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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2017, Vol. 37 Issue (5): 76-86    DOI: 10.13523/j.cb.20170510
技术与方法     
抑制西瓜蔓枯病菌的生防真菌筛选、鉴定及发酵条件优化
张旭辉1, 张红楠2, 李勇1, 汪文强1
1. 西南大学资源环境学院 重庆 400715;
2. 西南大学园艺园林学院 重庆 400715
Screening and Identification of Biocontrol Fungi against Didymella bryoniae and Optimization of Fermentation Conditions
ZHANG Xu-hui1, ZHANG Hong-nan2, LI Yong1, WANG Wen-qiang1
1. College of Resources and Environment, Southwest University, Chongqing 400715, China;
2. College of Horticulture and Landscape Architecture, Southwest University, Chongqing 400715, China
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摘要：

筛选对西瓜蔓枯病菌具有抑制作用的生防真菌，优化其发酵条件以提升发酵液抑菌效果。以平板对峙试验和摇瓶培养抑菌试验筛选拮抗真菌；根据形态学特征和18S rDNA序列分析进行菌株鉴定；通过扫描电镜观察对西瓜蔓枯病菌的寄生作用，以单因素试验和响应曲面法确定其最适发酵条件，在此基础上，通过盆栽试验研究其防病效果。结果表明：在所筛选到的9株生防真菌中M1菌株的抑菌率最高，其形态学特征与烟管菌（Bjerkandera adusta）相符，18S rDNA序列分析显示其与烟管菌在系统发育树上聚在一支，因此将菌株M1鉴定为黑管菌属（Bjerkandera）、烟管菌。在扫描电镜下观察到烟管菌能够直接穿透西瓜蔓枯病菌菌丝，对其发酵条件优化后确定了最佳条件组合：C/N为7.1，pH为7.4，装瓶量为44%，时间为19d，转速为180r/min，温度为29℃，在此条件下抑菌率为52.64%，高于未经优化的抑菌率。温室盆栽中对西瓜蔓枯病菌的防病效果达到74.3%，高于多菌灵处理。烟管菌作为生防真菌对西瓜蔓枯病菌具有较好的抑制作用，条件优化后进一步提升了抑菌效果，在生物防治中具有巨大应用潜力。
关键词： 拮抗;  西瓜蔓枯病菌;  抑菌率;  烟管菌;  条件优化    
Abstract:

In order to screen the biocontrol strain with antagonistic effects on Didymella bryoniae and enhance the inhibitory efficacy of the fermentation broth, antagonistic fungi was screened using the dual culture assay and shaking flask culture experiment. Subsequently, biocontrol strain was identified according to morphological character and 18S rDNA sequence analysis, and observation of parasitic effect of antagonistic strain on Didymella bryoniae was made by scanning electron microscope. The fermentation conditions were optimized according to single factor test and response surface methodology. In addition, the biocontrol efficacy of Bjerkandera adusta against Didymella bryoniae in plants was determined in greenhouse. The results reveal that the strain M1 has the highest inhibitory rates to Didymella bryoniae, reaching up to 59.56% and 49.56% in the dual culture assay and in shaking flask culture experiment separately. The morphological characteristics of M1 are consistent with those of Bjerkandera adusta and 18S rDNA sequence phylogenetic analysis reveal that M1 appears a sister lineage to Bjerkandera adusta and gathers together in a phylogenetic tree. So the strain M1 is identified as Bjerkandera adusta. Additionally, M1 is able to penetrate Didymella bryoniae mycelium directly. After optimized, the fermentation conditions are C/N of 7.1, pH of 7.4, the bottling quantity of 44%, fermentation time of 19d, the rotate speed of 180r/min, culture temperature of 29℃with the inhibitory rate of the fermentation broth increased from 49.56% to 52.64%. The biocontrol efficiency of Bjerkandera adusta against Didymella bryoniae is up to 74.3% in greenhouse, which is higher than that of treatment with carbendazim. These results demonstrate that as a biocontrol fungus, Bjerkandera adusta has an antagonistic effect on Didymella bryoniae, and also has potential value to develop into antiseptic agent in the field application.
Key words: Bjerkandera adusta    Didymella bryoniae    Antagonism    Condition optimization    Inhibitory rate
收稿日期: 2016-11-24 出版日期: 2017-05-25
ZTFLH:  Q815  
基金资助:

科技部农业科技成果转化资金资助项目（2013GB2F100396）
通讯作者: 李勇     E-mail: Liyongwf@swu.edu.cn
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引用本文:

张旭辉, 张红楠, 李勇, 汪文强. 抑制西瓜蔓枯病菌的生防真菌筛选、鉴定及发酵条件优化[J]. 中国生物工程杂志, 2017, 37(5): 76-86.

ZHANG Xu-hui, ZHANG Hong-nan, LI Yong, WANG Wen-qiang. Screening and Identification of Biocontrol Fungi against Didymella bryoniae and Optimization of Fermentation Conditions. China Biotechnology, 2017, 37(5): 76-86.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20170510    或    https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I5/76

[1] Keinath A P. From native plants in central Europe to cultivated crops worldwide:the emergence of Didymella bryoniae as a cucurbit pathogen. Hort Science, 2011, 46(4), 532-535.
[2] 李英. 瓜类蔓枯病菌的生物学特性和黄瓜抗病资源的筛选. 南京:南京农业大学, 园艺学院,2007. Li Y. Study on Biology Characteristics of Didymella bryoniae and Screening of Resistance Germplasm of Cucumber. Nanjing:Nanjing Agricultural University, College of Horticulture,2007.
[3] Kothera R I, Keinath A P, Dean R A, et al. AFLP analysis of a worldwide collection of Didymella bryoniae. Mycological Reserch. 2003, 107(2):297-304.
[4] 李雨, 王少秋, 谭蕊, 等. 西瓜蔓枯病有效药剂筛选及药效评价. 农药, 2016, 55(6):460-462. Li Y, Wang S Q, Tan R, et al. Evaluation of fungicides for control of gummy stem blight caused by Didymella bryoniae. Agrochemicals, 2016, 55(6):460-462.
[5] Keinath A P. Survival of Didymella bryoniae in buried watermelon vines in South Carolina. Plant Disease, 2002, 86(1):32-38.
[6] Sudisha J, Niranjana S R, Umesha S, et al. Transmission of seed-borne infection of muskmelon by Didymella bryoniae and effect of seed treatments on disease incidence and fruit yield. Biological Control, 2006, 37(2):196-205.
[7] Lou L N, Wang H Y, Qian C T, et al. Genetic mapping of gummy stem blight (Didymella bryoniae) resistance genes in Cucumis sativus-hystrix introgression lines. Euphytica, 2013, 192(3):359-369.
[8] Heydari A, Misaghi I J. The role of rhizosphere bacteria in herbicide-mediated increase in Rhizoctonia solani-induced cotton seedling damping-off. Plant and Soil, 2003, 257(2):391-396.
[9] Njoroge S M, Kabir Z, Martin F N, et al. Comparison of crop rotation for Verticillium wilt management and effect on Pythium species in conventional and organic strawberry production. Plant Disease, 2009, 93(5):519-527.
[10] Mancini V, Romanazzi G. Seed treatments to control seedborne fungal pathogens of vegetable crops. Pest Management Science, 2014, 70(6):860-868.
[11] Botelho, Gloria R, Mendon H, et al. Fluorescent Pseudomonads associated with the rhizosphere of crops-an overview. Brazilian Journal of Microbiology, 2006, 37(4):401-416.
[12] Abo-elyousr K A, Hashem M, Ali E H. Integrated control of cotton root disease by mixing fungal biocontrol agents and resistance inducers. Crop Protection, 2009, 28(4):295-301.
[13] Sudisha J, Niranjana S R, Umesha S, et al. Transmission of seed-borne infection of muskmelon by Didymella bryoniae and effect of seed treatments on disease incidence and fruit yield. Biological Control, 2006, 37(2), 196-205.
[14] Kaewkham T, Hynes R K, Siri B. The effect of accelerated seed ageing on cucumber germination following seed treatment with fungicides and microbial biocontrol agents for managing gummy stem blight by Didymella bryoniae. Biocontrol Science and Technology, 2016, 26(8):1048-1061.
[15] Seok K K, Seu Y B. Isolation of polyene antifungal antibiotics against gummy stem light caused by Didymella bryoniae. Korean Journal of Microbiology and Biotechnology, 2004, 32(3):238-242.
[16] Zhao J, Xue Q H, Shen G H, et al. Evaluation of Streptomyces spp. for biocontrol of gummy stem blight (Didymella bryoniae) and growth promotion of Cucumis melo L.. Biocontrol Science and Technology, 2012, 22(1):23-37.
[17] Martínez B, Pérez J, Infante D, et al. Antagonism of Trichoderma spp. isolates against Didymella bryoniae (Fuckel) Rehm. Artículo Original, 2013, 28(3):192-198.
[18] Jackson A M, Whipps J M, Llynch J M. Nutritional studies of four fungi with disease biocontrol potential. Enzyme and Microbial Technology, 1991, 13(91):456-461.
[19] Kredics L, Antal Z, Manczinger L, et al. Influence of environmental parameters on Trichoderma strains with biocontrol potential. Food Technology and Biotechnology, 2003, 41(7-8):37-42.
[20] 褚衍亮,王娜,余晗. 一株桑枝纤维降解菌株的鉴定及纤维素降解能力测定. 蚕业科学, 2015,41(1):127-133. Chu Y L, Wang N, Yu H. Identification of a Mulberry branch fiber degrading fungal strain and determination of its cellulose degrading activity. Science of Sericulture, 2015, 41(1):127-133.
[21] Mier N, Canete S, Klaebe A, et al. Insecticidal properties of mushroom and toadstool carpophores. Phytochemistry, 1996, 41(5):1293-1299.
[22] Stadler M, Sterner O. Production of bioactive secondary metabolites in the fruit bodies of macro fungi as a response to injury. Phytochemistry, 1998, 49(4):1013-1019.
[23] Domański S. Bjerkandera adusta on young Quercus rubra and Quercus robur injured by late spring frosts in the upper Silesia industrial district of Poland. European Journal of Forest Pathology, 1982, 12(6):406-413.
[24] 汪华, 喻大昭, 郭坚. 一株多孔烟管菌菌株高氏15号的鉴定及抑菌活性研究//陈万权,病虫害绿色防控与农产品质量安全--中国植物保护学会2015年学术年会论文集, 中国植物保护学会2015年学术年会, 长春, 2015, 北京:中国农业科学技术出版社, 2015:590. Wang H, Yu D Z, Guo J. Identification of Bjerkandera adusta 15 Strain and Study of Antimicrobial Activity//Chen W Q, Academic Annual Conference of the China Society for the Protection of Plants in 2015, Academic Annual Conference of the China Society for the Protection of Plants, Changchun, 2015, Beijing:China Agricultural Science and Technology Press, 2015:590.
[25] 方中达. 植病研究法. 第3版. 北京:中国农业出版社,2007:43-97. Fang Z D. Plant Pathology Research Methods. 3rd ed. Beijing:China Agriculture Press, 2007:43-97.
[26] 梁银, 张谷月, 王辰, 等. 一株拮抗放线菌的鉴定及其对黄瓜枯萎病的生防效应研究. 土壤学报, 2013, 50(4):810-817. Liang Y, Zhang G Y, Wang C, et al. Identification and biocontrol effect of a strain of actinomycete antagonistic to wilt disease of cucumber. Acta Pedologica Sinica, 2013, 50(4):810-817.
[27] 崔西苓. 耐盐碱木霉菌株的筛选鉴定、抗病促生及耐盐机理研究. 北京:中国农业科学院, 2014. Cui X L. Screening and Identification of Saline-Alkali Tolerant Trichoderma Strains and the Study on Disease Resistance, Growth Promotion and Salt-tolerant Mechanism. Beijing:Chinese Academy of Agricultural Sciences, 2014.
[28] Campanile G, Ruscellia, Luisi N. Antagonistic activity of endophytic fungi towards diplodia corticola assessed by in vitro and in planta tests. European Journal of Plant Pathology, 2007, 11(7):237-246.
[29] 魏景超. 真菌鉴定手册.上海:上海科学技术出版社,1979:367-378. Wei J C. Handbook of Fungal Identification. Shanghai:Shanghai Scientific and Technology Publishers, 1979:367-378.
[30] He Z, Wang J, Oh J, et al. Robust optimization for multiple responses using response surface methodology.Applied Stochastic Models in Business and Industry, 2010,26(2):157-171.
[31] 王新艳. 大丽轮枝菌致病相关突变体的筛选及致病基因VdCYP1功能初步研究. 北京:中国农业科学院, 2015. Wang Y X. Screening of Verticillium dahliae Pathogenicity-related Mutants and Functional Analysis of the Pathogenic Gene VdCYP1. Beijing:Chinese Academy of Agricultural Sciences, 2015.
[32] Smith V L, Wilcox W F, Harman G E. Potential for biological control of Phytophthora root and crown rots of apple by Trichoderma and Gliocladium spp.. Phytopathology, 1990, 80(9):880-885.
[33] 陈志敏, 顾钢, 陈顺辉, 等. 木霉菌对烟草疫霉的拮抗作用. 福建农林大学学报(自然科学版), 2009,38(3):234-237. Chen Z M, Gu G, Chen S H, et al. Antagonism of Trichoderma spp. to Phytophthora parasitica var. nicotianae. Journal of Fujian Agricultural and Forestry University (Natural Science Edition), 2009, 38(3):234-237.
[34] Eziashi E, Uma N, Adekunle A, et al. Biological control of Ceratocystis paradoxa causing black seed rot in oil palm sprouted seeds by Trichoderma species. Pakistan Journal of Biological Science, 2006, 9(10):1987-1990.
[35] Mala M P, Mukherjee B A, Horwitz C. Trichoderma-plant-pathogen interactions:advances in genetics of biological control. Indian Journal of Microbiology, 2012, 52(4):522-529.
[36] Ohberg H, Bang U. Biological control of clover rot on red clover by Coniothrium minitans under natural and controlled climatic conditions. Biocontrol Science and Technology, 2010, 20(1):25-36.
[37] 吴胜, 霍光华, 韩启灿, 等. 一株祼脚菇属菌株产抗菌活性物的基础液体培养碳、氮源等优化及其对柑橘青绿霉菌的作用. 中国生物工程杂志, 2016, 36(2):51-61. Wu S, Huo G H, Han Q C, et al. Optimization of carbon and nitrogen source of basic liquid culture of Gymnopus sp. producing anti-fungal activity substance and its effect on green and blue mold of citrus. China Biotechnology, 2016, 36(2):51-61.
[38] Nga N T, Giau N T, Long N T, et al. Rhizobacterially induced protection of watermelon against Didymella bryoniae. Journal of Applied Microbiology, 2010, 109(2):567-582.
[39] 祝新德, 冯镇泰, 许煜泉, 等. 荧光假单胞菌株M18防治甜瓜蔓枯病害. 上海交通大学学报, 2001,35(7):1062-1065. Zhu X D, Feng Z T, Xu Y Q, et al. Biocontroling of Melon Gummy Stem Blight (Mycosphaeralla melonis) using Pseudomonas fluorecens M18. Journal of Shanghai Jiao Tong University, 2001, 35(7):1062-1065.
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