菌株T-33抑菌活性物质及其对杨树烂皮病菌的抑制机理
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摘要
从国内外收集的29个木霉菌株中,筛选出对杨树烂皮病菌具有高效抑制作用的菌株T-33,研究了T-33抑菌活性物质的最佳提取工艺、稳定性、室内防治及生态安全性,研究了抑菌活性物质对杨树烂皮病菌的抑制机理并对T-33抑菌活性成分进行了分离纯化和结构鉴定,同时对木霉菌株T-33分类地位进行了分子生物学验证及生理学特性研究。取得以下成果：
(1)通过拮抗实验和抑菌实验从29个木霉菌株中,筛选出对杨树烂皮病菌具有显著抑制效果的菌株T-09、T-14和T-33。对3个菌株的18个不同提取物进行抑菌检测,获得高效提取物——木霉T-33的正丁醇提取物。该提取物对杨树烂皮病菌的生长抑制率为92%,孢子萌发抑制率可达100%。
(2)对木霉菌株T-33的分类地位进行了分子生物学验证,结果表明,木霉菌株T-33为绿色木霉(Trichoderma viride), rDNA ITS序列长度为583bP, GenBank登录号为JF823649。生理学特性研究结果表明：菌株T-33生长最适碳源为葡萄糖(用量为2%),最适氮源为黄豆粉(用量0.01%),最适初始pH值为6,最适培养温度25℃。室内木霉孢子萌发受季节的影响较大,其产孢规律与自然规律一致。
(3)强酸和强碱影响提取物的抑菌活性,高浓度的氧化剂和低浓度的还原剂对提取物的抑菌活性影响较大,高温(超过80℃)高压降低提取物的抑菌活性,Fe3+、Cu2+Al3+与Zn2+4种离子对提取物的抑菌活性影响较明显,Na+、K+、Mg2+与Ca2+四种离子对提取物抑菌活性的影响相对较小,紫外线对提取物的抑菌活性破坏较小。提取物的抑菌活性受贮存时间长短的影响,提取物适宜短期(7d)储存。
(4)研究了木霉菌T-33培养液提取物室内生物防治和生态安全性,结果表明,无论预防组还是治疗组,木霉菌T-33培养液提取物对杨树烂皮病原菌均表现出了显著的抑制效果。当天预防和治疗效果分别为90.82%和89.99%。提取物的LD50为3731.6mg/kg,95%可信限为2643.1-5268.6mg/kg,属于低毒。提取物在杨树烂皮病防治浓度范围内对生态环境安全,符合国家药物毒性的标准。
(5)通过木霉T-33培养液提取物对杨树烂皮病菌代谢途径中几种关键酶的影响研究,初步探讨了培养液提取物对杨树烂皮病的抑制机理。结果表明,木霉T-33培养液提取物可显著提高病原菌菌丝体的电导率和MDA含量,降低病原菌蛋白质含量,降低病原菌菌体的SDH、PK、HK、LDH、MDH与辅酶Ⅰ的活性,使Na+、K+-ATP酶、Mg2+-ATP和Ca2+-ATP酶的活性急剧下降,从而破坏糖、脂类与蛋白质相互间的代谢平衡。
(6)通过经典柱层析法、薄层层析法和半制备型高效液相色谱法对木霉菌株T-33抑菌活性物质进行了分离纯化,利用FT-IR红外光谱、紫外吸收光谱、LC-MS液质联机和核磁共振等检测手段对提取物中抑菌活性成分进行了定性分析和结构鉴定。结果表明抑菌活性成分为2、6-二叔丁基1、4苯醌,属于一种具有抗菌、消炎作用的植物代谢物。
Strain T-33was obtained as the high inhibiting strain on Cytospora chrysosperma, which is the pathogen of poplar canker, from29Thichoderma strains. The optimal extracting process, stability, control effects indoor and ecological safety of antifungal substances of strain T-33was studied. We also researched the inhibiting mechanism of antifungal substances on Cytospora chrysosperma. The structure of the antifungal substances was obtained by means of isolating, purifing and identification. The taxonomic position of strain T-33was verified by methods of molecular biology and physiological characteristics. The results are as follows.
(1) Three strains (T-09, T-14and T-33) were obtained as the high inhibiting strains on Cytospora chrysosperma by antagonistic and antifungal tests. The n-butanol extract from strain T-33has the most inhibiting effect on Cytospora chrysosperma among18extracts which extracted from three strains (T-09, T-14and T-33) by different organic solvents. The growth-inhibiting rate of n-butanol extract from strain T-33on Cytospora chrysosperma is92%, and the sprouting-inhibiting rate is100%.
(2) The taxonomic position of strain T-33was verified by methods of molecular biology and physiological characteristics. The results show that strain T-33is Trichoderma viride that the length of rDNA ITS sequences is583bp and Genbank accession number is JF823649. The results of physiological characteristics show that the optimal nutrition for strain T-33growth is: glucose (2%), soybean powder (0.01%), pH6. The optimal culture temperature is25℃Spores sprouting indoor were affected by seasons change, and the sporulation regularity indoor was as same as that in nature.
(3) Strong acid and strong alkali can significantly affect the antifungal activity of extract. High concentrations of oxidants and low concentrations of reducing agents can reduce the antifungal activity of extract obviously. And high temperature (more than80℃) and high pressure are the same. The antifungal activity of extract was more significantly impacted by Fe3+、Cu2+、Al3+and Zn2+and less by Na+、K+、Mg2+and Ca2+. UV had less disruptive to the extract. The antifungal activity of extract was reducing with the storage time, and the optimal storage time is in7days.
(4) The results of control indoor and ecological safety show that the n-butanol extract from strain T-33has significant inhibiting effect on poplar canker whether the prevention or treatment. The effects of prevention and treatment same day are90.82%and89.99%respectively. LD50of the extract is3731.6mg/kg,95%confidence limit is2643.1-5268.6mg/kg. So the extract has low toxicity. The concentration of extract using on biocontrol poplar canker is safety to ecological environment and comply with the standards of the national drug toxicity.
(5) The inhibiting mechanism of the extract on Cytospora chrysosperma was studied throught the affection of extract on several key enzymes in Cytospora chrysosperma metabolic pathways. The results show that the extract can significantly increase pathogen mycelium conductivity and MDA content; reduce pathogen protein content and enzyme activities (SDH, PK, HK, LDH, MDH and coenzyme I), also obviously decrease the activities of Na+, K+-ATP enzyme, Mg2+-ATP and Ca2+-ATP enzyme. Thus the metabolic balances among sugar, lipids and proteins are destroyed.
(6) The inhibiting substance is2,5-Cyclohexadiene-1,4-dione,2,6-bis(1,1-dimethylethyl) which is a kind of plant metabolites with antifungal and anti-inflammatory effects.
(1)通过拮抗实验和抑菌实验从29个木霉菌株中,筛选出对杨树烂皮病菌具有显著抑制效果的菌株T-09、T-14和T-33。对3个菌株的18个不同提取物进行抑菌检测,获得高效提取物——木霉T-33的正丁醇提取物。该提取物对杨树烂皮病菌的生长抑制率为92%,孢子萌发抑制率可达100%。
(2)对木霉菌株T-33的分类地位进行了分子生物学验证,结果表明,木霉菌株T-33为绿色木霉(Trichoderma viride), rDNA ITS序列长度为583bP, GenBank登录号为JF823649。生理学特性研究结果表明：菌株T-33生长最适碳源为葡萄糖(用量为2%),最适氮源为黄豆粉(用量0.01%),最适初始pH值为6,最适培养温度25℃。室内木霉孢子萌发受季节的影响较大,其产孢规律与自然规律一致。
(3)强酸和强碱影响提取物的抑菌活性,高浓度的氧化剂和低浓度的还原剂对提取物的抑菌活性影响较大,高温(超过80℃)高压降低提取物的抑菌活性,Fe3+、Cu2+Al3+与Zn2+4种离子对提取物的抑菌活性影响较明显,Na+、K+、Mg2+与Ca2+四种离子对提取物抑菌活性的影响相对较小,紫外线对提取物的抑菌活性破坏较小。提取物的抑菌活性受贮存时间长短的影响,提取物适宜短期(7d)储存。
(4)研究了木霉菌T-33培养液提取物室内生物防治和生态安全性,结果表明,无论预防组还是治疗组,木霉菌T-33培养液提取物对杨树烂皮病原菌均表现出了显著的抑制效果。当天预防和治疗效果分别为90.82%和89.99%。提取物的LD50为3731.6mg/kg,95%可信限为2643.1-5268.6mg/kg,属于低毒。提取物在杨树烂皮病防治浓度范围内对生态环境安全,符合国家药物毒性的标准。
(5)通过木霉T-33培养液提取物对杨树烂皮病菌代谢途径中几种关键酶的影响研究,初步探讨了培养液提取物对杨树烂皮病的抑制机理。结果表明,木霉T-33培养液提取物可显著提高病原菌菌丝体的电导率和MDA含量,降低病原菌蛋白质含量,降低病原菌菌体的SDH、PK、HK、LDH、MDH与辅酶Ⅰ的活性,使Na+、K+-ATP酶、Mg2+-ATP和Ca2+-ATP酶的活性急剧下降,从而破坏糖、脂类与蛋白质相互间的代谢平衡。
(6)通过经典柱层析法、薄层层析法和半制备型高效液相色谱法对木霉菌株T-33抑菌活性物质进行了分离纯化,利用FT-IR红外光谱、紫外吸收光谱、LC-MS液质联机和核磁共振等检测手段对提取物中抑菌活性成分进行了定性分析和结构鉴定。结果表明抑菌活性成分为2、6-二叔丁基1、4苯醌,属于一种具有抗菌、消炎作用的植物代谢物。
Strain T-33was obtained as the high inhibiting strain on Cytospora chrysosperma, which is the pathogen of poplar canker, from29Thichoderma strains. The optimal extracting process, stability, control effects indoor and ecological safety of antifungal substances of strain T-33was studied. We also researched the inhibiting mechanism of antifungal substances on Cytospora chrysosperma. The structure of the antifungal substances was obtained by means of isolating, purifing and identification. The taxonomic position of strain T-33was verified by methods of molecular biology and physiological characteristics. The results are as follows.
(1) Three strains (T-09, T-14and T-33) were obtained as the high inhibiting strains on Cytospora chrysosperma by antagonistic and antifungal tests. The n-butanol extract from strain T-33has the most inhibiting effect on Cytospora chrysosperma among18extracts which extracted from three strains (T-09, T-14and T-33) by different organic solvents. The growth-inhibiting rate of n-butanol extract from strain T-33on Cytospora chrysosperma is92%, and the sprouting-inhibiting rate is100%.
(2) The taxonomic position of strain T-33was verified by methods of molecular biology and physiological characteristics. The results show that strain T-33is Trichoderma viride that the length of rDNA ITS sequences is583bp and Genbank accession number is JF823649. The results of physiological characteristics show that the optimal nutrition for strain T-33growth is: glucose (2%), soybean powder (0.01%), pH6. The optimal culture temperature is25℃Spores sprouting indoor were affected by seasons change, and the sporulation regularity indoor was as same as that in nature.
(3) Strong acid and strong alkali can significantly affect the antifungal activity of extract. High concentrations of oxidants and low concentrations of reducing agents can reduce the antifungal activity of extract obviously. And high temperature (more than80℃) and high pressure are the same. The antifungal activity of extract was more significantly impacted by Fe3+、Cu2+、Al3+and Zn2+and less by Na+、K+、Mg2+and Ca2+. UV had less disruptive to the extract. The antifungal activity of extract was reducing with the storage time, and the optimal storage time is in7days.
(4) The results of control indoor and ecological safety show that the n-butanol extract from strain T-33has significant inhibiting effect on poplar canker whether the prevention or treatment. The effects of prevention and treatment same day are90.82%and89.99%respectively. LD50of the extract is3731.6mg/kg,95%confidence limit is2643.1-5268.6mg/kg. So the extract has low toxicity. The concentration of extract using on biocontrol poplar canker is safety to ecological environment and comply with the standards of the national drug toxicity.
(5) The inhibiting mechanism of the extract on Cytospora chrysosperma was studied throught the affection of extract on several key enzymes in Cytospora chrysosperma metabolic pathways. The results show that the extract can significantly increase pathogen mycelium conductivity and MDA content; reduce pathogen protein content and enzyme activities (SDH, PK, HK, LDH, MDH and coenzyme I), also obviously decrease the activities of Na+, K+-ATP enzyme, Mg2+-ATP and Ca2+-ATP enzyme. Thus the metabolic balances among sugar, lipids and proteins are destroyed.
(6) The inhibiting substance is2,5-Cyclohexadiene-1,4-dione,2,6-bis(1,1-dimethylethyl) which is a kind of plant metabolites with antifungal and anti-inflammatory effects.
引文
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