Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/89344
標題: 研發微生物源植物保護製劑防治甜椒疫病
Development of Microbial Agents for Controlling Phytophthora Blight of Bell Pepper
作者: Hao-Lun Lin
林皓崙
關鍵字: 甜椒
疫病
生物防治
Paenibacillus polymyxa
Bacillus amyloliquefaciens
游走孢子破裂
黃豆粉-蔗糖培養液
大豆油
槐葉蘋
Phytophthora blight
bell pepper
biological control
Paenibaciullus polymyxa
Bacillus amyloliquefaciens
zoospore lysis
soybean meal-sucrose medium
soybean oil
salvinia
引用: (1) 江嘉容。2011。檬果炭疽病的生物防治菌鑑定與其防病之雛型醱酵營養配方設計。國立中興大學植物病理學系碩士學位論文。76 頁。 (2) 呂理燊、高清文。1981。Phytophthora capsici 引起的甜椒及辣椒之疫病。植物保護學會會刊 23: 59-66。 (3) 李阿嬌。2000。彩色甜椒品種適應性與產銷概況。桃園區農業專訊 34: 17-21。 (4) 杜怡真。2012。以鹽輔助均相液液微萃取技術結合 HPLC-UV 快速偵測微生物發酵培養液中的表面素。國立中興大學化學系碩士學位論文。88 頁。 (5) 高郁琇。2013。萵苣黑斑病之鑑定及其微生物防治試驗。國立中興大學植物病理學系碩士學位論文。84 頁。 (6) 郭孚燿。2000。甜椒栽培技術。台中區農業改良場編印, 彰化。61 頁。 (7) 楊秀珠、余思葳。2012。甜椒、辣椒之病蟲害發生與管理。行政院農委會農業藥物毒物試驗所編印,台中。39 頁。 (8) 鄭倩芸。2012。具有防治十字花科蔬菜根瘤病的技術開發。國立中興大學植物病理學系碩士學位論文。79 頁。 (9) Agrios, G. N. 2005. Plant Pathology. 5th ed. Elsevier Academic Press, San Diego, CA,USA. 922 pp. (10) Ahmed, A. S., Sánchez, C. P., and Candela, M. E. 2000. Evaluation of induction of systemic resistance in pepper plants (Capsicum annuum) to Phytophthora capsici using Trichoderma harzianum and its relation with capsidiol accumulation. Eur. J. Plant Pathol. 106: 817-824. (11) Akgül, D., and Mirik, M. 2008. Biocontrol of Phytophthora capsici on pepper plants by Bacillus megaterium strains. J. Plant Pathol. 90: 29-34. (12) Ann, P.-J., Tsai, J.-N., Wong, I.-T., Hsieh, T.-F., and Lin, C.-Y. 2009. A simple technique, concentration and application schedule for using neutralized phosphorous acid to control Phytophthora diseases. Plant Pathol. Bull. 18: 155-165. (13) Ash, C., Priest, F. G., and Collins, M. D. 1993. Molecular identification of rRNA group 3 bacilli using a PCR probe test. Antonie Van Leeuwenhoek 64: 253-260. (14) Bowers, J. H., Papavizas, G. C., and Johnston, S. A. 1990. Effect of soil mperature and soil-water matric potential on the survival of Phytophthora capsici in natural soil. Plant Dis. 74: 771-777. (15) Carrillo, C., Teruel, J. A., Aranda, F. J., and Ortiz, A. 2003. Molecular mechanism of membrane permeabilization by the peptide antibiotic surfactin. Biochim. Biophys. Acta 1611: 91-97. (16) Chae, D., De Jin, R., Hwangbo, H., Kim, Y., Kim, Y., Park, R., Krishnan, H., and Kim, K. 2006. Control of late blight (Phytophthora capsici ) in pepper plant with a compost containing multitude of chitinase-producing bacteria. BioControl 51: 339-351. (17) Chen, J.-R., Wang, T.-C., and Chung, W.-H. 2010. Evaluation on metalaxyl sensitivity of Phytophthora capsici isolates collected from pepper in Taiwan. Plant Pathol. Bull. 19: 271-279. (18) Chen, J.-T., and Huang, J.-W. 2010. Antimicrobial activity of edible mushroom culture filtrates on plant pathogens. Plant Pathol. Bull. 19: 261-270. (19) Chun, J., and Bae, K. 2000. Phylogenetic analysis of Bacillus subtilis and related taxa based on partial gyrA gene sequences. Antonie Van Leeuwenhoek 78: 123- 127. (20) Daffonchio, D., Borin, S., Consolandi, A., Mora, D., Manachini, P. L., and Sorlini, C. 1998. 16S-23S rRNA internal transcribed spacers as molecular markers for the species of the 16S rRNA group I of the genus Bacillus. FEMS Microbiol. Lett. 163: 229-236. (21) Erwin, D. C., and Ribeiro, O. K. 1996. Phytophthora Diseases Worldwide. American Phytopathological Society, St. Paul, MN, USA. 562 pp. (22) Ezziyyani, M., Requena, M. E., Egea-Gilabert, C., and Candela, M. E. 2007. Biological control of Phytophthora root rot of pepper using Trichoderma harzianum and Streptomyces rochei in combination. J. Phytopathol. 155: 342- 349. (23) Gürtler, V., and Stanisich, V. A. 1996. New approaches to typing and identification of bacteria using the 16S-23S rDNA spacer region. Microbiology 142: 3-16. (24) Gevens, A. J., Donahoo, R. S., Lamour, K. H., and Hausbeck, M. K. 2007. Characterization of Phytophthora capsici from Michigan surface irrigation water. Phytopathology 97: 421-428. (25) Granke, L. L., and Hausbeck, M. K. 2010. Effects of temperature, concentration, age, and algaecides on Phytophthora capsici zoospore infectivity. Plant Dis. 94: 54-60. (26) Granke, L. L., Quesada-Ocampo, L., Lamour, K., and Hausbeck, M. K. 2012. Advances in research on Phytophthora capsici on vegetable crops in the United States. Plant Dis. 96: 1588-1600. (27) Hausbeck, M. K., and Lamour, K. H. 2004. Phytophthora capsici on vegetable crops: research progress and management challenges. Plant Dis. 88: 1292-1303. (28) Hiradate, S., Yoshida, S., Sugie, H., Yada, H., and Fujii, Y. 2002. Mulberry anthracnose antagonists (iturins) produced by Bacillus amyloliquefaciens RC- 2. Phytochemistry 61: 693-698. (29) Jiang, Z.-Q., Guo, Y.-H., Li, S.-M., Qi, H.-Y., and Guo, J.-H. 2006. Evaluation of biocontrol efficiency of different Bacillus preparations and field application methods against Phytophthora blight of bell pepper. Biol. Control 36: 216-223. (30) Joo, G. J. 2005. Production of an anti-fungal substance for biological control of Phytophthora capsici causing phytophthora blight in red-peppers by Streptomyces halstedii. Biotechnol. Lett. 27: 201-205. (31) Jung, W.-J., Jin, Y.-L., Kim, Y.-C., Kim, K.-Y., Park, R.-D., and Kim, T.-H. 2004. Inoculation of Paenibacillus illinoisensis alleviates root mortality, activates of lignification-related enzymes, and induction of the isozymes in pepper plants infected by Phytophthora capsici. Biol. Control 30: 645-652. (32) Jung, W. J., Jin, Y. L., Park, R. D., Kim, K. Y., Lim, K. T., and Kim, T. H. 2006. Treatment of Paenibacillus illinoisensis suppresses the activities of antioxidative enzymes in pepper roots caused by Phytophthora capsici infection. World J. Microbiol. Biotechnol. 22: 901-907. (33) Kim, B. S., Lee, J. Y., and Hwang, B. K. 2000. In vivo control and in vitro antifungal activity of rhamnolipid B, a glycolipid antibiotic, against Phytophthora capsici and Colletotrichum orbiculare. Pest Manag. Sci. 56: 1029-1035. (34) Kim, H. S., Sang, M. K., Jeun, Y.-C., Hwang, B. K., and Kim, K. D. 2008. Sequential selection and efficacy of antagonistic rhizobacteria for controlling Phytophthora blight of pepper. Crop Prot. 27: 436-443. (35) Kim, S. G., Khan, Z., Jeon, Y. H., and Kim, Y. H. 2009. Inhibitory effect of Paenibacillus polymyxa GBR-462 on Phytophthora capsici causing Phytophthora blight in chili pepper. J. Phytopathol. 157: 329-337. (36) Kim, S. G., Jang, Y., Kim, H. Y., Koh, Y. J., and Kim, Y. H. 2010. Comparison of microbial fungicides in antagonistic activities related to the biological control of Phytophthora blight in chili pepper caused by Phytophthora capsici. Plant Pathol. J. 26: 340-345. (37) Kostman, J. R., Edlind, T. D., LiPuma, J. J., and Stull, T. L. 1992. Molecular epidemiology of Pseudomonas cepacia determined by polymerase chain reaction ribotyping. J. Clin. Microbiol. 30: 2084-2087. (38) Lamour, K. 2009. Phytophthora capsici: sex, selection, and the wealth of variation. Pages 165-177 in: Oomycete Genetics and Genomics: Diversity, Interactions and Research Tools, K. Lamour; and S. Kamoun, eds. John Wiley & Sons, Inc., New Jersey, USA. (39) Lamour, K. H., and Hausbeck, M. K. 2000. Mefenoxam insensitivity and the sexual stage of Phytophthora capsici in Michigan cucurbit fields. Phytopathology 90: 396-400. (40) Lamour, K. H., Stam, R., Jupe, J., and Huitema, E. 2012. The oomycete broad‐host‐ range pathogen Phytophthora capsici. Mol. Plant Pathol. 13: 329-337. (41) Lee, K., Kamala-Kannan, S., Sub, H., Seong, C., and Lee, G. 2008. Biological control of Phytophthora blight in red pepper (Capsicum annuum L.) using Bacillus subtilis. World J. Microbiol. Biotechnol. 24: 1139-1145. (42) Leonian, L. H. 1922. Stem and fruit blight of peppers caused by Phytophthora capsici sp. nov. Phytopathology 12: 401-408. (43) Lim, J. H., and Kim, S. D. 2010. Biocontrol of Phytophthora blight of red pepper caused by Phytophthora capsici using Bacillus subtilis AH18 and B. licheniformis K11 formulations. J. Korean Soc. Appl. Biol. Chem. 53: 766- 773. (44) Lue, Y. S., Deng, W. L., Wu, Y. F., Cheng, A. S., Hsu, S. T., and Tzeng, K. C. 2010. Characterization of Xanthomonas associated with bacterial spot of tomato and pepper in Taiwan. Plant Pathol. Bull. 19: 181-190. (45) Mao, W., Lewis, J. A., Lumsden, R. D., and Hebbar, K. P. 1998. Biocontrol of selected soilborne diseases of tomato and pepper plants. Crop Prot. 17: 535-542. (46) Mei, X. L., Zhao, Q. Y., Tan, S. Y., Xu, Y. C., Shen, B., and Shen, Q. R. 2010. Screening, identification, and biocontrol effect of antagonistic bacteria against Phytophthora capsici (in Chinese). Chin. J. Appl. Ecol. 21: 2652-2658. (47) Mercier, J., and Manker, D. C. 2005. Biocontrol of soil-borne diseases and plant growth enhancement in greenhouse soilless mix by the volatile-producing fungus Muscodor albus. Crop Prot. 24: 355-362. (48) Moyne, A. L., Shelby, R., Cleveland, T. E., and Tuzun, S. 2001. Bacillomycin D: an iturin with antifungal activity against Aspergillus flavus. J. Appl. Microbiol. 90: 622-629. (49) Nguyen, X. H., Naing, K. W., Lee, Y. S., Tindwa, H., Lee, G. H., Jeong, B. K., Ro, H.- M., Kim, S. J., Jung, W. J., and Kim, K. Y. 2012. Biocontrol potential of Streptomyces griseus H7602 against root rot disease (Phytophthora capsici) in pepper. Plant Pathol. J. 28: 282-289. (50) Oh, B.-T., Hur, H., Lee, K.-J., Shanthi, K., Soh, B.-Y., Lee, W.-J., Myung, H., and Kamala-Kannan, S. 2011. Suppression of Phytophthora blight on pepper (Capsicum annuum L.) by bacilli isolated from brackish environment. Biocontrol Sci. Technol. 21: 1297-1311. (51) Ongena, M., Jourdan, E., Adam, A., Paquot, M., Brans, A., Joris, B., Arpigny, J.-L., and Thonart, P. 2007. Surfactin and fengycin lipopeptides of Bacillus subtilis as elicitors of induced systemic resistance in plants. Environ. Microbiol. 9: 1084-1090. (52) Ongena, M., and Jacques, P. 2008. Bacillus lipopeptides: versatile weapons for plant disease biocontrol. Trends Microbiol. 16: 115-125. (53) Ozyilmaz, U., and Benlioglu, K. 2013. Enhanced biological control of hytophthora blight of pepper by biosurfactant-producing Pseudomonas. Plant Pathol. J. 29: 418-426. (54) Parra, G., and Ristaino, J. B. 2001. Resistance to mefenoxam and metalaxyl among field isolates of Phytophthora capsici causing Phytophthora blight of bell pepper. Plant Dis. 85: 1069-1075. (55) Peypoux, F., Bonmatin, J. M., and Wallach, J. 1999. Recent trends in the biochemistry of surfactin. Appl. Microbiol. Biotechnol. 51: 553-563. (56) Pires, M. N., and Seldin, L. 1997. Evaluation of Biolog system for identification of strains of Paenibacillus azotofixans. Antonie Van Leeuwenhoek 71: 195-200. (57) Priest, F. G., Goodfellow, M., Shute, L. A., and Berkeley, R. C. W. 1987. Bacillus amyloliquefaciens sp. nov., nom. rev. Int. J. Syst. Bacteriol. 37: 69-71. (58) Raaijmakers, J. M., De Bruijn, I., Nybroe, O., and Ongena, M. 2010. Natural functions of lipopeptides from Bacillus and Pseudomonas: more than surfactants and antibiotics. FEMS Microbiol. Rev. 34: 1037-1062. (59) Robles-Yerena, L., Rodríguez-Villarreal, R. A., Ortega-Amaro, M. A., Fraire- Velázquez, S., Simpson, J., Rodríguez-Guerra, R., and Jiménez-Bremont, J. F. 2010. Characterization of a new fungal antagonist of Phytophthora capsici. Scient. Horticult. 125: 248-255. (60) Sang, M. K., Chun, S.-C., and Kim, K. D. 2008. Biological control of Phytophthora blight of pepper by antagonistic rhizobacteria selected from a sequential screening procedure. Biol. Control 46: 424-433. (61) Sang, M. K., and Kim, K. D. 2012. The volatile-producing Flavobacterium johnsoniae strain GSE09 shows biocontrol activity against Phytophthora capsici in pepper. J. Appl. Microbiol. 113: 383-398. (62) Sanogo, S., and Ji, P. 2012. Integrated management of Phytophthora capsici on solanaceous and cucurbitaceous crops: current status, gaps in knowledge and research needs. Can. J. Plant Pathol. 34: 479-492. (63) Segarra, G., Avilés, M., Casanova, E., Borrero, C., and Trillas, I. 2013. Effectiveness of biological control of Phytophthora capsici in pepper by Trichoderma asperellum strain T34. Phytopathologia Mediterranea 52: 77-83. (64) Sid Ahmed, A., Pérez-Sánchez, C., Egea, C., and Candela, M. E. 1999. Evaluation of Trichoderma harzianum for controlling root rot caused by Phytophthora capsici in pepper plants. Plant Pathol. 48: 58-65. (65) Sid Ahmed, A., Ezziyyani, M., Pérez Sánchez, C., and Candela, M. E. 2003. Effect of chitin on biological control activity of Bacillus spp. and Trichoderma harzianum against root rot disease in pepper (Capsicum annuum) plants. Eur. J. Plant Pathol. 109: 633-637. (66) Stanghellini, M. E., and Tomlinson, J. A. 1987. Inhibitory and lytic effects of a nonionic surfactant on various asexual stages in the life-cycle of Pythium and Phytophthora species. Phytopathology 77: 112-114. (67) Stanghellini, M. E., and Miller, R. M. 1997. Biosurfactants: Their identity and potential efficacy in the biological control of zoosporic plant pathogens. Plant Dis. 81: 4-12. (68) Sunwoo, J. Y., Lee, Y. K., and Hwang, B. K. 1996. Induced resistance against Phytophthora capsici in pepper plants in response to DL-β-amino-n-butyric acid. Eur. J. Plant Pathol. 102: 663-670. (69) Vanittanakom, N., Loeffler, W., Koch, U., and Jung, G. 1986. Fengycin-a novel antifungal lipopeptide antibiotic produced by Bacillus subtilis F-29-3. J. Antibiotics 39: 888-901. (70) Wang, L.-T., Lee, F.-L., Tai, C.-J., and Kasai, H. 2007. Comparison of gyrB gene sequences, 16S rRNA gene sequences and DNA–DNA hybridization in the Bacillus subtilis group. Int. J. Syst. Evol. Microbiol. 57: 1846-1850. (71) Wang, Q., Ma, Y., Yang, H., and Chang, Z. 2014. Effect of biofumigation and chemical fumigation on soil microbial community structure and control of pepper Phytophthora blight. World J. Microbiol. Biotechnol. 30: 507-518. (72) Weisburg, W. G., Barns, S. M., Pelletier, D. A., and Lane, D. J. 1991. 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173: 697-703. (73) Whipps, J. M. 1987. Effect of media on growth and interactions between a range of soil-borne glasshouse pathogens and antagonistic fungi. New Phytol. 107: 127-142. (74) Xu, D., and Côté, J.-C. 2003. Phylogenetic relationships between Bacillus species and related genera inferred from comparison of 3' end 16S rDNA and 5' end 16S– 23S ITS nucleotide sequences. Int. J. Syst. Evol. Microbiol. 53: 695-704. (75) Yu, G. Y., Sinclair, J. B., Hartman, G. L., and Bertagnolli, B. L. 2002. Production of iturin A by Bacillus amyloliquefaciens suppressing Rhizoctonia solani. Soil Biol. Biochem. 34: 955-963.
摘要: 由甜椒疫病菌 Phytophthora capsici Leonian 引起的甜椒疫病是栽培甜椒過程的重要病害之一,尤其在高溫多濕的氣候條件下,常造成甜椒生產的經濟損失。當前農民於田間大量施用農業化學藥劑,致有環境汙染與食品安全等的疑慮,因此開發替代的防治方法確有其必要性。本研究主要目的在於建立一套評估甜椒疫病之生物防治菌的模式,祈有助於研發甜椒疫病的微生物源植物保護製劑。首先,從槐葉蘋(Salvinia molesta D.Mitch.)分離 85 株細菌菌株置於營養瓊脂培養基平板上,分別與甜椒疫病菌進行對峙培養試驗,結果發現 SM64 與SM69 兩菌株具有最佳抑制疫病菌菌絲生長的功效,其抑制率分別為 71.8%與70.1%。進一步利用 Biolog、16S rRNA 及 16S-23S 核糖體核酸內轉錄區間(16S-23S rRNA ITS)序列比對等分析,將 SM64 與 SM69 菌株分別鑑定為Paenibacillus polymyxa (Prazmowski) Ash et al.及 Bacillus amyloliquefaciens (ex Fukumoto) Priest et al.。利用牛肉煎汁培養液(NB)培養 SM64 與 SM69 菌株四天後的醱酵液,分別處理甜椒疫病菌的游走孢子懸浮液,結果發現兩菌株均可破壞甜椒疫病菌之游走孢子,使其濃度降低 59-98%左右。此外,以培養在含有2% (w/v)蔗糖之 NB 及 3% (w/v)豆漿培養液之 SM64 與 SM69 的培養菌液,分別處理甜椒切離葉後,結果兩菌株皆可百分之百抑制疫病菌的感染。在載玻片評選 SM64 與 SM69 菌株抑制游走孢子發芽的最佳醱酵培養基,發現兩菌株於黃豆粉-糖蜜培養液(SM)培養四日後,僅 SM69 菌株可顯著降低游走孢子之發芽百分率,SM64 之 SM 醱酵稀釋液則無法抑制游走孢子發芽,惟 SM69 之一百倍醱酵稀釋液仍可抑制 38%之游走孢子發芽。利用高壓液相層析儀(HPLC)分析SM69 菌株之代謝產物,發現 SM69 菌株具有產生 iturin A 及 surfactin 類之抑菌物質的能力。在黃豆粉-蔗糖培養液(SSu)添加 2.5% (v/v)大豆油後,其 200 倍醱酵稀釋液可顯著的使甜椒罹病等級自對照組之 4 級降至 1 級。在溫室中,將SM69 菌株在 SSu 與在 SSu-大豆油之 200 倍醱酵稀釋液分別施用於甜椒植株,結果發現於甜椒植株接種疫病菌後一日再施用 SSu-大豆油醱酵液具有最佳防治病害的效果,其可使植株罹病等級由對照組 3 級罹病度降至 1.6 級。綜合甜椒切離葉與甜椒幼苗之防治試驗結果,顯示 B. amyloliquefaciens SM69 菌株具有潛力研發成為防治甜椒疫病之微生物源植物保護製劑。
Phytophthora blight of bell pepper caused by Phytophthora capsici Leonian is one of the most important diseases occurred in pepper cultivation. It causes dramatical economic loss of bell pepper production in warm and humid conditions. The most applied strategy for disease management is frequent application of chemical pesticides which results in environmental pollution and food safety issues. The purpose of this study was to develop microbial agents for controlling Phytophthora blight of bell pepper. Out of the 85 isolates originated from salvinia (Salvinia molesta D.Mitch.) plants, two isolates, namely SM64 and SM69, showed strong inhibitory effect on the mycelial growth of P. capsici Pc28089 on nutrient agar plate, with the inhibition of 71.8% and 70.1%, respectively. SM64 and SM69 were respectively identified as Paenibacillus polymyxa (Prazmowski) Ash et al. and Bacillus amyloliquefaciens (ex Fukumoto) Priest et al. according to the results of biolog assay, and the sequences of partial 16S rRNA , and 16S-23S rRNA internal transcribed spacer. When the pathogen was treated with 4-day-old culture liquid of SM64 or SM69 in nutrient broth (NB), the zoospores were destroyed and their concentrations were markedly reduced by 59-98%. The culture liquid of SM64 or SM69 in NB amended with 2% (w/v) sucrose or 3% (w/v) soybean meal medium (SMM) could completely suppress disease development on detached pepper leaves. Screening of the best medium for culturing SM64 and SM69 was conducted by treating zoospores of P. capsici with 4-day-old culture liquid on sterile slides. The culture liquid of SM69 in soybean meal-molasses medium (SM) significantly reduced zoospore germination of the pathogen, but SM64 in SM culture liquid did not inhibit zoospore germination. However, the SM69 culture liquid at 100-fold dilution could still reduce zoospore germination by 38%. Analyzing antifungal substances from fermentation filtrate of SM69 by high pressure liquid chromatography (HPLC), iturin A-like and surfactin-like compounds could be detected. The 200-fold diluted culture liquid of SM69 in soybean meal-sucrose medium (SSu) amended with 2.5% (v/v) soybean oil significantly reduced the disease index from 4 to 1 compared to the control. After spraying 200-fold diluted culture liquid of SM69 in SSu medium or SSu amended with 2.5% (v/v) soybean oil on bell pepper seedlings at greenhouse condition, treatment of culture liquid of SM69 in SSu medium amended with soybean oil, sprayed at one day after-inoculation, showed the best disease suppression effect among all treatments, resulting in reduction of disease index from 3 of the control to 1.6. Based on results from assays on detached pepper leaves or pepper seedlings, it suggested that the bacterium, B. amyloliquefaciens isolate SM69, has great potential to be developed into microbial agent for controlling Phytophthora blight of bell pepper.
URI: http://hdl.handle.net/11455/89344
文章公開時間: 2018-07-16
Appears in Collections:植物病理學系

文件中的檔案:

取得全文請前往華藝線上圖書館



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.