防治草莓灰霉病酵母菌株筛选及防病机制研究
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摘要
论文研究了利用酵母菌防治草莓灰霉病的基础问题,包括酵母菌分离、筛选、鉴定、防病机制和防病潜力等。取得的研究结果如下：
1.筛选到7株对草莓灰霉病有显著防效的酵母菌菌株,其编号为C410、C23、C25、W4682、YW1、YCXT2和YCXT3。
2.明确了7株酵母菌的分类属性。菌株C410、C23和C25属于间型假丝酵母(Candida intermedia),菌株YCXT2和YCXT3属于准红锁掷孢酵母(Sporidiobolus pararoseus),菌株W4682属于汉逊狄巴利酵母(Debaryomyces hansenii),菌株YW1属于出芽短梗霉(Aureobasidium pullulans)。
3.证实菌株C410、W4682、YCXT3和YW1可通过竞争机制产生对灰霉菌的防治效果。这些酵母菌的无细胞发酵液对灰葡萄孢菌菌丝生长及孢子萌发没有抑制作用。用不同浓度的各酵母菌菌株细胞悬浮液(105-107 cells/ml)浸泡草莓果实后,草莓灰霉病的发病情况显著(P<0.01)低于清水浸泡处理。此外,试验证实菌株C410、W4682、YCXT3和YW1均可在草莓果实表面定殖。
4.证实菌株C410、W4682、YCXT3和YW1均能挥发性物质(volatile organic compounds, VOCs),对灰葡萄孢菌菌丝生长和孢子萌发具有抑菌作用。结果还表明：菌株C410和YCXT3产生的VOCs对储藏期草莓灰霉病具有明显的防治效果。
5.基本明确了菌株C410和YCXT3产生的VOCs成分,并明确了部分成分的抗真菌活性。在菌株C410产生的VOCs中,2-nonanone；pentanoic acid,4-methyl-, ethyl ester; 1,3.5,7-cyclooctatetraene; 1-butanol,3-methyl-, acetate; acetic acid, pentyl ester; hexanoic acid, ethyl ester和1-butanol,3-methyl-等对灰霉菌菌丝生长和孢子萌发具有抑制作用。在菌株YCXT3产生挥发性物质中,2-ethyl-1-hexanol对灰霉菌菌丝生长和孢子萌发具有抑制作用。
6.证实菌株C410和YW1的细胞悬浮液对大棚草莓灰霉病的发生具有显著地防治效果。在采收前用菌株C410接种草莓果实,在收获后这些草莓果实灰霉病发生显著降低。
A basic study was conducted to isolate yeasts from strawberry plants, to screen yeast strains effective for suppression of the gray mould pathogen Botrytis cinerea, to identify the selected yeast strains and to characterize the efficacy and the mechanisms of the yeast strains. Results are summarized below:
1. The yeast strains were isolated from healthy strawberry plants, which collected from Hubei, Hunan and other areas in China. A total of 1151 yeast strains were isolated. Through plate dual culture screening, in vitro leaves and fruits screening,7 yeast strains, designated as C410, C23, C25, W4682, YW1, YCXT2 and YCXT3 were selected.
2. The 7 selected yeast strains were identified by observing the colony characteristics, spore morphology and reproduction mode, determining the physiological characteristics and analyze the DNA sequence of the internal transcribed spacer of ribosomal DNA. Strains C410, C23 and C25 were identified as Candida intermedia (GenBank Ace. No. GQ913345). Strains YCXT2 and YCXT3 were identified as Sporidiobolus pararoseus (GenBank Ace. No. GQ913347). Strain W4682 was identified as Debaryomyces hansenii (GenBank Ace. No. GQ913348). Strain YW1 was identified as Aureobasidium pullulans (GenBank Ace. No. GQ913346).
3. The cell-free culture filtrates of strains C410, W4682, YCXT3 and YW1 could not inhibit either mycelial growth or conidial germination of Botrytis cinerea, suggesting that the four yeast strains could not produce non-volatile antifungal substances. Different concentrations of yeast cell suspensions have significantly effect to control strawberry fruit rot caused by B. cinerea. All the four yeast strains could colonize the surface of strawberry fruits. The results suggest that the four yeast strains inhibit infection of strawberry fruits through competition for nutritients and space.
4. The biocontrol potential of the volatile organic compounds (VOCs) produced by four yeast strains (C410, W4682, YCXT3 and YW1) was assessed. The results showed that the VOCs produced by four yeast strains could significantly inhibit the mycelial growth of B. cinerea. The VOCs produced by strains C410 and YCXT3 were effectiove in suppression of the mycelial growth and conidial germination of B. cinerea, and in control of strawberry fruit rot caused by B. cinerea.
5. The VOVs produced by strains C410 and YCXT3 were identified by gas chromatography-mass spectrometry (GC-MS). The effect of the VOCs of strains C410 and YCXT3 on suppression of mycelial growth and conidial germination of B. cinerea was tested. Results showed that there were 49 compounds released by strain C410 on yeast extract peptone dextrose agar (YEPDA). Synthetic chemicals of 1,3,5,7-cyclooctatetraene; 3-methyl-l-butanol; 2-nonanone; pentanoic acid,4-methyl-, ethyl ester; 3-methyl-1-butanol, acetate; acetic acid, pentyl ester; and hexanoic acid, ethyl ester were highly inhibitory to conidial germination and mycelial growth of B. cinerea. There were 38 compounds released by strain YCXT3on YEPDA. The synthetic chemical 2-ethyl-1-hexanol was highly inhibitory to conidial germination and mycelial growth of B. cinerea.
6. Four yeast strains (C410, W4682, YCXT3 and YW1) could effectively control the potted seedling rot of strawberry. The cell suspension of yeast strains C410 and YW1 could significantly control fruit rot of strawberry by sprayed the cell suspension of yeast strains in shed. When the cell suspension of yeast strains inoculated on strawberry fruits before harvest, the incidence of postharvest fruit rot of strawberry were reduced, significantly.
All the results mentioned above laid a solid foundation for utilization of the yeast strains C410, W4682, YCXT3 and YW1 for control gray mold disease of strawberry caused by Botrytis cinerea.
1.筛选到7株对草莓灰霉病有显著防效的酵母菌菌株,其编号为C410、C23、C25、W4682、YW1、YCXT2和YCXT3。
2.明确了7株酵母菌的分类属性。菌株C410、C23和C25属于间型假丝酵母(Candida intermedia),菌株YCXT2和YCXT3属于准红锁掷孢酵母(Sporidiobolus pararoseus),菌株W4682属于汉逊狄巴利酵母(Debaryomyces hansenii),菌株YW1属于出芽短梗霉(Aureobasidium pullulans)。
3.证实菌株C410、W4682、YCXT3和YW1可通过竞争机制产生对灰霉菌的防治效果。这些酵母菌的无细胞发酵液对灰葡萄孢菌菌丝生长及孢子萌发没有抑制作用。用不同浓度的各酵母菌菌株细胞悬浮液(105-107 cells/ml)浸泡草莓果实后,草莓灰霉病的发病情况显著(P<0.01)低于清水浸泡处理。此外,试验证实菌株C410、W4682、YCXT3和YW1均可在草莓果实表面定殖。
4.证实菌株C410、W4682、YCXT3和YW1均能挥发性物质(volatile organic compounds, VOCs),对灰葡萄孢菌菌丝生长和孢子萌发具有抑菌作用。结果还表明：菌株C410和YCXT3产生的VOCs对储藏期草莓灰霉病具有明显的防治效果。
5.基本明确了菌株C410和YCXT3产生的VOCs成分,并明确了部分成分的抗真菌活性。在菌株C410产生的VOCs中,2-nonanone；pentanoic acid,4-methyl-, ethyl ester; 1,3.5,7-cyclooctatetraene; 1-butanol,3-methyl-, acetate; acetic acid, pentyl ester; hexanoic acid, ethyl ester和1-butanol,3-methyl-等对灰霉菌菌丝生长和孢子萌发具有抑制作用。在菌株YCXT3产生挥发性物质中,2-ethyl-1-hexanol对灰霉菌菌丝生长和孢子萌发具有抑制作用。
6.证实菌株C410和YW1的细胞悬浮液对大棚草莓灰霉病的发生具有显著地防治效果。在采收前用菌株C410接种草莓果实,在收获后这些草莓果实灰霉病发生显著降低。
A basic study was conducted to isolate yeasts from strawberry plants, to screen yeast strains effective for suppression of the gray mould pathogen Botrytis cinerea, to identify the selected yeast strains and to characterize the efficacy and the mechanisms of the yeast strains. Results are summarized below:
1. The yeast strains were isolated from healthy strawberry plants, which collected from Hubei, Hunan and other areas in China. A total of 1151 yeast strains were isolated. Through plate dual culture screening, in vitro leaves and fruits screening,7 yeast strains, designated as C410, C23, C25, W4682, YW1, YCXT2 and YCXT3 were selected.
2. The 7 selected yeast strains were identified by observing the colony characteristics, spore morphology and reproduction mode, determining the physiological characteristics and analyze the DNA sequence of the internal transcribed spacer of ribosomal DNA. Strains C410, C23 and C25 were identified as Candida intermedia (GenBank Ace. No. GQ913345). Strains YCXT2 and YCXT3 were identified as Sporidiobolus pararoseus (GenBank Ace. No. GQ913347). Strain W4682 was identified as Debaryomyces hansenii (GenBank Ace. No. GQ913348). Strain YW1 was identified as Aureobasidium pullulans (GenBank Ace. No. GQ913346).
3. The cell-free culture filtrates of strains C410, W4682, YCXT3 and YW1 could not inhibit either mycelial growth or conidial germination of Botrytis cinerea, suggesting that the four yeast strains could not produce non-volatile antifungal substances. Different concentrations of yeast cell suspensions have significantly effect to control strawberry fruit rot caused by B. cinerea. All the four yeast strains could colonize the surface of strawberry fruits. The results suggest that the four yeast strains inhibit infection of strawberry fruits through competition for nutritients and space.
4. The biocontrol potential of the volatile organic compounds (VOCs) produced by four yeast strains (C410, W4682, YCXT3 and YW1) was assessed. The results showed that the VOCs produced by four yeast strains could significantly inhibit the mycelial growth of B. cinerea. The VOCs produced by strains C410 and YCXT3 were effectiove in suppression of the mycelial growth and conidial germination of B. cinerea, and in control of strawberry fruit rot caused by B. cinerea.
5. The VOVs produced by strains C410 and YCXT3 were identified by gas chromatography-mass spectrometry (GC-MS). The effect of the VOCs of strains C410 and YCXT3 on suppression of mycelial growth and conidial germination of B. cinerea was tested. Results showed that there were 49 compounds released by strain C410 on yeast extract peptone dextrose agar (YEPDA). Synthetic chemicals of 1,3,5,7-cyclooctatetraene; 3-methyl-l-butanol; 2-nonanone; pentanoic acid,4-methyl-, ethyl ester; 3-methyl-1-butanol, acetate; acetic acid, pentyl ester; and hexanoic acid, ethyl ester were highly inhibitory to conidial germination and mycelial growth of B. cinerea. There were 38 compounds released by strain YCXT3on YEPDA. The synthetic chemical 2-ethyl-1-hexanol was highly inhibitory to conidial germination and mycelial growth of B. cinerea.
6. Four yeast strains (C410, W4682, YCXT3 and YW1) could effectively control the potted seedling rot of strawberry. The cell suspension of yeast strains C410 and YW1 could significantly control fruit rot of strawberry by sprayed the cell suspension of yeast strains in shed. When the cell suspension of yeast strains inoculated on strawberry fruits before harvest, the incidence of postharvest fruit rot of strawberry were reduced, significantly.
All the results mentioned above laid a solid foundation for utilization of the yeast strains C410, W4682, YCXT3 and YW1 for control gray mold disease of strawberry caused by Botrytis cinerea.
引文
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