病原物的分离纯化[J].金陵科技学院学报,2009(3):59-63.
[14]刘梅,徐同.木霉的营养生长及发酵条件[J].云南农业大学学报,2000,15(3):263-278.
[15]Papavizas G C,Dunn M T,Lewis J A.Liquid fermentation technology for experimental production of biocontrol fungi[J].Phytopathology,1984,74:1171-1175.
[16]Jackson A M,Whipps J M,Lynch J M.Effects of temperature,pH and water potential on grow th of four fungi with disease biocontrol potential[J].World J.Microbiol.Bio technol,1991,7:494-501.
[17]Lewis J A,Papavizas G C.Production of chlamydospores and co nidia by Trichoderma spp.in liquid and solid growth media[J].Soil Biol Biochem,1983,15:351-357.
[18]王慧中,赵培洁,陈卫辉.哈茨木霉浅层液体培养适宜条件的研究[J].浙江农业学报,1995,7(1):61-62.
[19]王慧中,赵培洁,陈卫辉.哈茨木霉浅层液体培养最适pH的研究[J].江西农业大学学报,1995,17(1):77-79.
[20]陈卫辉,赵培洁,王慧中,等.哈茨木霉液体培养技术研究[J].江西农业大学学报,1998,20(2):170-174.
[21]赵培洁,王慧中,陈卫辉.利用虫草头孢菌发酵废液培养哈茨木霉[J].中国生物防治,1995,11(1):48.
[22]赵培洁,王慧中,陈卫辉.哈茨木霉浅层液体培养最适采收时间的研究[J].植物病理学报,1995,25(3):220.
[23]魏林,梁志怀,罗赫荣,等.木霉代谢产物对豇豆种子活力及幼苗生理特性的影响[J].湖南农业学,2004(6):14-18.
[24]张旭东,刘云龙.木霉生防菌对植物生长的影响[J].云南农业大学学报,2001,16(4):299-303.
[25]程玲娟,徐秉良,梁巧兰.深绿木霉对几种牧草草种发芽率的影响[J].草原与草坪,2008,6:28-31.
[26]Hanson L E,Howell C R.Elicitors of plant defense reponse from biocontrol strains of Trichodermaviren[J].Phytopathology,2004,94(8):171-176.
[27]Sriram S,Manasa S B,Savitha M J.Potential use of elicitors from Trichoderma in induced systemic resistance for the management of Phytophthora capsici in red pepper[J].Journal Biology Control,2009,23(4):449-456.
[28]Woo S L,Scala F,Ruocco M,et al.The molecular biology of the interactions between Trichoderma spp,phytopathogenic fungi,and plants[J].Phytopathology,2006,96:181-185.