红豆草黑腐病菌毒素的研究
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摘要
本试验旨在通过研究红豆草黑腐病病菌的生物学特性和病原菌产生毒素的条件及其毒素的组成成分,为以后利用红豆草黑腐病病原菌粗毒素,合成与该毒素相拮抗的化学物质,更有效地防治该病害。
试验从田间具有典型病斑的发病红豆草叶片上分离病菌并培养鉴定。通过对病原形态的鉴定和致病性测定结果表明,该病菌菌落等径生长,初无色,后渐变为青褐色至黑褐色,边缘整齐;菌丝细长,分枝,有隔膜,初为无色,渐变为浅褐色;分生孢子梗直或向一侧略弯曲,褐色,分生孢子常单生或短链生,倒棍棒状、长椭圆形,具横隔膜3～7个,纵隔膜0～3个,斜隔膜0～2个。喙及假喙柱状,淡褐色,分隔。与Alternaria属相关的文献资料对照,将其鉴定为Alternaria tenuis Nees.(细链格孢)。
生物学测定结果表明,供试的6种液体培养基:马铃薯葡萄糖琼脂培养液、马铃薯葡萄糖琼脂叶片汁液培养液、马铃薯蔗糖琼脂培养液、改良Fries培养液、查彼培养液及PSK培养液中,病菌在PSK培养液中生长速率最快;适宜菌丝生长的时间为11~13d,而菌丝干重在第13天达到最大值;菌丝生长的适宜温度范围为20～30℃,最适温度为25℃,低于15℃或者高于35℃均不适宜生长;菌丝在光暗交替并且振荡的条件下生长最快;在pH值3～11范围内均能生长,以pH 4生长最好。
本试验研究了产生粗毒素的培养条件。结果表明,在试验设计的上述6种液体培养基中,产毒能力最强的为PSK液体培养基,适宜培养时间为13d,培养温度为15℃,适宜pH值为6,培养方式为连续24 h黑暗及振荡培养。选择甘肃省红豆草种子,以种子萌发抑制法对粗毒素进行生物检测,表明培养得到的粗毒素具有一定的毒性。
采用PSK培养液在适宜条件下培养黑腐病菌,经乙酸乙酯萃取、浓缩、纯化,得到较纯的毒素样品。经生物测定发现,该毒素具有一定的毒性。利用气相色谱-质谱联用仪(HP6890-5973I)测定该毒素组分。结果表明,红豆草细链格孢菌主要组成成分为乙酸-N-羟基琥珀酰亚胺酯,相对含量为67.02%。
The aim of experimentation was to study the biology characteristic of the Alternaria tenuis Nees., the condition of phytotoxin-producity and the composing component of the toxin, to synthesize chemical substances which had antagonism for the toxin, and by means of the method of using poisons as antidotes to control the disease.
In the experiment, the pathogen was isolated from pathogenic and typical spots of sainfoin leaves in the field. The morphologic and pathogenic identification of the pathogen showed that the disease colony was equal diameter growth, the early colorless, gradually became greenish brown and dark brown, regular edge; the mycelia were slender, had branch and speta, the early colorless, gradually became light brown; conidiophores were straight or little curved, brown. The conidia usually growed in theshape of single or short chain, clavate or ovate, 3～7 transverse, 0～4 longitudinal septa, and 0~2 inclined septa. Their beaks were columnar, hazel and septation. In view of the morphology and comparison with other concerned Alternaria species, the pathogens were identified as Alternaria tenuis Nees.
The test of biology showed that the growth speed of the pathogen was the fastest on PSK liquid culture medium in six kinds of culture media(PD liquid culture Medium, PD and the juice of sainfoin leaves liquid culture Medium, PS liquid cultrue Medium, Improved Fries liquid culture Medium, Czapek liquid culture Medium and PSK liquid culture medium).The suitable time for mycelium growth was in the range of 11~13d, and mycelial dry weight reached maximum on 13th day. The appropriate temperature for mycelium growth was in the range of 20～30℃, the optimal temperature for mycelium growth was 25℃. It was not suitable for mycelium growth under 15℃or over 35℃. The mycelium growth speed was the fastest under the alternate condition of light and dark,shaking. The mycelium could grow between pH 5 and pH 12. And the mycelial dry weight was the largest on pH 8.
The condition of phytotoxin-producity capability was studied in the experiment. The results showed that PSK liquid medium was the best for producing phytotoxin in six kinds of phytotoxin-producity culture media which were tested in the experimentation. The optimal conditions for producing phytotoxin were cultured period 13d, cultured temperature 15℃, PH 6, continual 24h dark and shaking. Sainfoin seed germination was selected to test the toxicity of crude toxin. The result showed that the crude toxin which was cultured had definite toxicity.
Alternaria tenuis was cultured on PSK liquid medium under suitable conditions, and culturefiltrates was extracted by ethyl acetate, and concentration, purification. Found by bioassay, the phytotoxin had toxicity. Using gas chromatograph-mass spectrometer determinated toxin components. The results showed that the main substains of Alternaria tenuis of onobrychis viciaefolia was Acetic acid 2,5-dioxo-1-pyrrolidinyl ester, the relative content was 67.02%.
试验从田间具有典型病斑的发病红豆草叶片上分离病菌并培养鉴定。通过对病原形态的鉴定和致病性测定结果表明,该病菌菌落等径生长,初无色,后渐变为青褐色至黑褐色,边缘整齐;菌丝细长,分枝,有隔膜,初为无色,渐变为浅褐色;分生孢子梗直或向一侧略弯曲,褐色,分生孢子常单生或短链生,倒棍棒状、长椭圆形,具横隔膜3～7个,纵隔膜0～3个,斜隔膜0～2个。喙及假喙柱状,淡褐色,分隔。与Alternaria属相关的文献资料对照,将其鉴定为Alternaria tenuis Nees.(细链格孢)。
生物学测定结果表明,供试的6种液体培养基:马铃薯葡萄糖琼脂培养液、马铃薯葡萄糖琼脂叶片汁液培养液、马铃薯蔗糖琼脂培养液、改良Fries培养液、查彼培养液及PSK培养液中,病菌在PSK培养液中生长速率最快;适宜菌丝生长的时间为11~13d,而菌丝干重在第13天达到最大值;菌丝生长的适宜温度范围为20～30℃,最适温度为25℃,低于15℃或者高于35℃均不适宜生长;菌丝在光暗交替并且振荡的条件下生长最快;在pH值3～11范围内均能生长,以pH 4生长最好。
本试验研究了产生粗毒素的培养条件。结果表明,在试验设计的上述6种液体培养基中,产毒能力最强的为PSK液体培养基,适宜培养时间为13d,培养温度为15℃,适宜pH值为6,培养方式为连续24 h黑暗及振荡培养。选择甘肃省红豆草种子,以种子萌发抑制法对粗毒素进行生物检测,表明培养得到的粗毒素具有一定的毒性。
采用PSK培养液在适宜条件下培养黑腐病菌,经乙酸乙酯萃取、浓缩、纯化,得到较纯的毒素样品。经生物测定发现,该毒素具有一定的毒性。利用气相色谱-质谱联用仪(HP6890-5973I)测定该毒素组分。结果表明,红豆草细链格孢菌主要组成成分为乙酸-N-羟基琥珀酰亚胺酯,相对含量为67.02%。
The aim of experimentation was to study the biology characteristic of the Alternaria tenuis Nees., the condition of phytotoxin-producity and the composing component of the toxin, to synthesize chemical substances which had antagonism for the toxin, and by means of the method of using poisons as antidotes to control the disease.
In the experiment, the pathogen was isolated from pathogenic and typical spots of sainfoin leaves in the field. The morphologic and pathogenic identification of the pathogen showed that the disease colony was equal diameter growth, the early colorless, gradually became greenish brown and dark brown, regular edge; the mycelia were slender, had branch and speta, the early colorless, gradually became light brown; conidiophores were straight or little curved, brown. The conidia usually growed in theshape of single or short chain, clavate or ovate, 3～7 transverse, 0～4 longitudinal septa, and 0~2 inclined septa. Their beaks were columnar, hazel and septation. In view of the morphology and comparison with other concerned Alternaria species, the pathogens were identified as Alternaria tenuis Nees.
The test of biology showed that the growth speed of the pathogen was the fastest on PSK liquid culture medium in six kinds of culture media(PD liquid culture Medium, PD and the juice of sainfoin leaves liquid culture Medium, PS liquid cultrue Medium, Improved Fries liquid culture Medium, Czapek liquid culture Medium and PSK liquid culture medium).The suitable time for mycelium growth was in the range of 11~13d, and mycelial dry weight reached maximum on 13th day. The appropriate temperature for mycelium growth was in the range of 20～30℃, the optimal temperature for mycelium growth was 25℃. It was not suitable for mycelium growth under 15℃or over 35℃. The mycelium growth speed was the fastest under the alternate condition of light and dark,shaking. The mycelium could grow between pH 5 and pH 12. And the mycelial dry weight was the largest on pH 8.
The condition of phytotoxin-producity capability was studied in the experiment. The results showed that PSK liquid medium was the best for producing phytotoxin in six kinds of phytotoxin-producity culture media which were tested in the experimentation. The optimal conditions for producing phytotoxin were cultured period 13d, cultured temperature 15℃, PH 6, continual 24h dark and shaking. Sainfoin seed germination was selected to test the toxicity of crude toxin. The result showed that the crude toxin which was cultured had definite toxicity.
Alternaria tenuis was cultured on PSK liquid medium under suitable conditions, and culturefiltrates was extracted by ethyl acetate, and concentration, purification. Found by bioassay, the phytotoxin had toxicity. Using gas chromatograph-mass spectrometer determinated toxin components. The results showed that the main substains of Alternaria tenuis of onobrychis viciaefolia was Acetic acid 2,5-dioxo-1-pyrrolidinyl ester, the relative content was 67.02%.
引文
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