放线菌K5的种类鉴定及其对棉花枯萎病菌的拮抗和防病作用
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摘要
虽然许多地区种植的棉花为抗枯萎病品种,但病害仍较为严重。本论文从生物防治原理出发,研究对棉花枯萎病菌(Fusarium oxysporum f. sp. vasinfectum)具有拮抗作用的链霉菌,期望为棉花枯萎病的综合治理提供有效途径。
取不同生态条件下的土样若干,用稀释分离法获得放线菌207株。以棉花枯萎病菌等多种病原菌为目标菌,抑菌带法测定,共筛选到32株具有拮抗活性的放线菌菌株,占所分离总数的15.46%。其中K5对棉花枯萎病菌的拮抗作用最强,抑菌带宽为8mm,同时对其他病菌也有一定的拮抗作用,如水稻稻瘟病菌(Pyricularia grisea)等。
最有利于K5菌株拮抗作用发挥的培养条件是:利用大豆粉葡萄糖培养基,培养基pH为6.5-7.0,10%接种量,温度为28-31℃,120r/min振荡培养,时间为7d。
K5培养滤液可以破坏枯萎病菌菌丝形态,使菌丝畸形、细胞壁破损、甚至细胞崩解。培养滤液处理9h即可见病菌菌丝细胞异常,17h出现细胞崩解。不同浓度培养滤液对病菌作用的差异表现在,高浓度可以缩短严重症状出现的时间。当培养滤液稀释100倍时,对菌丝形态仍具有破坏作用。培养滤液可抑制病菌菌丝生长、分生孢子的产生,最高抑制率分别达88.41%和77.45%;对分生孢子萌发和芽管生长的抑制率分别是98.62%和49.72%。随浓度下降,滤液的抑制作用减弱,但100倍稀释液对菌丝生长、产孢和分生孢子萌发仍具有一定的抑制作用。
盆栽条件下,K5菌株的发酵滤液对棉花苗期枯萎病的防治效果达46.50%,与多菌灵浸种处理的防病效果相似。放线菌K5对棉苗不仅没有不良影响,反而有一定的促生作用,幼苗株高和鲜重均高于其他各处理。
K5菌株可以产生基质内菌丝和气生菌丝,气生菌丝形成孢子长链,孢子椭圆形至球形,表面光滑。K5在高氏一号培养基上生长旺盛,气生菌丝灰红色,基质菌丝黄褐色,不产生可溶性色素;能较好利用麦芽糖、半乳糖,一般利用葡萄糖和鼠李糖,不能利用蔗糖、阿拉伯糖、果糖等;可以使明胶液化,牛奶胨化,淀粉水解;可以产生硫化氢和黑色素。以上培养性状、生理生化特征都与玫瑰产色链霉菌(Streptomyces roseochromogenus)相近。K5菌株16S rDNA序列测定,与玫瑰产色链霉菌的同源性高达99.63%,遗传距离为12.4。因此,认为K5菌株应该是玫瑰产色链霉菌(S. roseochromogenus)。
Although disease-resistant varieties of cotton were planted in many areas, Fusarium wilt of cotton occurred widely and severely. This paper studied a Streptomyces which has antagonism against Fusarium oxysporum f. sp. vasinfectum in order to provide an effective way for integrated management of Fusarium wilt of cotton.
207 strains of actinomycetes were isolated from some soil samples collected from various areas with different ecology conditions. From them 32 strains of actinomycetes with antagonism against many pathogens such as F. oxysporum f. sp. vasinfectum were obtained by the inhibition zone method, which accounted for 15.46% of total strains. K5 strain exhibited the strongest antagonism against F. oxysporum f. sp. vasinfectum among all strains and the size of inhibition zone to pathogen was 8mm. Meanwhile, it also showed antagonistic effect on some other pathogenic fungi, such as Pyricularia grisea et al.
The better culture conditons for K5 to exert its antagonism were listed as follows: The strains was cultured for 7 days at 28-31℃in the oybean and glucose medium, in which pH value was 6.5-7.0, 10% inoculation amount, and shaking at 120r/min.
The culture filtrate of K5 revealed obvious damage and inhibitory effects on the mycelial morphology and growth of F. oxysporum f. sp. vasinfectum. When the pathogen was cultured in amended medium with K5’culture filtrate, it was observed by microscope that the hypha became abnormal, cell wall was destroied, which could lead to leakage protoplasm from the wound and even lysis of hypha . The mycelium cultured for 9h in above amended medium present non-normal, and for 17 hours the cells of mycelium were destroyed. There was some difference in mycelium treated by culture filtrate with different concentration. The hyphal cell lysis occurred soon at the high concentration of the culture filtrate. When the culture filtrate was diluted 100 times, the culture filtrate could also destroy the hypha. The culture filtrate could inhibit the growth of hypha and sporulation, the rates of inhibition were 88.41% and 77.45%, respectively; the inhibitory rates to conidial germination and growth of germ-tube were 98.62% and 49.72%, respectively. As the dilution of the filtrate was increased, the inhibitory effect on pathogen of the filtrate could decline. Although the culture filtrate was diluted 100 times, it still showed inhibition to the growth of hypha, sporulation and conidial germination.
The results of pot tests showed that the control effect of the ferment filtration of K5 on seedling Fusarium wilt of cotton was 46.50%, which resembled those of chemical fungicide carbendazim. Not only K5 had no any damage on cotton seedlings, but also it could promote the cottons’growth, the seedling height and weight were significantly higher than that of control plants.
K5 could produce substrate mycelia and aerial mycelia. The aerial mycelia formed catenarian spore-filaments, the shape of spore was oval or round and the surface of spore was smooth. K5 grew well in Gause’s No.1 medium, the color of aerial mycelia was grey-red and the color of substrate mycelia was filemot, and there was no soluble pigment. K5 could well make good use of maltose, galactose, the ability of using glucose and rhamnose was moderate while it could not use sucrose, arabinose and fructose. Some physiology phenomena of the strain such as gelatin liquefaction, milk peptonization, starch hydrolysis, and melanin-like substance production could be observed. Its characteristics of morphology, physiology and biochemics were similar to those of Streptomyces roseochromogenus. K5’s 16S rDNA sequences analysis were achieved, the overall similarity value between K5 and S. Roseochromogenus was 99.63% and the hereditary distance was 12.4. According to above experiment, the strain K5 was identified to be S. roseochromogenus.
取不同生态条件下的土样若干,用稀释分离法获得放线菌207株。以棉花枯萎病菌等多种病原菌为目标菌,抑菌带法测定,共筛选到32株具有拮抗活性的放线菌菌株,占所分离总数的15.46%。其中K5对棉花枯萎病菌的拮抗作用最强,抑菌带宽为8mm,同时对其他病菌也有一定的拮抗作用,如水稻稻瘟病菌(Pyricularia grisea)等。
最有利于K5菌株拮抗作用发挥的培养条件是:利用大豆粉葡萄糖培养基,培养基pH为6.5-7.0,10%接种量,温度为28-31℃,120r/min振荡培养,时间为7d。
K5培养滤液可以破坏枯萎病菌菌丝形态,使菌丝畸形、细胞壁破损、甚至细胞崩解。培养滤液处理9h即可见病菌菌丝细胞异常,17h出现细胞崩解。不同浓度培养滤液对病菌作用的差异表现在,高浓度可以缩短严重症状出现的时间。当培养滤液稀释100倍时,对菌丝形态仍具有破坏作用。培养滤液可抑制病菌菌丝生长、分生孢子的产生,最高抑制率分别达88.41%和77.45%;对分生孢子萌发和芽管生长的抑制率分别是98.62%和49.72%。随浓度下降,滤液的抑制作用减弱,但100倍稀释液对菌丝生长、产孢和分生孢子萌发仍具有一定的抑制作用。
盆栽条件下,K5菌株的发酵滤液对棉花苗期枯萎病的防治效果达46.50%,与多菌灵浸种处理的防病效果相似。放线菌K5对棉苗不仅没有不良影响,反而有一定的促生作用,幼苗株高和鲜重均高于其他各处理。
K5菌株可以产生基质内菌丝和气生菌丝,气生菌丝形成孢子长链,孢子椭圆形至球形,表面光滑。K5在高氏一号培养基上生长旺盛,气生菌丝灰红色,基质菌丝黄褐色,不产生可溶性色素;能较好利用麦芽糖、半乳糖,一般利用葡萄糖和鼠李糖,不能利用蔗糖、阿拉伯糖、果糖等;可以使明胶液化,牛奶胨化,淀粉水解;可以产生硫化氢和黑色素。以上培养性状、生理生化特征都与玫瑰产色链霉菌(Streptomyces roseochromogenus)相近。K5菌株16S rDNA序列测定,与玫瑰产色链霉菌的同源性高达99.63%,遗传距离为12.4。因此,认为K5菌株应该是玫瑰产色链霉菌(S. roseochromogenus)。
Although disease-resistant varieties of cotton were planted in many areas, Fusarium wilt of cotton occurred widely and severely. This paper studied a Streptomyces which has antagonism against Fusarium oxysporum f. sp. vasinfectum in order to provide an effective way for integrated management of Fusarium wilt of cotton.
207 strains of actinomycetes were isolated from some soil samples collected from various areas with different ecology conditions. From them 32 strains of actinomycetes with antagonism against many pathogens such as F. oxysporum f. sp. vasinfectum were obtained by the inhibition zone method, which accounted for 15.46% of total strains. K5 strain exhibited the strongest antagonism against F. oxysporum f. sp. vasinfectum among all strains and the size of inhibition zone to pathogen was 8mm. Meanwhile, it also showed antagonistic effect on some other pathogenic fungi, such as Pyricularia grisea et al.
The better culture conditons for K5 to exert its antagonism were listed as follows: The strains was cultured for 7 days at 28-31℃in the oybean and glucose medium, in which pH value was 6.5-7.0, 10% inoculation amount, and shaking at 120r/min.
The culture filtrate of K5 revealed obvious damage and inhibitory effects on the mycelial morphology and growth of F. oxysporum f. sp. vasinfectum. When the pathogen was cultured in amended medium with K5’culture filtrate, it was observed by microscope that the hypha became abnormal, cell wall was destroied, which could lead to leakage protoplasm from the wound and even lysis of hypha . The mycelium cultured for 9h in above amended medium present non-normal, and for 17 hours the cells of mycelium were destroyed. There was some difference in mycelium treated by culture filtrate with different concentration. The hyphal cell lysis occurred soon at the high concentration of the culture filtrate. When the culture filtrate was diluted 100 times, the culture filtrate could also destroy the hypha. The culture filtrate could inhibit the growth of hypha and sporulation, the rates of inhibition were 88.41% and 77.45%, respectively; the inhibitory rates to conidial germination and growth of germ-tube were 98.62% and 49.72%, respectively. As the dilution of the filtrate was increased, the inhibitory effect on pathogen of the filtrate could decline. Although the culture filtrate was diluted 100 times, it still showed inhibition to the growth of hypha, sporulation and conidial germination.
The results of pot tests showed that the control effect of the ferment filtration of K5 on seedling Fusarium wilt of cotton was 46.50%, which resembled those of chemical fungicide carbendazim. Not only K5 had no any damage on cotton seedlings, but also it could promote the cottons’growth, the seedling height and weight were significantly higher than that of control plants.
K5 could produce substrate mycelia and aerial mycelia. The aerial mycelia formed catenarian spore-filaments, the shape of spore was oval or round and the surface of spore was smooth. K5 grew well in Gause’s No.1 medium, the color of aerial mycelia was grey-red and the color of substrate mycelia was filemot, and there was no soluble pigment. K5 could well make good use of maltose, galactose, the ability of using glucose and rhamnose was moderate while it could not use sucrose, arabinose and fructose. Some physiology phenomena of the strain such as gelatin liquefaction, milk peptonization, starch hydrolysis, and melanin-like substance production could be observed. Its characteristics of morphology, physiology and biochemics were similar to those of Streptomyces roseochromogenus. K5’s 16S rDNA sequences analysis were achieved, the overall similarity value between K5 and S. Roseochromogenus was 99.63% and the hereditary distance was 12.4. According to above experiment, the strain K5 was identified to be S. roseochromogenus.
引文
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