生防菌盾壳霉产生抗真菌物质及其特性和防病潜力研究
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摘要
油菜菌核病是由核盘菌[Sclerotinia sclerotiorum(Lib.)de Bary]引起的一种世界性分布的重要病害。重寄生菌盾壳霉(Coniothyrium minitans)是一种应用前景广阔的生防菌。前人对盾壳霉寄生核盘菌的机制及其防病潜力进行了深入研究。McQuilken et al.(2003)报道了盾壳霉抑制核盘菌菌丝生长的抗生机制，并从盾壳霉培养物中分离和鉴定出抗真菌物质(Antifungal substance，简称AFS)macrosphelideA。但对其产生的规律、基本特性、能否抑制核盘菌菌核及子囊孢子萌发、是否能够产生防病效果等问题还不明确。针对这些问题，我们开展了盾壳霉产生AFS及其特性和防病潜力研究。
用改良的查彼培养液(MCD)培养盾壳霉，制备含AFS的盾壳霉培养物滤液(MCD_(cm))。在马铃薯葡萄糖琼脂培养基(PDA)上测定其对核盘菌菌丝生长、菌核萌发和子囊孢子萌发的抑制作用。结果表明：盾壳霉AFS对核盘菌菌丝生长具有明显的抑制作用。随MCD_(cm)(含AFS)浓度(x)提高，抑菌活性(y)增强。两者之间的关系符合方程：y=19.268Ln(x)+44.152(R~2=0.9703，P＜0.01)。被抑制的核盘菌菌丝原生质出现颗粒化及外渗，菌丝细胞膨大，甚至溃解等异常现象。用MCD_(cm)处理核盘菌菌核之后，菌核萌发延迟，但最终能完全萌发。说明盾壳霉AFS对核盘菌菌核没有致死效应，但有一定程度的毒害作用；在含有MCD_(cm)的PDA平板上，核盘菌子囊孢子能够萌发，培养12h的萌发率达到99.4％，但芽管生长受到明显抑制，出现细胞膨大和生长受阻等症状。
采用MCD培养液和马铃薯葡萄糖培养液(potato dextrose broth，简称PDB)振荡培养(20℃，200r／min)的方法研究了菌株和培养基种类对盾壳霉产生AFS的影响。结果表明：盾壳霉野生菌株Chy-1及其突变菌株Uv-2003能够产生AFS，而Chy-1的另外两个突变菌株Uv-202和Uv-1731却不能产生AFS。在MCD培养液中，培养盾壳霉6d后即检测到AFS活性，培养12d～15d后，培养液对核盘菌菌丝生长的抑制率分别达到86.4％和89.7％。在3d～15d培养期间，培养液的pH值从4.6下降为3.6。在PDB培养液中，培养3d～15d后，培养液对核盘菌菌丝生长的抑制率低于17.8％。培养6d后，培养液的pH值从5.9(0d)上升至7.5，培养15d后，pH值下降为6.0。
分别以MCD和PDB为基础，研究碳源、氮源、磷酸盐和环境pH值对盾壳霉产生AFS的影响。在碳源试验中，以MCD为基础时，供试7种碳源中，盾壳霉利用6种碳源(可溶性淀粉、蔗糖、麦芽糖、葡萄糖、乳糖和果糖)能产生AFS。葡萄糖和果糖对盾壳霉产生AFS最为适宜，而山梨醇则不适宜。以PDB为基础时，7种供试碳源中，果糖和乳糖对盾壳霉产生AFS适宜，其它5种碳源
Sclerotinia stem rot caused by Sclerotinia sclerotiorum (Lib) de Bary is an important disease of oilseed rape with a worldwide distribution. Coniothyrium minitans Campbell is an effective biocontrol agent of S. sclerotiorum. The mycoparasitism of C. minitans on S. sclerotiorum was profoundly studied and exploited as a mechanism in biological control. McQuilken et al. (2003) reported that C. minitans could produce antifungal substances (shorted as AFS_(cm)) and an inhibitory metabolite named macrosphelide A was identified. However, the antifungal mechanisms involved in interaction between C. minitans and S. sclerotiorum are still unknown and need to be clarified. The purpose of this thesis is aimed at answering questions including: (1) the effect of AFS_(cm) on germination of sclerotia or ascospores of S. sclerotiorum; (2) effect of nutritional factors on production of AFS_(cm); (3) basic properties about the AFS_(cm); and (4) efficacy of the AFS_(cm) in suppression of the infection by S. sclerotiorum.
The modified Czapek-Dox (MCD) was used to incubate C. minitans and the AFS-containing cultural filtrate (MCD_(cm)) was used to test its inhibitory effect on mycelial growth of S. sclerotiorum, on germination of sclerotia and ascospores of this fungus. The results demonstrated that MCD_(cm) did inhibit mycelial growth (MG) of S. sclerotiorum and the relationship between the concentration of MCD_(cm) (x) amended in potato dextrose agar (PDA) and the percentage of inhibition of MG (y) fitted the equation : y=19.268Ln(x)+44.152 (R~2=0.9703, P<0.01). The inhibited mycelia of 5. sclerotiorum showed severe abnormal symptoms including cytoplasma granulation and outward leakage, cellular swelling and collapse. Treartment of S. sclerotiorum sclerotia with AFS_(cm) resulted in a delay in myceliogenic germination implying that the AFS_(cm) was toxic to sclerotial cells, however, without any lethal effect. On PDA plates with and without the amendment of AFS_(cm), ascospores of S. sclerotiorum germinated at 99.4% and 98.8%, respectively, after incubation for 12 h. However, growth of the germ tubes were suppressed by the formation of swollen cells in the presence of AFS_(cm).
Strains of C. minitans, and media MCD/PDB were compared for production of AFS under the shaking incubation conditions (20 ℃ and 200r/min). The results showed that the wild-type strain Chy-1 of C. minitans and its mutated strainUv-2003 were able to produce AFS, whereas other two mutated strains Uv-202 and Uv-1731 was unable to produce AFS. Production of AFS differed greatly in the media MCD and
用改良的查彼培养液(MCD)培养盾壳霉，制备含AFS的盾壳霉培养物滤液(MCD_(cm))。在马铃薯葡萄糖琼脂培养基(PDA)上测定其对核盘菌菌丝生长、菌核萌发和子囊孢子萌发的抑制作用。结果表明：盾壳霉AFS对核盘菌菌丝生长具有明显的抑制作用。随MCD_(cm)(含AFS)浓度(x)提高，抑菌活性(y)增强。两者之间的关系符合方程：y=19.268Ln(x)+44.152(R~2=0.9703，P＜0.01)。被抑制的核盘菌菌丝原生质出现颗粒化及外渗，菌丝细胞膨大，甚至溃解等异常现象。用MCD_(cm)处理核盘菌菌核之后，菌核萌发延迟，但最终能完全萌发。说明盾壳霉AFS对核盘菌菌核没有致死效应，但有一定程度的毒害作用；在含有MCD_(cm)的PDA平板上，核盘菌子囊孢子能够萌发，培养12h的萌发率达到99.4％，但芽管生长受到明显抑制，出现细胞膨大和生长受阻等症状。
采用MCD培养液和马铃薯葡萄糖培养液(potato dextrose broth，简称PDB)振荡培养(20℃，200r／min)的方法研究了菌株和培养基种类对盾壳霉产生AFS的影响。结果表明：盾壳霉野生菌株Chy-1及其突变菌株Uv-2003能够产生AFS，而Chy-1的另外两个突变菌株Uv-202和Uv-1731却不能产生AFS。在MCD培养液中，培养盾壳霉6d后即检测到AFS活性，培养12d～15d后，培养液对核盘菌菌丝生长的抑制率分别达到86.4％和89.7％。在3d～15d培养期间，培养液的pH值从4.6下降为3.6。在PDB培养液中，培养3d～15d后，培养液对核盘菌菌丝生长的抑制率低于17.8％。培养6d后，培养液的pH值从5.9(0d)上升至7.5，培养15d后，pH值下降为6.0。
分别以MCD和PDB为基础，研究碳源、氮源、磷酸盐和环境pH值对盾壳霉产生AFS的影响。在碳源试验中，以MCD为基础时，供试7种碳源中，盾壳霉利用6种碳源(可溶性淀粉、蔗糖、麦芽糖、葡萄糖、乳糖和果糖)能产生AFS。葡萄糖和果糖对盾壳霉产生AFS最为适宜，而山梨醇则不适宜。以PDB为基础时，7种供试碳源中，果糖和乳糖对盾壳霉产生AFS适宜，其它5种碳源
Sclerotinia stem rot caused by Sclerotinia sclerotiorum (Lib) de Bary is an important disease of oilseed rape with a worldwide distribution. Coniothyrium minitans Campbell is an effective biocontrol agent of S. sclerotiorum. The mycoparasitism of C. minitans on S. sclerotiorum was profoundly studied and exploited as a mechanism in biological control. McQuilken et al. (2003) reported that C. minitans could produce antifungal substances (shorted as AFS_(cm)) and an inhibitory metabolite named macrosphelide A was identified. However, the antifungal mechanisms involved in interaction between C. minitans and S. sclerotiorum are still unknown and need to be clarified. The purpose of this thesis is aimed at answering questions including: (1) the effect of AFS_(cm) on germination of sclerotia or ascospores of S. sclerotiorum; (2) effect of nutritional factors on production of AFS_(cm); (3) basic properties about the AFS_(cm); and (4) efficacy of the AFS_(cm) in suppression of the infection by S. sclerotiorum.
The modified Czapek-Dox (MCD) was used to incubate C. minitans and the AFS-containing cultural filtrate (MCD_(cm)) was used to test its inhibitory effect on mycelial growth of S. sclerotiorum, on germination of sclerotia and ascospores of this fungus. The results demonstrated that MCD_(cm) did inhibit mycelial growth (MG) of S. sclerotiorum and the relationship between the concentration of MCD_(cm) (x) amended in potato dextrose agar (PDA) and the percentage of inhibition of MG (y) fitted the equation : y=19.268Ln(x)+44.152 (R~2=0.9703, P<0.01). The inhibited mycelia of 5. sclerotiorum showed severe abnormal symptoms including cytoplasma granulation and outward leakage, cellular swelling and collapse. Treartment of S. sclerotiorum sclerotia with AFS_(cm) resulted in a delay in myceliogenic germination implying that the AFS_(cm) was toxic to sclerotial cells, however, without any lethal effect. On PDA plates with and without the amendment of AFS_(cm), ascospores of S. sclerotiorum germinated at 99.4% and 98.8%, respectively, after incubation for 12 h. However, growth of the germ tubes were suppressed by the formation of swollen cells in the presence of AFS_(cm).
Strains of C. minitans, and media MCD/PDB were compared for production of AFS under the shaking incubation conditions (20 ℃ and 200r/min). The results showed that the wild-type strain Chy-1 of C. minitans and its mutated strainUv-2003 were able to produce AFS, whereas other two mutated strains Uv-202 and Uv-1731 was unable to produce AFS. Production of AFS differed greatly in the media MCD and
引文
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