臺灣博碩士論文加值系統

稻熱病是由稻熱病菌 (Magnaporthe grisea)感染水稻所致，稻熱病菌是屬於子囊菌門糞殼菌綱的植物性病原真菌，能對水稻造成嚴重感染，並造成經濟上的損失，目前已知的稻熱病廣泛的分布於熱帶以及溫帶地區。現今控制稻熱病的方法是施用化學農藥，鑒於稻熱病嚴重的危害性，本研究嘗試發展一套生物防治的方法。本實驗室從不同環境中篩選出許多細菌，利用對峙試驗去測試這些細菌對於稻熱病菌的拮抗性。其中，由水稻栽培環境得到的一隻伯克氏菌 (Burkholderia sp. B2 strain，簡稱B2菌)，其對於稻熱病菌有顯著的抑制效果。為了更進一步地瞭解B2菌抑制稻熱病菌的方式，我們從B2菌所產生的抗菌物質方面開始著手研究。從兩個面向來看，抗菌物質可分為氣態抗菌物質以及液態抗菌物質。氣態抗菌物質的發現是經由B2菌固態培養後所產氣體對稻熱病菌有抑制生長的效果，因此，藉由GC/MS/MS分析B2菌所產的揮發性有機化合物(Volatile Organic Compounds; VOCs)，並利用GC/MS/MS圖譜推測出可能對稻熱病菌具有毒性的揮發性有機化合物，針對這些揮發性有機化合物與稻熱病進行薰蒸試驗。
另一方面，由B2菌分泌出來的抗菌物質即歸類於液態抗菌物質中。在對峙試驗中，藉由掃描式電子顯微鏡可以觀察到B2菌分泌出的抗菌物質會造成稻熱病菌菌絲死亡。B2菌的培養基上清液對於稻熱病菌有生長抑制的效果。為了確認液態抗菌物質的身分，利用分子篩的方式將B2菌培養基上清液分為不同載留分子量(Molecular weight cut off; MWCO)的分層，再將不同分層的培養基上清液與稻熱病菌進行對峙試驗。本實驗使用1 kDa、3kDa、10kDa此三種大小的分子篩膜，找出培養基上清液中抗菌物質的分子量範圍界於3kDa與10kDa之間，最終目的希望能夠確認抗菌物質的身分。

Rice blast is caused by the infection of Magnaporthe grisea, which belongs to Ascomycota of Sordariomycetes. Rice blast could lead to immensely economic loss in tropical and temperate regions. Application of fungicide is the major measure to reduce the incidence of rice blast nowadays. Therefore, developing a biological method is desired in views of the adverse effects of rice blast and using fungicide. In this study, bacteria were isolated from different environments and sources and tested for their activity against the growth of M. grisea by confrontation assay. A Burkholderia strain (abbreviated as B2 strain), which was isolated from rice cultivated environment, showed significant inhibition against M. grisea. In order to investigate the inhibitory mechanism of B2 strain, we set out to search the antifungal substances produced by this bacterial strain. Toxic gaseous substances were released to inhibit the growth of M. grisea when B2 strain was cultivated on potato dextrose agar. Therefore, GC/MS/MS was used to analyze the volatile organic compounds (VOCs) produced by B2 strain. Fumigation test was held to verify a putative toxic VOC for its antagonistic activity. In the confrontation assay, the antifungal substance secreted by the B2 strain caused the dead hyphae of M. grisea , which could be observed by scanning electron microscopy (SEM). The antifungal substances secreted in the medium by B2 strain were also tested. The supernatant of the culture of B2 strain had an inhibition effect on the growth of M. grisea. To identify the antifungal substance, molecular sieves with different molecular weight cut-off (MWCO) value such as 1 kDa, 3 kDa, 10 kDa will be used to separate the supernatant into different fractions, and each of the fractions will be tested in confrontation assay so as the approximate molecular mass of the antifungal substance was determined in a range from 3 kDa to 10 kDa. Finally, figuring out the identity of the antifungal substance is the wish of this experiment.

摘要 i
Abstract ii
目錄 iii
表目錄 iv
圖目錄 v
第 1 章 前言 1
第 2 章 材料與方法 4
一、 供試菌株 4
二、 供試病原真菌 4
三、 培養基 4
(一)液態培養基 4
(二)固態培養基 5
四、 對峙試驗 (Confrontation assays) 6
紙錠 6
受試菌株培養 6
受試病原真菌 6
五、 利用熱場發射掃描式電子顯微鏡觀察受抑制菌絲之型態 6
六、 不同培養天數Burkholderia sp. B2對真菌的拮抗 6
七、 Burkholderia sp. B2 培養基裡有效抗菌成分其主要分子量大小範圍 7
八、 Burkholderia sp. B2 於固態培養狀態下其分泌的可擴散抗菌物質是否可通過透析膜 7
九、 細菌產氰酸(hydrogen cyanide，HCN)測試 7
十、 探討不同固態配養基影響細菌所產揮發性有機化合物與其對真菌的拮抗能力 8
十一、 觀察受到揮發性有機化合物所抑制的菌絲其外貌型態 8
十二、 細菌所產發性有機化合物成分分析 8
十三、 測試VOC dameter對真菌的抑制能力 9
第 3 章 結果 10
一、 篩選拮抗植物病原真菌的環境菌 10
二、 Burkholderia sp. B2分泌的抗真菌物質 10
三、 Burkholderia B2與Pseudomonas B6所產的揮發性有機物其拮抗真菌之效 11
第 4 章 討論 13
第 5 章 參考文獻 15
第 6 章 圖與表 20

Alias Bin Abdullah, S.I.a.K.A. 2006. Estimate of rice consumption in asian countries and the world towards 2050.
Altindag, M., Sahin, M., Esitken, A., Ercisli, S., Guleryuz, M., Donmez, M.F., and Sahin, F. 2006. Biological control of brown rot (Moniliana laxa Ehr.) on apricot (Prunus armeniaca L. cv. Hacıhaliloğlu) by Bacillus, Burkholdria, and Pseudomonas application under in vitro and in vivo conditions. Biological Control 38:369-372.
Ashkani, S., Yusop, M.R., Shabanimofrad, M., Harun, A.R., Sahebi, M., and Latif, M.A. 2015. Genetic analysis of resistance to rice blast: a study on the inheritance of resistance to the blast disease pathogen in an F3 population of rice. Journal of Phytopathology 163:300-309.
Avalos, M., van Wezel, G.P., Raaijmakers, J.M., and Garbeva, P. 2018. Healthy scents: microbial volatiles as new frontier in antibiotic research? Current Opinion in Microbiology 45:84-91.
Bradbury, M.G., Egan, S.V., and Bradbury, J.H. 1999. Picrate paper kits for determination of total cyanogens in cassava roots and all forms of cyanogens in cassava products. Journal of the Science of Food and Agriculture 79:593-601.
Bussaban, B., Lumyong, S., Lumyong, P., Seelanan, T., Park, D., McKenzie, E., and Hyde, K. 2005. Molecular and morphological characterization of Pyricularia and allied genera.
Chaiharn, M., Chunhaleuchanon, S., and Lumyong, S. 2009. Screening siderophore producing bacteria as potential biological control agent for fungal rice pathogens in Thailand. World Journal of Microbiology and Biotechnology 25:1919-1928.
Cooper, M., Tavankar, G.R., and Williams, H.D. 2003. Regulation of expression of the cyanide-insensitive terminal oxidase in Pseudomonas aeruginosa Microbiology 149:1275-1284.
Couch, B.C., and Kohn, L.M. 2002. A multilocus gene genealogy concordant with host preference indicates segregation of a new species, Magnaporthe oryzae, from M. grisea. Mycologia 94:683-693.
Dean, R., Van Kan, J.A.L., Pretorius, Z.A., Hammond-Kosack, K.E., Di Pietro, A., Spanu, P.D., Rudd, J.J., Dickman, M., Kahmann, R., Ellis, J., and Foster, G.D. 2012. The Top 10 fungal pathogens in molecular plant pathology. Molecular Plant Pathology 13:414-430.
Dos Passos, J.F.M., da Costa, P.B., Costa, M.D., Zaffari, G.R., Nava, G., Boneti, J.I., de Oliveira, A.M.R., and Passaglia, L.M.P. 2014. Cultivable bacteria isolated from apple trees cultivated under different crop systems: Diversity and antagonistic activity against Colletotrichum gloeosporioides. Genet Mol Biol 37:560-572.
Dufresne, M., and Osbourn, A.E. 2001. Definition of tissue-specific and general requirements for plant infection in a phytopathogenic fungus. Molecular Plant-Microbe Interactions 14:300-307.
Effmert, U., Kalderás, J., Warnke, R., and Piechulla, B. 2012. Volatile mediated interactions between bacteria and fungi in the soil. Journal of Chemical Ecology 38:665-703.
Elshafie, H.S., Camele, I., Racioppi, R., Scrano, L., Iacobellis, N.S., and Bufo, S.A. 2012. In vitro antifungal activity of Burkholderia gladioli pv. agaricicola against some phytopathogenic fungi. Int J Mol Sci 13:16291-16302.
Garbeva, P., Hordijk, C., Gerards, S., and de Boer, W. 2014a. Volatiles produced by the mycophagous soil bacterium Collimonas. FEMS Microbiology Ecology 87:639-649.
Garbeva, P., Hordijk, C., Gerards, S., and de Boer, W. 2014b. Volatile-mediated interactions between phylogenetically different soil bacteria. Front Microbiol 5:289-289.
Giorgio, A., De Stradis, A., Lo Cantore, P., and Iacobellis, N.S. 2015. Biocide effects of volatile organic compounds produced by potential biocontrol rhizobacteria on Sclerotinia sclerotiorum. Front Microbiol 6:1056.
Groenhagen, U., Baumgartner, R., Bailly, A., Gardiner, A., Eberl, L., Schulz, S., and Weisskopf, L. 2013. Production of bioactive volatiles by different Burkholderia ambifaria strains. Journal of Chemical Ecology 39:892-906.
Hirooka, T., and Ishii, H. 2013. Chemical control of plant diseases. Journal of General Plant Pathology 79:390-401.
Hosseyni-Moghaddam, M., and Soltani, J. 2013. An investigation on the effects of photoperiod, aging and culture media onvegetative growth and sporulation of rice blast pathogen Pyricularia oryzae. Progress in Biological Sciences 3:135-143.
Huang, C.-J., Tsay, J.-F., Chang, S.-Y., Yang, H.-P., Wu, W.-S., and Chen, C.-Y. 2012. Dimethyl disulfide is an induced systemic resistance elicitor produced by Bacillus cereus C1L. Pest Management Science 68:1306-1310.
Hunziker, L., Bönisch, D., Groenhagen, U., Bailly, A., Schulz, S., and Weisskopf, L. 2015. Pseudomonas strains naturally associated with potato plants produce volatiles with high potential for inhibition of phytophthora infestans. Applied and Environmental Microbiology 81:821.
Innovations., M.B. (2018). Agriculture products (Marrone Bio Innovations: Marrone Bio Innovations).
Kanchiswamy, C.N., Malnoy, M., and Maffei, M.E. 2015. Bioprospecting bacterial and fungal volatiles for sustainable agriculture. Trends in Plant Science 20:206-211.
Kato, H. 2001. Rice blast disease.
Khush, G.S. 2005. What it will take to feed 5.0 billion rice consumers in 2030. Plant Molecular Biology 59:1-6.
Komárek, M., Čadková, E., Chrastný, V., Bordas, F., and Bollinger, J.-C. 2010. Contamination of vineyard soils with fungicides: A review of environmental and toxicological aspects. Environment International 36:138-151.
Kyndt, T., Fernandez, D., and Gheysen, G. 2014. Plant-parasitic nematode infections in rice: Molecular and Cellular Insights. Annual Review of Phytopathology 52:135-153.
Lemfack, M.C., Nickel, J., Dunkel, M., Preissner, R., and Piechulla, B. 2014. mVOC: a database of microbial volatiles. Nucleic Acids Res 42:D744-D748.
Lemfack, M.C., Gohlke, B.-O., Toguem, S.M.T., Preissner, S., Piechulla, B., and Preissner, R. 2018. mVOC 2.0: a database of microbial volatiles. Nucleic Acids Res 46:D1261-D1265.
Li, X., Li, Y., Wang, R., Wang, Q., and Lu, L. 2019. Toxoflavin produced by Burkholderia gladioli from Lycoris aurea Is a new broad-spectrum fungicide. Applied and Environmental Microbiology 85:e00106-00119.
Lo Cantore, P., Giorgio, A., and Iacobellis, N.S. 2015. Bioactivity of volatile organic compounds produced by Pseudomonas tolaasii. Front Microbiol 6:1082-1082.
Luque-Almagro, V.M., Huertas, M.-J., Martínez-Luque, M., Moreno-Vivián, C., Roldán, M.D., García-Gil, L.J., Castillo, F., and Blasco, R. 2005. Bacterial degradation of cyanide and its metal complexes under alkaline conditions. Applied and environmental microbiology 71:940-947.
Maffei, M.E. 2010. Sites of synthesis, biochemistry and functional role of plant volatiles. South African Journal of Botany 76:612-631.
Maffei, M.E., Gertsch, J., and Appendino, G. 2011. Plant volatiles: Production, function and pharmacology. Natural Product Reports 28:1359-1380.
Moebius, N., Ross, C., Scherlach, K., Rohm, B., Roth, M., and Hertweck, C. 2012. Biosynthesis of the respiratory toxin bongkrekic acid in the pathogenic bacterium Burkholderia gladioli. Chemistry & Biology 19:1164-1174.
Morath, S.U., Hung, R., and Bennett, J.W. 2012. Fungal volatile organic compounds: A review with emphasis on their biotechnological potential. Fungal Biology Reviews 26:73-83.
Moshi, A.P., and Matoju, I. 2017. The status of research on and application of biopesticides in Tanzania. Review. Crop Protection 92:16-28.
Muthayya, S., Sugimoto, J.D., Montgomery, S., and Maberly, G.F. 2014. An overview of global rice production, supply, trade, and consumption. Annals of the New York Academy of Sciences 1324:7-14.
Ortega-Campayo, V., Pérez-Martín, M., and Sesma, A. 2018. Magnaporthe and its relatives. eLS:1-11.
Pimentel, D., and Burgess, M. 2014. Environmental and economic benefits of reducing pesticide use. Pages 127-139 in: Integrated Pest Management: Pesticide Problems, Vol.3, D. Pimentel and R. Peshin, eds. Springer Netherlands, Dordrecht.
Pooja, K., and Katoch, A. 2014. Past, present and future of rice blast management. Plant Science Today; Vol 1 No 3 (2014)DO - 10.14719/pst.2014.1.3.24.
Quan, C.S., Zheng, W., Liu, Q., Ohta, Y., and Fan, S.D. 2006. Isolation and characterization of a novel Burkholderia cepacia with strong antifungal activity against Rhizoctonia solani. Applied Microbiology and Biotechnology 72:1276-1284.
Recep, K., Fikrettin, S., Erkol, D., and Cafer, E. 2009. Biological control of the potato dry rot caused by Fusarium species using PGPR strains. Biological Control 50:194-198.
Sesma, A., and Osbourn, A.E. 2004. The rice leaf blast pathogen undergoes developmental processes typical of root-infecting fungi. Nature 431:582-586.
Skamnioti, P., and Gurr, S.J. 2009. Against the grain: safeguarding rice from rice blast disease. Trends in Biotechnology 27:141-150.
Song, L., Jenner, M., Masschelein, J., Jones, C., Bull, M.J., Harris, S.R., Hartkoorn, R.C., Vocat, A., Romero-Canelon, I., Coupland, P., Webster, G., Dunn, M., Weiser, R., Paisey, C., Cole, S.T., Parkhill, J., Mahenthiralingam, E., and Challis, G.L. 2017. Discovery and biosynthesis of gladiolin: A Burkholderia gladioli antibiotic with promising activity against Mycobacterium tuberculosis. Journal of the American Chemical Society 139:7974-7981.
Steiner, I. (2015). Quantifiation of total cyanide content in kernels of stone fruits.
Šubík, J., and Behúň, M. 1974. Effect of bongkrekic acid on growth and metabolism of filamentous fungi. Archives of Microbiology 97:81-88.
Suprapta, D.N. 2012. Potential of microbial antagonists as biocontrol agents against plant fungal pathogens.
Verginer, M., Leitner, E., and Berg, G. 2010. Production of volatile metabolites by grape-associated microorganisms. Journal of Agricultural and Food Chemistry 58:8344-8350.
Wilson, R.A., and Talbot, N.J. 2009. Under pressure: investigating the biology of plant infection by Magnaporthe oryzae. Nature Reviews Microbiology 7:185.
Yoon, M.-Y., Cha, B., and Kim, J.-C. 2013. Recent trends in studies on botanical fungicides in agriculture. Plant Pathol J 29:1-9.
Zlosnik, J.E.A., Tavankar, G.R., Bundy, J.G., Mossialos, D., apos, Toole, R., and Williams, H.D. 2006. Investigation of the physiological relationship between the cyanide-insensitive oxidase and cyanide production in Pseudomonas aeruginosa 152:1407-1415.
Zou, C.-S., Mo, M.-H., Gu, Y.-Q., Zhou, J.-P., and Zhang, K.-Q. 2007. Possible contributions of volatile-producing bacteria to soil fungistasis. Soil Biology and Biochemistry 39:2371-2379.