結合即時定量PCR及孢子收集技術發展稻熱病監測預測模式

Applying quantitative real-time PCR and spore trapping techniques for the development of a rice blast monitoring and forecasting model

10.6342/NTU.2014.02568

黃啓銘

稻熱病 ； Magnaporthe oryzae ； 即時定量聚合酶鏈鎖反應 ； 流行病學 ； 孢子收集器 ； 預測模式 ； 高解析度解離分析 ； Avirulence基因 ； Rice blast ； Magnaporthe oryzae ； Quantitative real-time PCR (qPCR) ； Epidemiology ； Spore trap ； Forecasting model ； High resolution melt (HRM) analysis ； Avirulence gene

臺灣大學植物病理與微生物學研究所學位論文

2014年

碩士

鍾嘉綾

繁體中文

由病原真菌Magnaporthe oryzae所造成之稻熱病，為水稻在全球各產區的最重要的病害之一。臺灣於1970年代已有稻熱病預測模式之相關研究，但目前稻熱病之監測預警仍仰賴植保人員按時赴田間視察發病狀況，再依結果發布警報通知農民加強防治。本研究著眼於開發不同於傳統目視調查法之新監測技術，透過發展便於採集田間稻熱病菌Magnaporthe oryzae分生孢子與定量之技術，配合定期收集田間發病情形及氣象因子建立稻熱病預測模式。本研究已完成旋風式孢子收集器、空飄孢子核酸萃取技術的開發，並以稻熱病菌Magnaporthe infection structure specific protein (mif23) gene為模版，設計出適用於即時定量PCR (quantitative real-time PCR, qPCR) 且能專一性增幅稻熱病菌核酸之引子對。本研究開發之SYBR Green qPCR定量法，最低可偵測到4 copy numbers之稻熱病菌DNA，但能精確及穩定偵測的極限則約10 copy numbers；若以TaqMan系統進行定量，其精確偵測之極限約為4 copy numbers。為發展預測系統，本研究自2012年第一期作起，於農試所嘉義分所、嘉義溪口農場、以及全臺共七個農業改良場選定之田區等處設置監測點，收集包含每日田間稻熱病菌孢子、每日氣象數據與每週發病程度等資料 (部分監測點並未收集到完整之資料)。初步分析發現，本技術確實可在田間發現病徵前偵測到孢子，且田間孢子數量在發病度提升時大幅增加，至抽穗期間則下降。另一方面，本研究同時模擬孢子實際收集狀況，期望了解最適合保存樣本之方式，結果發現樣本應避免UVB照射，收取後先以CTAB緩衝液懸浮，再保存於室溫或4℃冷藏，但應儘速在兩週內完成核酸萃取，以避免樣本降解。接著以2012-2013年農試所嘉義分所、嘉義溪口農場監測點之資料，配合預測日前1-14天或7-14天之累積氣象資料，初步建立預測能力良好之模式，包含可針對臺稉9號、臺南11號或臺中192號等單一品種進行預測之模式，以及可運用於多種品種之模式。模式建立過程中發現，孢子數並未如預期為一顯著因子，其原因除了氣象因子 (如相對濕度及降雨時數等) 對於稻熱病發展趨勢之影響力可能較大之外，推測亦與孢子收集及病害調查所採取之方式、範圍、頻率等有關。最後，為能將空飄孢子收集技術擴大運用於病原菌生理小種之判別，針對稻熱病菌pex31 (Avr-Pik/kp/km) 基因，開發出能鑑別A、D、A+D及C type等不同型態對偶基因之高解析度解離分析 (High Resolution Melt, HRM) 新技術，其正確鑑別之極限約為25顆空飄孢子。本研究所開發之空飄孢子收集、定量及Avirulence基因型判別技術，未來若配合適當之引子對，將可應用於其他病害之偵測或監測。初步建立之稻熱病監測預測模式，則仍須透過多年度、不同地點之氣象及發病度等資料之持續累積與驗證，才能改進其預測能力並使之逐步符合病害預警之實務需求。

Rice blast, caused by Magnaporthe oryzae, is one of the most devastating diseases of rice worldwide. In Taiwan, despite the attempt of developing rice blast forecasting model(s) in 1970s, nationwide disease monitoring and notification has long been relying on periodic surveys by trained plant protection personnel. The objective of this study is to first develop an approach which allows the collection and quantification of M. oryzae conidia (airborne inoculum) in the field. A modified blast disease forecasting model, using the amount of conidia along with several weather factors (including temperature, humidity, rainfall, etc.) as parameters, will then be established. We have successfully developed a cyclone-based spore trap and a standard sample processing protocol for extracting DNA from collected airspores. Using quantitative real-time PCR (qPCR) technology and a specific primer pair designed based on a Magnaporthe infection structure specific protein (mif23) gene, the amount of M. oryzae conidia can be easily quantified. While detection limit for the SYBR Green qPCR assay can be as low as 4 copy numbers of M. oryzae gDNA, the limit for reliable and accurate quantification is 10 copy numbers. For the TaqMan assay, the limit for reliable and accurate quantification is 4 copy numbers. Aiming to build a forecasting model, airspore samples, weather data, and disease severity ratings have been periodically collected from ten monitoring stations located at the paddy field and upland field blast nurseries at Chiayi Agricultural Experiment Station, a field site at Chiayi Sikou Farm, and seven field sites chosen by seven District Agricultural Research and Extension Stations in Taiwan (missing data exist for some of the monitoring stations). With our newly-developed spore trap and qPCR technique, M. oryzae spores can be detected before the appearance of leaf blast. It was observed that during the whole season, the amount of spores first increased while the field plants were commonly infected, and it then dropped after the stage of panicle development. In order to improve the handling and storage of airspore samples, we tested the effects of different treatments on the preservation of spore DNA. The optimized way would be: to avoid UV light exposure while sampling, to suspend the sample with CTAB buffer after collection, to store the sample at room temperature or 4℃, and to finish DNA extraction within two weeks. For disease modeling, we developed preliminary rice blast forecasting models for specific rice cultivars (TK9, TN11 and TC192) and multiple cultivars, on the basis of cumulative meteorological data from 1-14 or 7-14 days prior to the prediction day. It was found that the "number of spores" was not considered a significant parameter in most of the models, indicating that weather parameters such as relative humidity and hours of rainfall may be key factors favoring rice blast development. The approaches, sampling ranges and frequencies of the spore trapping and disease ratings may also have some effect on the result. Finally, to make the spore trapping technique applicable for characterization of pathogen physiological races, we developed a high resolution melt (HRM) technique which was proved to be powerful for the detection of the A, D, A+D, and C types of alleles at the pex31 (Avr-pik/kp/km) gene in M. oryzae. The differentiation limit for the HRM analysis is 25 airspores. In the future, with the use of other specific primer pairs, the spore trapping, qPCR, and HRM techniques develop in this study can be widely applied for the monitoring and detection of various airborne diseases. Since the data used for modeling in our study were from the monitoring stations at the Chiayi blast nursery and Sikou Farm, it is important to know that before the forecasting models can be widely applied, more weather data and disease severity data from multiple years, cultivars, and locations are required for model training, validation, and improvement.

1. 4. 陳繹年、林宗俊、陳純葳、賴明信、卓緯玄、廖大經、張芳瑜。2013。國際稻米研究所之稻熱病抗性檢定評估技術研習心得分享。農業試驗所技術服務　94:17-22。
   連結：
2. 6. 蔡武雄。2007。稻熱病。植物保護圖鑑系列8－水稻保護。第263-270頁。林慶元、洪士程、徐保雄、施錫彬、陳治官、黃益田、劉清和、劉達修、蔣永正、蔣慕琰、鄭清煥、羅幹成編。行政院農業委員會動植物防疫檢疫局。臺北。
   連結：
3. 10. Arauz, L. F., Neufeld, K. N., Lloyd, A. L., and Ojiambo, P. S. 2010. Quantitative models for germination and infection of Pseudoperonospora cubensisin response to temperature and duration of leaf wetness. Phytopathology 100(9):959-967.
   連結：
4. 11. Arny, C. J., and Rowe, R. C. 1991. Effect of temperature and duration of surface wetness on spore production and infection of cucumbers by Didymella bryoniae. Phytopathology 81:206-209.
   連結：
5. 12. Ashizawa, T., Sasahara, M., Ohba, A., Hori, T., Ishikawa, K., Sasaki, Y., Kuroda, T., Harasawa, R., Zenbayashi, K. S., and Koizumi, S. 2005. Evaluation of a leaf blast simulation model (BLASTMUL) for rice multilines in different locations and cultivars, and effective blast control using the mode. Pages 477-479. in: Proceedings of the World Rice Research Conference - Rice is life: scientific perspectives for the 21st century. K. Toriyama, K. L. Heong, and B. Hardy, eds. International Rice Research Institute, Japan.
   連結：
6. 13. Barnes, C. W., Szabo, L. J., and Bowersox, V. C. 2009. Identifying and quantifying Phakopsora pachyrhizi spores in rain. Phytopathology 99(4):328-338.
   連結：
7. 14. Berruyer, R., Poussier, S., Kankanala, P., Mosquera, G., and Valent, B. 2006. Quantitative and qualitative influence of inoculation methods on in planta growth of rice blast fungus. Phytopathology 96(4):346-355.
   連結：
8. 16. Bock, C. H., and Cotty, P. J. 2006. Methods to sample air borne propagules of Aspergillus flavus. European Journal of Plant Pathology 114(4):357-362.
   連結：
9. 18. Broome, J. C., English, J. T., Marois, J. J., Latorre, B. A., and Aviles, J. C. 1995. Development of an infection model for Botrytis bunch rot of grapes based on wetness duration and temperature. Phytopathology 85:97-102.
   連結：
10. 19. Calderon, C., Ward, E., Freeman, J., and McCartney, A. 2002. Detection of airborne fungal spores sampled by rotating-arm and hirst-type traps using PCR assays. Aerosol Science 33:289-296.
    連結：
11. 20. Calvero Jr, S. B., Coakley, S. M., and Teng, P. S. 1996. Development of empirical forecasting models for rice blast based on weather factors. Plant Pathology 45:667-678.
    連結：
12. 21. Carisse, O., and Lefebvre, A. 2011. A model to estimate the amount of primary inoculum of Elsinoe ampelina. Plant Disease 95(9):1167-1171.
    連結：
13. 22. Carisse, O., Bourgeois, G., and Duthie, J. A. 2000. Influence of temperature and leaf wetness duration on infection of strawberry leaves by Mycosphaerella fragariae. Phytopathology 90:1120-1125.
    連結：
14. 23. Carisse, O., Tremblay, D. M., Levesque, C. A., Gindro, K., Ward, P., and Houde, A. 2009. Development of a TaqMan real-time PCR assay for quantification of airborne conidia of Botrytis squamosa and management of Botrytis leaf blight of onion. Phytopathology 99(11):1273-1280.
    連結：
15. 24. Chadha, S., and Gopalakrishna, T. 2006. Detection of Magnaporthe grisea in infested rice seeds using polymerase chain reaction. Journal of Applied Microbiology 100(5):1147-1153.
    連結：
16. 25. Chen, B. T., Feather, G. A., Maynard, A., and Rao, C. Y. 2004. Development of a personal sampler for collecting fungal spores. Aerosol Science and Technology 38(9):926-937.
    連結：
17. 27. Cheng, J. C., Huang, C. L., Lin, C. C., Chen, C. C., Chang, Y. C., Chang, S. S., and Tseng, C. P. 2006. Rapid detection and identification of clinically important bacteria by high-resolution melting analysis after broad-range ribosomal RNA real-time PCR. Clinical Chemistry 52(11):1997-2004.
    連結：
18. 28. Chien, C. C. 1974. Studies on the physiologic races of rice blast fungus, Pyricularia oryzae Cav. Journal of Agricultural Research of China 23(1):16-37.
    連結：
19. 29. Chien, C. C., Hsieh, L. C., and Chang, Y. C. 1989. Studies on the break-down of resistance in rice cultivar Tainung 70 to rice blast. Journal of Agricultural Research of China 38(1):72-79.
    連結：
20. 30. Chien, C. C., Tsai, W. H., Yang, Y. Z., and Liu, C. 1984. Studies on the Epidemiology of rice blast disease in central areas of Taiwan. Journal of Agricultural Research of China 33(32):169-180.
    連結：
21. 31. Chindamporn, A., Iwaguch, S.-I., Nakagawa, Y., Homma, M., and Tanaka, K. 1993. Clonal size-variation of rDNA cluster region on chromosome XI1 of Saccharomyces cerevisiae. Journal of General Microbiology 139:1409-1415.
    連結：
22. 32. Chung, C.-L., Jamann, T., Longfellow, J., and Nelson, R. 2010. Characterization and fine-mapping of a resistance locus for northern leaf blight in maize bin 8.06. Theor Appl Genet 121:205–227.
    連結：
23. 34. Cortes, C., and Vapnik, V. 1995. Support-Vector Networks. Machine Learning 20:273-297.
    連結：
24. 35. 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(9):683-693.
    連結：
25. 38. El Jarroudi, M., Delfosse, P., Maraite, H., Hoffmann, L., and Tychon, B. 2009. Assessing the accuracy of simulation model for septoria leaf blotch disease progress on winter wheat. Plant Disease 93(10):983-992.
    連結：
26. 41. Fourniera, P., Gaillardin, C., Persuy, M.-A., Klootwijk, J., and Heerikhuizen, H. V. 1986. Heterogeneity in the ribosomal family of the yeast Yarrowia lipolytica: genomic organization and segregation studies. Gene 42:273-282.
    連結：
27. 42. Fraaije, B. A., Cools, H. J., Fountaine, J., Lovell, D. J., Motteram, J., West, J. S., and Lucas, J. A. 2005. Role of ascospores in further spread of qoi-resistant cytochromeballeles (g143a) in field populations of Mycosphaerella graminicola. Phytopathology 95(8):933-941.
    連結：
28. 43. Garber, R. C., Turgeon, B. G., Selker, E. U., and Yoder, O. C. 1988. Organization of ribosomal RNA genes in the fungus Cochliobolus heterostrophus. Current Genetics 14:573-582.
    連結：
29. 44. Ghanbarnia, K., Dilantha Fernando, W. G., and Crow, G. 2009. Developing rainfall- and temperature-based models to describe infection of canola under field conditions caused by pycnidiospores of Leptosphaeria maculans. Phytopathology 99(7):879-886.
    連結：
30. 45. Greer, C. A., and Webster, R. K. 2001. Occurrence, distribution, epidemiology, cultivar reaction, and management of rice blast disease in California. Plant Disease 85(10):1096-1102.
    連結：
31. 46. Gregory, P. H. 1952. Spore content of the atmosphere near the ground. Nature 170:475.
    連結：
32. 47. Hastie, T., Tibshirani, R., and Friedman, J. 2009. The Elements of Statistical Learning: Data Mining, Inference, and Prediction. Pages 745. 2 ed. Springer, US.
    連結：
33. 48. Hirst, J. M. 1952. An automatic volumetric spore trap. Annals of Applied Biology 39:259-265.
    連結：
34. 49. Ho, J.-H. 2008. Remote sensing technique and agriculture application. Bulletin of Taichung District Agricultural Improvement Station 98:69-80.
    連結：
35. 50. Howlett, B. J., Rolls, B. D., and Cozijnsen, A. J. 1997. Organisation of ribosomal DNA in the ascomycete Leptosphaeria maculans. Microbiological Research 152:261-267.
    連結：
36. 52. IRRI. 2002. Standard evalution system for rice. Pages 15. International Rice Research Institute, Philippine.
    連結：
37. 55. Jarosz, N., Loubet, B., Durand, B., McCartney, A., Foueillassar, X., and Huber, L. 2003. Field measurements of airborne concentration and deposition rate of maize pollen. Agricultural and Forest Meteorology 119(1-2):37-51.
    連結：
38. 56. Jiang, J.-A., Tseng, C.-L., Lu, F.-M., Yang, E.-C., Wu, Z.-S., Chen, C.-P., Lin, S.-H., Lin, K.-C., and Liao, C.-S. 2008. A GSM-based remote wireless automatic monitoring system for field information: A case study for ecological monitoring of the oriental fruit fly, Bactrocera dorsalis (Hendel). Computers and Electronics in Agriculture 62(2):243-259.
    連結：
39. 57. Johnson, J. B., and Omland, K. S. 2004. Model selection in ecology and evolution. Trends in Ecology & Evolution 19(2):101-108.
    連結：
40. 58. Kachroo, P., Leong, S. A., and Chattoo, B. B. 1994. Pot2, an inverted repeat transposon from the rice blast fungus Magnaporthe grisea. Molecular Genetics and Genomics 245:339-348.
    連結：
41. 61. Khang, C. H., Park, S.-Y., Lee, Y.-H., Valent, B., and Kang, S. 2008. Genome organization and evolution of the AVR-Pita avirulence gene family in the Magnaporthe grisea species complex. MPMI 21(5):658-670.
    連結：
42. 63. Kim, C. K., and Kim, C. H. 1991. Predicting rice blast outbreaks in Korea. Pages 53-68. in: Rice blast modeling and forecasting. P. S. Teng, L. R. Pollard, and G. Argosino, eds. International Rice Research Institute, Philippines.
    連結：
43. 65. Kim, S. B., Huo, X., and Tsui, K.-L. 2009. A finite-sample simulation study of cross validation in tree-based models. Information Technology and Management 10(4):223-233.
    連結：
44. 66. Kirkpatrick, N. C., Blacker, H. P., Woods, W. G., Gasser, R. B., and Noormohammadi, A. H. 2009. A polymerase chain reaction-coupled high-resolution melting curve analytical approach for the monitoring of monospecificity of avian Eimeria species. Avian Pathology 38(1):13-19.
    連結：
45. 67. Kobayashi, J. 1984. Studies on epidemic of rice leaf blast Pyricularia oryzae Cav. in its early stage. Bull. Akita Agric. Exp. Stn. 26:1-84.
    連結：
46. 69. Kriss, A. B., Paul, P. A., and Madden, L. V. 2010. Relationship between yearly fluctuations in Fusarium head blight intensity and environmental variables: a Window-pane analysis. Phytopathology 100(8):784-797.
    連結：
47. 73. Lalancette, N., and Hickey, K. D. 1986. An apple powdery mildew model based on plant growth, primary inoculum, and fungicide concentration. Phytopathology 76:1176-1182.
    連結：
48. 74. Landschoot, S., Waegeman, W., Audenaert, K., Vandepitte, J., Haesaert, G., and De Baets, B. 2012. Toward a Reliable Evaluation of Forecasting Systems for Plant Diseases: A Case Study Using Fusarium Head Blight of Wheat. Plant Disease 96(6):889-896.
    連結：
49. 75. Latorre, B. A., Rioja, M. E., Lillo, C., and Munoz, M. 2002. The effect of temperature and wetness duration on infection and a warning system for European canker (Nectria galligena) of apple in Chile. Crop Protection 21:285-291.
    連結：
50. 76. Lee, Y. H., and Dean, R. A. 1993. Stage-specific gene expression during appressorium formation of Magnaporthe grisea. Experimental Mycology 17:215-222.
    連結：
51. 77. Li, H., Shu, C., He, X., Gao, J., Liu, R., and Huang, D. 2012. Detection and identification of vegetative insecticidal proteins vip3 genes of Bacillus thuringiensis strains using polymerase chain reaction-high resolution melt analysis. Current Microbiology 64(5):463-468.
    連結：
52. 78. Li, Q., Kong, B.-H., Fan, J.-H., Cai, H., Fu, Y., and Chen, H.-R. 2011. Early detection of rice blast by TaqMan real-time flourescence quantitative polymerase chain reaction. Acta Phytopathologica Sinica 4(12):118-123.
    連結：
53. 79. Liu, J., Wang, X., Mitchell, T., Hu, Y., Liu, X., Dai, L., and Wang, G.-L. 2010. Recent progress and understanding of the molecular mechanisms of the rice-Magnaporthe oryzae interaction. Molecular Plant Pathology 11(3):419-427.
    連結：
54. 80. Luo, Y., Ma, Z., Reyes, H. C., Morgan, D., and Michailides, T. J. 2007. Quantification of airborne spores of Monilinia fructicola in stone fruit orchards of California using real-time PCR. European Journal of Plant Pathology 118(2):145-154.
    連結：
55. 81. Macher, J., Chen, B., and Rao, C. 2008a. Chamber evaluation of a personal, bioaerosol cyclone sampler. Journal of Occupational and Environmental Hygiene 5(11):702-712.
    連結：
56. 82. Macher, J., Chen, B., and Rao, C. 2008b. Field evaluation of a personal, bioaerosol cyclone sampler. Journal of Occupational and Environmental Hygiene 5(11):724-734.
    連結：
57. 83. Madden, L. V. 2006. Botanical epidemiology: some key advances and its continuing role in disease management. European Journal of Plant Pathology 115(1):3-23.
    連結：
58. 84. Maleszka, R., and Clark-Walker, G. D. 1990. Magnification of the rDNA cluster in Kluyveromyces lactis. Molecular Genetics and Genomics 223:342-344.
    連結：
59. 86. Maron, P.-A., Lejon, D. P. H., Carvalho, E., Bizet, K., Lemanceau, P., Ranjard, L., and Mougel, C. 2005. Assessing genetic structure and diversity of airborne bacterial communities by DNA fingerprinting and 16S rDNA clone library. Atmospheric Environment 39(20):3687-3695.
    連結：
60. 87. May, K. R. 1945. The cascade impactor: an instrument for sampling coarse aerosols. J. Sci. Instrum. 22:187-195.
    連結：
61. 88. McCartney, H. A., and Aylor, D. E. 1987. Relative contributions of sedimentation and impaction to deposition of particles in a crop canopy. Agricultural and Forest Meteorology 40:343-358
    連結：
62. 89. Mohapatra, N. K., Mukherjee, A. K., Rao, A. V. S., and Nayak, P. 2008. Disease progress curves in the rice blast pathosystem compared with the logistic and gompertz models. ARPN Journal of Agricultural and Biological Science 3 (1):28-37.
    連結：
63. 90. Monroe, J. S., Santini, J. B., and Latin, R. 1997. A model defining the relationship between temperature and leaf wetness duration, and infection of watermelon by Colletotrichum orbiculare. Plant Disease 81:739-742.
    連結：
64. 91. Mousanejad, S., Alizadeh, A., and Safaie, N. 2009. Effect of weather factors on spore population dynamics of rice blast fungus in Guilan province. Journal of Plant Protection Research 49(3):319-329.
    連結：
65. 92. Myers, R. H. 1990. Classical and modern regression with applications. Pages 488. 2 ed. PWS-KENT Publishing Company, Boston.
    連結：
66. 93. Nemoto, F., and Ishiguro, K. 2004. Computer simulation approaches for rice blast disease forecasting in Japan. Pages 289-296. in: Rice Blast: Interaction with Rice and Control. S. Kawasaki, ed. Kluwer Academic Publishers, Japan.
    連結：
67. 94. Ong, D. C. T., Yam, W. C., Siu, G. K. H., and Lee, A. S. G. 2010. Rapid detection of Rifampicin- and Isoniazid-resistant Mycobacterium tuberculosis by high-resolution melting analysis. Journal of Clinical Microbiology 48(4):1047-1054.
    連結：
68. 95. Orbach, M. J., Farrall, L., Sweigard, J. A., Chumley, F. G., and Valent, B. 2000. A telomeric avirulence gene determines efficacy for the rice blast resistance gene Pi-ta. The Plant Cell 12: 2019-2032.
    連結：
69. 96. Ou, S. H. 1985. Rice diseases. Pages 380. Commonwealth Mycological Institute, England.
    連結：
70. 97. Parker, P. E., Bock, C. H., and Gottwald, T. R. 2005. Comparison of techniques to sample Xanthomonas axonopodis pv.citriin windblown spray. Plant Disease 89(12):1324-1330.
    連結：
71. 98. Pasero, P., and Marilley, M. 1993. Size variation of rDNA clusters in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Molecular Genetics and Genomics 236:448-452.
    連結：
72. 99. Paul, P. A., and Munkvold, G. P. 2005. Regression and artificial neural network modeling for the prediction of gray leaf spot of maize. Phytopathology 95(4):388-396.
    連結：
73. 100. Peccia, J., and Hernandez, M. 2006. Incorporating polymerase chain reaction-based identification, population characterization, and quantification of microorganisms into aerosol science: A review. Atmospheric Environment 40(21):3941-3961.
    連結：
74. 101. Pinnschmidt, H. O., Klein-Gebbinck, H. W., Bonman, J. M., and Kranz, J. 1993. Comparison of aerial concentration, deposition and infectiousness of condia of Pyricularia grisea by spore-sampling techniques. Phytopathology 83:1182-1189.
    連結：
75. 102. Qi, M., and Yang, Y. 2002. Quantification of Magnaporthe grisea during infection of rice plants using real-time polymerase chain reaction and northern blot phosphoimaging analyses. Phytopathology 92:870-876.
    連結：
76. 104. Rogstad, S. H. 1992. Saturated NACl-CTAB solution as a means of field preservation of leaves for DNA analyses. Taxon 41:701-708.
    連結：
77. 105. Savary, S., Nelson, A., Willocquet, L., Pangga, I., and Aunario, J. 2012. Modeling and mapping potential epidemics of rice diseases globally. Crop Protection 34:6-17.
    連結：
78. 106. Schweigkofler, W., O'Donnell, K., and Garbelotto, M. 2004. Detection and quantification of airborne conidia of Fusarium circinatum, the causal agent of pine pitch canker, from Two California sites by using a real-time PCR approach combined with a simple spore trapping method. Applied and Environmental Microbiology 70(6):3512-3520.
    連結：
79. 107. Slany, M., Vanerkova, M., Nemcova, E., Zaloudikova, B., Ruzicka, F., and Freiberger, T. 2010. Differentiation of Staphylococcus spp. by high-resolution melting analysis. Canadian Journal of Microbiology 56(12):1040-1049.
    連結：
80. 108. Stergiopoulos, I., and de Wit, P. J. G. M. 2009. Fungal effector proteins. Annual Review of Phytopathology 47(1):233-263.
    連結：
81. 111. Suzuki, H. 1975. Meteorological factors in the epidemiology of rice blast. Annual Review of Phytopathology 13:239-256.
    連結：
82. 112. Talbot, N. J. 2003. On the trail of a cereal killer: Exploring the biology of Magnaporthe grisea. Annual Review of Microbiology 57(1):177-202.
    連結：
83. 116. Toyoda, S., and Suzuki, N. 1952. Histochemical studies on the lesions of rice blast caused by Piricularia oryzae Cav. I. Some observations on the sporulations on lesions of different types occurring on leaves of the same variety. Ann. Phytopathol. Soc. Jpn 17:1-4.
    連結：
84. 117. Tsai, W.-H. 1978. Epidemiological studies of rice blast disease observations of 1976 and 1977. Journal of Agricultural Research of China 27(4):355-362.
    連結：
85. 118. Tsai, W.-H. 1980. Effect of air temperature and number of rainy days on rice leaf blast. Journal of Agricultural Research of China 29(1):21-25.
    連結：
86. 120. Tsai, W. H., Huang, Y. T., Yu, C. M., Leu, W. T., Yang, Y. Z., Liang, W. J., Lee, S. C., Tsai, T. W., and Chien, C. C. 1978. Epidemiological studies of rice blast disease observations in 1977. Journal of Agricultural Research of China 27(3):237-244.
    連結：
87. 121. Tseng, M. N., Chung, P. C., and Tzean, S. S. 2011. Enhancing the stress tolerance and virulence of an entomopathogen by metabolic engineering of dihydroxynaphthalene melanin biosynthesis genes. Applied and Environmental Microbiology 77(13):4508-4519.
    連結：
88. 122. Tyler, B., Chuma, I., Isobe, C., Hotta, Y., Ibaragi, K., Futamata, N., Kusaba, M., Yoshida, K., Terauchi, R., Fujita, Y., Nakayashiki, H., Valent, B., and Tosa, Y. 2011. Multiple translocation of the AVR-Pita effector gene among chromosomes of the rice blast fungus Magnaporthe oryzae and related species. PLoS Pathogens 7(7):e1002147.
    連結：
89. 123. Vossen, R. H. A. M., Aten, E., Roos, A., and den Dunnen, J. T. 2009. High-resolution melting analysis (HRMA)-more than just sequence variant screening. Human Mutation 30(6):860-866.
    連結：
90. 126. West, J. S., Atkins, S. D., Emberlin, J., and Fitt, B. D. L. 2008. PCR to predict risk of airborne disease. Trends in Microbiology 16(8):380-387.
    連結：
91. 127. Wong, M.-H., Henderson, J., and Drenth, A. 2013. Identification and differentiation of Phyllosticta species causing freckle disease of banana using high resolution melting (HRM) analysis. Plant Pathology 62(1285-1293).
    連結：
92. 128. Wu, L., Damicone, J. P., Duthie, J. A., and Melouk, H. A. 1999. Effects of temperature and wetness duration on infection of peanut cultivars by Cercospora arachidicola. Phytopathology 89:653-659.
    連結：
93. 129. Yang, Y. Z., Cin, C. S., and Chang, D. C. 1982. Field studies on the epidemiology of rice blast in Taichung area. Bulletin of Taichung District Agricultural Improvement Station 6:28-40.
    連結：
94. 130. Yoshida, K., Saitoh, H., Fujisawa, S., Kanzaki, H., Matsumura, H., Tosa, Y., Chuma, I., Takano, Y., Win, J., Kamoun, S., and Terauchi, R. 2009. Association genetics reveals three novel avirulence genes from the rice blast fungal pathogen Magnaporthe oryzae. The Plant Cell 21(5):1573-1591.
    連結：
95. 132. Zhang, N., Zhao, S., and Shen, Q. 2011. A six-gene phylogeny reveals the evolution of mode of infection in the rice blast fungus and allied species. Mycologia 103(6):1267-1276.
    連結：
96. 133. Zhou, G., Whong, W. Z., Ong, T., and Chen, B. 2000. Development of a fungus-specific PCR assay for detecting low-level fungi in an indoor environment. Molecular and Cellular Probes 14(6):339-348.
    連結：
97. 1. 江昭皚、陳家榜、莊欽龍。2013。「害蟲」來了我知道！－智慧型遠距農業害蟲動態監測與預報系統。臺大校友雙月刊。第24-29頁。國立臺灣大學。台北。
98. 2. 柯勇。1998。作物病害與防治。1版。藝軒出版社。臺北。550頁。
99. 3. 張義璋。2001。水稻稻熱病之防治。臺北。網址：http://kmweb.coa.gov.tw/techcd/。
100. 5. 蔡武雄。1991。水稻葉稻熱病發生預測。稻作病害研討會專刊。第15-30頁。蔡財旺、蔡武雄、簡錦忠、張義璋編。臺灣省農業試驗所。臺中。
101. 7. Agrios, G. N. 2005. Plant Pathology. Pages 922. 5 ed. Academic Press, USA.
102. 8. Andersen, A. A. 1958. New sampler for the collection, sizing and enumeration of viable airborne particles. Journal of Bacteriology 76:471-484.
103. 9. Arai, N., and Yoshino, R. 1987. Studies on the sporulation of rice blast fungus. (1) Relation between sporulation and temperature. Ann. Phytopathol. Soc. Jpn 53:371-372.
104. 15. Biloni, M., Rodolfi, M., and Picco, A. M. 2006. SIRBINT, a new simulation model to forecast rice blast disease. Italian Journal of Agrometeorology 3:58-62.
105. 17. Bouman, B. A. M., Kropff, M. J., Tuong, T. P., Wopereis, M. C. S., Berge, H. F. M. t., and Laar, H. H. v. 2001. ORYZA2000: modeling lowland rice. Pages 235. Edited by B. Hardy. (Philippines): International Rice Research Institute, and Wageningen: Wageningen University and Research Centre.
106. 26. Chen, R. S. 2009. Applications and perspectives of molecular markers on the population genetics of Mangnaporthe oryzae. in: Proceedings of Symposium on Achievements and Perspectives of Rice Protection in Taiwan.
107. 33. Coakley, S. M., and Line, R. F. 1982. Prediction of stripe rust epidemics on winter wheat using statistical models. (Abstr.) Phytopathology 72(7):1006.
108. 36. Crook, B. 1995a. Inertial samplers: biological perspectives. Pages 247-267. in: Bioaerosols Handbook. C. S. Cox, and C. M. Wathers, eds. Lewis Publishers, Boca Raton.
109. 37. Crook, B. 1995b. Non-inertial samplers: biological perspectives. Pages 269-283. in: Bioaerosols Handbook. C. S. Cox, and C. M. Wathers, eds. Lewis Publishers, Boca Raton.
110. 39. El Refaei, M. I. 1977. Epidemiology of rice blast disease in the tropics with special reference to leaf wetness in relation to disease development., Indian Agricultural Research Institute, New Delhi.
111. 40. Elazegui, F. A., Castilla, N. P., Nieva, L. P., and Cruz, C. M. V. 2009. Rice Knowledge Bank. Philippines. From: http://www.knowledgebank.irri.org/ipm/rice-blast.html.
112. 51. Hu, Z.-L., Cai, H., Fan, J.-H., and Chen, H.-R. 2009. Establishment of real-time fluorescent PCR method with TaqMan probe for Magnaporthe grisea. Journal of Yunnan Agricultural University 24(3):349-353.
113. 53. Ishiguro, K., and Hashimoto, A. 1991. Computer-based forecasting of rice blast epidemics in Japan. Pages 39-52. in: Rice blast modeling and forecasting. P. S. Teng, L. R. Pollard, and G. Argosino, eds. International Rice Research Institute, Philippines.
114. 54. Japan Meteorological Agency. 2012. Japan Meteorological Agency. Japan. From: http://www.jma.go.jp/jma/en/Activities/observations.html.
115. 59. Kaundal, R., Kapoor, A. S., and Raghava, G. P. S. 2006a. RB-Pred. India. From: http://www.imtech.res.in/raghava/rbpred/submit.html.
116. 60. Kaundal, R., Kapoor, A. S., and Raghava, G. P. S. 2006b. Machine learning techniques in disease forecasting: a case study on rice blast prediction. BMC Bioinformatics 7(1):485.
117. 62. Kim, C. K. 2001. Epidemiology of rice blast disease in Korea. Pages 119-129. in: Major Fungal Diseases of Rice Recent Advances. S. Sreenivasaprasad, and R. Johnson, eds. Kluwer Academic Publishers, Netherland.
118. 64. Kim, C. K., and Yoshino, R. 2000. Sporulation of Pyricularia grisea at different growth stages of rice in the field. Plant Pathol. J. 16(3):147-150.
119. 68. Koizumi, S., and Kato, H. 1991. Dynamic simulation of blast epidemics using a multiple canopy spore dispersal model. Pages 75-88. in: Rice blast modeling and forecasting. P. S. Teng, L. R. Pollard, and G. Argosino, eds. International Rice Research Institute, Philippines.
120. 70. Kutner, M. H., Nachtsheim, C. J., Neter, J., and Li, W. 2005. Applied Linear Statistical Models. Pages 1396. 5 ed. McGraw-Hill, New York.
121. 71. Lacey, J., and Venette, J. 1995. Outdoor air sampling techniques. Pages 407-472. in: Bioaerosols Handbook. C. S. Cox, and C. M. Wathers, eds. Lewis Publishers, Boca Raton.
122. 72. Lacey, M. E., and West, J. S. 2006. The Air Spora. Pages 156. Springer, Boston.
123. 85. Manibhushanrao, K., and Krishnan, P. 1991. Epidemiology of blast (EBIPLA): a simulation model and forecasting system for tropical rice in India. Pages 31-38. in: Rice blast modeling and forecasting. P. S. Teng, L. R. Pollard, and G. Argosino, eds. International Rice Research Institute, Philippines.
124. 103. Reddy, C. S., Susheela, K., Kapoor, A. S., Kaundal, R., Krishnaiah, N. V., Mishra, B., Ramakrishna, Y. S., Prasad, Y. G., Reddy, D. Y., and Prabhakar, M. 2004. Forewarning Rice Blast in India. Pages 46. Directorate of Rice Research, India.
125. 109. Surin, A., Arunyanart, P., Rojanahusdin, W., Munkong, S., Dhitikiattipong, R., and Disthaporn, S. 1991. Using empirical blast models to establish disease management recommendations in Thailand. Pages 69-74. in: Rice blast modeling and forecasting. P. S. Teng, L. R. Pollard, and G. Argosino, eds. International Rice Research Institute, Philippines.
126. 110. Suzuki, H. 1965. The falling of Piricularia oryzae-spore by rain. Proc. Assoc. Plant Prot., North Jpn. 16:135-137.
127. 113. TeBeest, D. O., Guerber, C., and Ditmore, M. 2007. Rice blast. The Plant Health Instructor.
128. 114. Teng, P. S. 1994. The epidemiological basis for blast management. Pages 409-433. in: Rice blast disease. R. S. Zeigler, S. A. Leong, and P. S. Teng, eds. International Rice Research Institute, Philippines.
129. 115. Teng, P. S., Klein-Gebbinck, H. W., and Pinnschmidt, H. 1991. An analysis of the blast pathosystem to guide modeling and forecasting. Pages 1-30. in: Rice blast modeling and forecasting. P. S. Teng, L. R. Pollard, and G. Argosino, eds. International Rice Research Institute, Philippines.
130. 119. Tsai, W. H. 2009. The review on the studies of rice blast. Pages 1-12. in: Proceedings of Symposium on Achievements and Perspectives of Rice Protection in Taiwan.
131. 124. Wakefield, A. E. 1996. DNA Sequences Identical to Pneumocystis carinii f. sp. carinii and Pneumocystis carinii f. sp. hominis in Samples of Air Spora. Journal of Clinical Microbiology 34(7):1754-1759.
132. 125. Wang, T.-K., Tasi, W.-H., Fang, S.-J., and Yen, C.-F. 2009. The monitoring and coordinated management of rice pests and its future prospects. Pages 13-28. in: Proceedings of Symposium on Achievements and Perspectives of Rice Protection in Taiwan.
133. 131. Yoshino, R. 1971. Ecological studies on the infection in rice blast epidemics. I. Infection rates and hyphal growth in epidermal cells. Proc. Assoc. Plant Prot. Hokuriku 19:14-17.