喜树内生真菌的分离及其对植物病原菌的抑菌活性研究
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摘要
植物内生真菌能够产生结构新颖、功能特殊的次生代谢产物,是新化合物、新药物的潜在资源,在农业和医药行业中具有重要的应用前景。本文从喜树内生真菌中筛选得到一株对植物病原菌具有抑菌活性的菌株,编号G-4,并对其进行了生物学特性测定及发酵液抑菌活性研究。主要结果如下:
1.采用组织分离法,以PDA为分离培养基,从喜树的枝条、叶片、果实和根部分离得到28株内生真菌。通过对峙试验,测定了28株内生真菌对茄褐纹病菌(Phomopsis vexans(Sacc.et Syd)Harter )、番茄枯萎病菌( Fusarium oxysporum f. sp. lycopersici)、苹果腐烂病菌(Valsa mali miyabe et Yamada)、枣炭疽病菌(Gloelsporium fructigenum Blerk)、梨黑星病菌(Venturia pirina)、葡萄灰霉病菌(Botrytis cinerea)6种植物病原菌的拮抗作用,结果表明28株内生真菌对6种供试病原菌均有不同程度的抑制作用,其中G-4菌株作用最明显,对6种病原菌抑菌率均达到60%以上,对苹果腐烂病菌(Valsa mali miyabe et )高达89.68%。
2.对G-4菌株生物学特性测定结果表明:在麦芽侵粉培养基上G-4菌丝生长迅速;在28℃条件下G-4菌株生长最佳;最适pH范围为6.4-8,pH为2.4时菌丝不长;果糖作为碳源时,菌丝生长速度最快,在无糖条件菌落亦可生长。
3.采用菌丝生长速率法测定了G-4菌株发酵液对6种供试植物病原菌抑菌活性,结果显示G-4菌株发酵液经过滤处理和高温处理对6种供试病原菌均有一定的抑制作用。其中过滤处理对苹果腐烂病菌(Valsa mali miyabe et Yamada)的抑制率最高,72h抑菌率为95.04%,高温处理对苹果腐烂病菌(Valsa mali miyabe et Yamada)的抑制率达94.44%。
4.通过发酵3d、4d、5d、6d、7d的G-4菌株发酵液对苹果腐烂病原菌和茄褐纹病原菌的抑菌率的测定,结果表明G-4菌株发酵液对苹果腐烂和茄褐纹病菌的抑菌率随着发酵天数的增加而增大。发酵液对苹果腐烂和茄褐纹病菌菌丝毒力测定结果表明,培养48h二者的EC50为236.43mg/L和169.06mg/L。另外,4℃条件下保存G-4菌株发酵液不同保存时间不会影响其抑菌活性。
5.发酵液抑制孢子萌发试验,结果显示4h、8h、12h、24h的抑制率分别为38.00%、43.97%、56.06%、46.93%,其中12h抑制率最高。此外,在发酵液对苹果腐烂病的保护及治疗作用的活体试验中,可以看到各浓度下的G-4菌株发酵液对苹果腐烂病均有一定效果,480mg/L浓度下对苹果腐烂病的治疗效果为69.5%,保护效果为87.4%。
The plant endophyte can metabolize many interesting secondary metabolites with novel structure and distinctive activities,which is an important source of new molecular skeleton and drugs,and has good prospects in the agricultural and medical application. The fermentation product of the strain G-4 isolated from the root of Camptotheca Acuminata Decne has strong antifungal activity on various plant pathogens.Biological characteristics and fermentation liquid of strain G-4 was studied.The main results are as follows:
1.Used tissue isolation method on potato dextrose agar,28 strains of endophytic fungi were isolated from the stems, leaves, fruits and roots of Camptotheca acuminata Decne. The results of plat-confront indicated that 28 strains of endophytic fungi had some inhibition on the six plant pathogens: Phomopsis vexans(Sacc.et Syd)Harter、Fusarium oxysporum f. sp. lycopersici、Valsa mali miyabe et Yamada、Gloelsporium fructigenum Blerk、Venturia pirina、Botrytis cinerea.the strain G-4 has a significant antifungal activity on pathogens, the inhibition rates were all more than 60% to the six plant pathogens,and the most rate was 89.68% to the Valsa mali miyabe et.
2.Biological characteristics of strain G-4 was studied, the result showed that malt powder was the best medium for its growth; the strain G-4 grew well when the temperature was 28℃and the pH was the range 6.4 from 8; The major carbon sources was fructose,the stain G-4 can grow with no carbon.
3.Used hypha growth rate method,the antifungal activity of fermentation liquid by high temperature and filtration treatment showed that the effect was remarkable to the six plant pathogens. The inhibition rate of high temperature treatment was the most 95.04% to Valsa mali at 72h,and the filtration treatment was 94.44%.
4.The antifungal activity of the fermentation liquid on the third、fourth、fifth、sixth and seventh day to Phomopsis vexans and Valsa mali was sdudied.The result indicate that the inhibition rate increased with the time of ferment. The EC50 of the strain G-4 fermentation liquid against the mycelia growth of Valsa mali and Phomopsis vexans was 236.43mg/L and 169.06mg/L when the mycelia growed 48h.Otherwise,the different storage time can not influence the antifungal activity of fermentation liquid in the condition of 4℃.
5.The inhibition of the strain G-4 fermentation filtrate against spore germination of Valsa mali was investigated.The results indicated that at 4h、8h、12h and 24h after treatiing,the inhibition rate of the spore germination was 38.00%、43.97%、56.06% and 46.93%,respectively,with the inhibition rate at 12h after treating was the highest.In addition,by the in vivo experiment of protectiveaction and therapeutic action against canker of apple fruit.we can say that the effect was obviously with the different concentration of the fermentation filtrate.The protective efficacy was 61.9% and the therapeutic efficacy was 81.6% against canker of apple fruit at the concentration of 480mg/L.
1.采用组织分离法,以PDA为分离培养基,从喜树的枝条、叶片、果实和根部分离得到28株内生真菌。通过对峙试验,测定了28株内生真菌对茄褐纹病菌(Phomopsis vexans(Sacc.et Syd)Harter )、番茄枯萎病菌( Fusarium oxysporum f. sp. lycopersici)、苹果腐烂病菌(Valsa mali miyabe et Yamada)、枣炭疽病菌(Gloelsporium fructigenum Blerk)、梨黑星病菌(Venturia pirina)、葡萄灰霉病菌(Botrytis cinerea)6种植物病原菌的拮抗作用,结果表明28株内生真菌对6种供试病原菌均有不同程度的抑制作用,其中G-4菌株作用最明显,对6种病原菌抑菌率均达到60%以上,对苹果腐烂病菌(Valsa mali miyabe et )高达89.68%。
2.对G-4菌株生物学特性测定结果表明:在麦芽侵粉培养基上G-4菌丝生长迅速;在28℃条件下G-4菌株生长最佳;最适pH范围为6.4-8,pH为2.4时菌丝不长;果糖作为碳源时,菌丝生长速度最快,在无糖条件菌落亦可生长。
3.采用菌丝生长速率法测定了G-4菌株发酵液对6种供试植物病原菌抑菌活性,结果显示G-4菌株发酵液经过滤处理和高温处理对6种供试病原菌均有一定的抑制作用。其中过滤处理对苹果腐烂病菌(Valsa mali miyabe et Yamada)的抑制率最高,72h抑菌率为95.04%,高温处理对苹果腐烂病菌(Valsa mali miyabe et Yamada)的抑制率达94.44%。
4.通过发酵3d、4d、5d、6d、7d的G-4菌株发酵液对苹果腐烂病原菌和茄褐纹病原菌的抑菌率的测定,结果表明G-4菌株发酵液对苹果腐烂和茄褐纹病菌的抑菌率随着发酵天数的增加而增大。发酵液对苹果腐烂和茄褐纹病菌菌丝毒力测定结果表明,培养48h二者的EC50为236.43mg/L和169.06mg/L。另外,4℃条件下保存G-4菌株发酵液不同保存时间不会影响其抑菌活性。
5.发酵液抑制孢子萌发试验,结果显示4h、8h、12h、24h的抑制率分别为38.00%、43.97%、56.06%、46.93%,其中12h抑制率最高。此外,在发酵液对苹果腐烂病的保护及治疗作用的活体试验中,可以看到各浓度下的G-4菌株发酵液对苹果腐烂病均有一定效果,480mg/L浓度下对苹果腐烂病的治疗效果为69.5%,保护效果为87.4%。
The plant endophyte can metabolize many interesting secondary metabolites with novel structure and distinctive activities,which is an important source of new molecular skeleton and drugs,and has good prospects in the agricultural and medical application. The fermentation product of the strain G-4 isolated from the root of Camptotheca Acuminata Decne has strong antifungal activity on various plant pathogens.Biological characteristics and fermentation liquid of strain G-4 was studied.The main results are as follows:
1.Used tissue isolation method on potato dextrose agar,28 strains of endophytic fungi were isolated from the stems, leaves, fruits and roots of Camptotheca acuminata Decne. The results of plat-confront indicated that 28 strains of endophytic fungi had some inhibition on the six plant pathogens: Phomopsis vexans(Sacc.et Syd)Harter、Fusarium oxysporum f. sp. lycopersici、Valsa mali miyabe et Yamada、Gloelsporium fructigenum Blerk、Venturia pirina、Botrytis cinerea.the strain G-4 has a significant antifungal activity on pathogens, the inhibition rates were all more than 60% to the six plant pathogens,and the most rate was 89.68% to the Valsa mali miyabe et.
2.Biological characteristics of strain G-4 was studied, the result showed that malt powder was the best medium for its growth; the strain G-4 grew well when the temperature was 28℃and the pH was the range 6.4 from 8; The major carbon sources was fructose,the stain G-4 can grow with no carbon.
3.Used hypha growth rate method,the antifungal activity of fermentation liquid by high temperature and filtration treatment showed that the effect was remarkable to the six plant pathogens. The inhibition rate of high temperature treatment was the most 95.04% to Valsa mali at 72h,and the filtration treatment was 94.44%.
4.The antifungal activity of the fermentation liquid on the third、fourth、fifth、sixth and seventh day to Phomopsis vexans and Valsa mali was sdudied.The result indicate that the inhibition rate increased with the time of ferment. The EC50 of the strain G-4 fermentation liquid against the mycelia growth of Valsa mali and Phomopsis vexans was 236.43mg/L and 169.06mg/L when the mycelia growed 48h.Otherwise,the different storage time can not influence the antifungal activity of fermentation liquid in the condition of 4℃.
5.The inhibition of the strain G-4 fermentation filtrate against spore germination of Valsa mali was investigated.The results indicated that at 4h、8h、12h and 24h after treatiing,the inhibition rate of the spore germination was 38.00%、43.97%、56.06% and 46.93%,respectively,with the inhibition rate at 12h after treating was the highest.In addition,by the in vivo experiment of protectiveaction and therapeutic action against canker of apple fruit.we can say that the effect was obviously with the different concentration of the fermentation filtrate.The protective efficacy was 61.9% and the therapeutic efficacy was 81.6% against canker of apple fruit at the concentration of 480mg/L.
引文
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