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

黃啓銘

doi:10.6342/NTU.2014.02568

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结合即时定量PCR及孢子收集技术发展稻热病监测预测模式

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

黄啓铭 , 硕士　　导师：锺嘉绫　　

繁体中文 DOI： 10.6342/NTU.2014.02568

稻热病； Magnaporthe oryzae ；即时定量聚合酶链锁反应；流行病学；孢子收集器；预测模式；高解析度解离分析； Avirulence基因； Rice blast ； Magnaporthe oryzae ； Quantitative real-time PCR (qPCR) ； Epidemiology ； Spore trap ； Forecasting model ； High resolution melt (HRM) analysis ； Avirulence gene

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摘要 │ 参考文献 (133) │ 国际计量

摘要〈TOP〉

由病原真菌Magnaporthe oryzae所造成之稻热病，为水稻在全球各产区的最重要的病害之一。台湾於1970年代已有稻热病预测模式之相关研究，但目前稻热病之监测预警仍仰赖植保人员按时赴田间视察发病状况，再依结果发布警报通知农民加强防治。本研究着眼於开发不同於传统目视调查法之新监测技术，透过发展便於采集田间稻热病菌Magnaporthe oryzae分生孢子与定量之技术，配合定期收集田间发病情形及气象因子建立稻热病预测模式。本研究已完成旋风式孢子收集器、空飘孢子核酸萃取技术的开发，并以稻热病菌Magnaporthe infection structure specific protein (mif23) gene为模版，设计出适用於即时定量PCR (quantitative real-time PCR, qPCR) 且能专一性增幅稻热病菌核酸之引子对。本研究开发之SYBR Green qPCR定量法，最低可侦测到4 copy numbers之稻热病菌DNA，但能精确及稳定侦测的极限则约10 copy numbers；若以TaqMan系统进行定量，其精确侦测之极限约为4 copy numbers。为发展预测系统，本研究自2012年第一期作起，於农试所嘉义分所、嘉义溪口农场、以及全台共七个农业改良场选定之田区等处设置监测点，收集包含每日田间稻热病菌孢子、每日气象数据与每周发病程度等资料 (部分监测点并未收集到完整之资料)。初步分析发现，本技术确实可在田间发现病徵前侦测到孢子，且田间孢子数量在发病度提升时大幅增加，至抽穗期间则下降。另一方面，本研究同时模拟孢子实际收集状况，期望了解最适合保存样本之方式，结果发现样本应避免UVB照射，收取後先以CTAB缓冲液悬浮，再保存於室温或4℃冷藏，但应尽速在两周内完成核酸萃取，以避免样本降解。接着以2012-2013年农试所嘉义分所、嘉义溪口农场监测点之资料，配合预测日前1-14天或7-14天之累积气象资料，初步建立预测能力良好之模式，包含可针对台稉9号、台南11号或台中192号等单一品种进行预测之模式，以及可运用於多种品种之模式。模式建立过程中发现，孢子数并未如预期为一显着因子，其原因除了气象因子 (如相对湿度及降雨时数等) 对於稻热病发展趋势之影响力可能较大之外，推测亦与孢子收集及病害调查所采取之方式、范围、频率等有关。最後，为能将空飘孢子收集技术扩大运用於病原菌生理小种之判别，针对稻热病菌pex31 (Avr-Pik/kp/km) 基因，开发出能监别A、D、A+D及C type等不同型态对偶基因之高解析度解离分析 (High Resolution Melt, HRM) 新技术，其正确监别之极限约为25颗空飘孢子。本研究所开发之空飘孢子收集、定量及Avirulence基因型判别技术，未来若配合适当之引子对，将可应用於其他病害之侦测或监测。初步建立之稻热病监测预测模式，则仍须透过多年度、不同地点之气象及发病度等资料之持续累积与验证，才能改进其预测能力并使之逐步符合病害预警之实务需求。

并列摘要〈TOP〉

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.

参考文献 ( 133 ) 〈TOP〉

4. 陈绎年、林宗俊、陈纯葳、赖明信、卓纬玄、廖大经、张芳瑜。2013。国际稻米研究所之稻热病抗性检定评估技术研习心得分享。农业试验所技术服务　94:17-22。
连结：
6. 蔡武雄。2007。稻热病。植物保护图监系列8－水稻保护。第263-270页。林庆元、洪士程、徐保雄、施锡彬、陈治官、黄益田、刘清和、刘达修、蒋永正、蒋慕琰、郑清焕、罗干成编。行政院农业委员会动植物防疫检疫局。台北。
连结：
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.
连结：
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.
连结：
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.
连结：

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引用含有DOI的文献

DOI可强化引用精确性、增强学术圈连结，并给予使用者跨平台的良好使用经验，目前在全世界已有超过五千万个对象申请DOI。 如想对DOI的使用与概念有进一步了解，请参考 （ ） 。

DOI可强化引用精确性、增强学术圈连结，并给予使用者跨平台的良好使用经验，目前在全世界已有超过五千万个对象申请DOI。如想对DOI的使用与概念有进一步了解，请参考（）。