水稻紋枯病菌 (Rhizoctonia solani) 會造成水稻紋枯病 (sheath blight of rice)。此病原菌為土傳性病原菌，能以菌核及菌絲殘存於土壤中，可自莖基部侵入水稻植株並於葉鞘部大量繁殖，進而造成水稻發生枯萎的虎斑狀病徵，為全球水稻產業上一大重要限制因子。為了預防此病害發生造成水稻產業上的經濟損失，發展一個具備專一、快速、且能夠現地進行檢測之診斷工具用以檢測田間之R. solani，就此病害管理而言是重要策略之一。本研究擬開發一個水稻紋枯病菌之現地檢測技術，用以檢測R. solani。根據試驗結果，本研究已開發出快速萃取試劑，可用於田間檢體之核酸萃取，並完成專一性引子對SMS RS1-F/ SMS RS1-R及GMRS-3/ITS1靈敏度及專一性測試。本研究以四種不同萃取系統進行比較，結果顯示自動萃取系統搭配專一性引子對SMS RS1-F/ SMS RS1-R之檢出率為100%，為四種萃取系統中最穩定；而使用快速萃取試劑次之，其檢出率為96%，此結果證實本研究所開發的快速萃取試劑確實能有效地用於檢測紋枯病。透過同日間分析 (intraday assay) 及異日間分析 (interday assay) 測試結果，證實本研究所開發的不同萃取系統皆能夠穩定地進行水稻檢體的核酸萃取。以不同病程之水稻檢體進行萃取之in-planta試驗結果顯示，以自動萃取系統進行檢體核酸萃取可獲得較穩定的檢出率，平均檢出率高於80%。未來擬進一步針對田間帶菌土壤進行萃取測試，使此檢測系統更能落實於水稻田間紋枯病菌之現地檢測工作中。

Sheath blight of rice (ShB) caused by Rhizoctonia solani is a major limiting factor to rice production. This soil-borne fungal pathogen can survive in the soil by sclerotia and mycelia. They are the primary inoculum in rice fields. The primary inoculum has ability to infect the rice sheath from the stem base and multiply in the tissues causing lesion symptoms on sheath. In order to prevent the outbreak of rice sheath blight and to reduce the economic loss of rice yield, it’s critically important to develop a rapid detection method with specific, fast, and on-site advantages for disease management. In this study, the rapid extraction reagent which can be used for on-site detection was developed. The specificity and sensitivity of the novel primers set SMS RS1-F/SMS RS1-R and the reference-primer set GMRS-3/ITS1 were tested. In addition, this study developed the four different extraction protocols for rice sheath infected by R. solani. Moreover, the intraday- and interday-assays were performed for the reproducibility evaluation of the detection methods developed in this study. The results of reproducibility evaluation indicated that all developed detection protocols were suitable to be used for diseased rice sheath. In the future, an on-site will be developed for field soil to make the detection system to be more completely.

參考文獻
陳思豪 (2007) 台灣水稻紋枯病病原菌Rhizoctonia solani AG1-IA菌株之多樣性。國立中興大學植物病理學研究所第37屆畢業碩士論文，78頁。
游俊明 (1984) 台灣水稻菌核性病害之研究1 .水稻疑似紋枯病及紋枯病樣病斑上分離之幾種菌核病菌。桃園區農業改良場研究彙報 2: 37-44。
Agrios, G. N. (2005) Vascular wilts caused by ascomycetes and deuteromycetes (mitosporic fungi), p. 74. In: G. N. Agrios (ed.), Plant Pathol. Dana Dreibelbis, UK. 922 pp.
Arakawa, M., and K. Inagaki (2014) Molecular markers for genotyping anastomosis groups and understanding the population biology of Rhizoctonia species. J. Gen. Plant Pathol. 80: 401-407.
Bernardes-de-Assis, J., M. Storari, M. Zala, W. Wang, D. Jiang, L. ShiDong, M. Jin, B. A. McDonald, and P. C. Ceresini (2009) Genetic structure of populations of the rice-infecting pathogen Rhizoctonia solani AG-1 IA from China. Phytopathology 99: 1090-1099.
Butler, E. E., and C. E. Bracker (1970) Morphology and cytology of Rhizoctonia solani. p. 32-53 In: Parmeter Jr. J. R., (ed.) Biology and Pathology of Rhizoctonia solani. University of California Press, Berkeley, U. S. A. 255 pp.
Çebi Kılıçoğlu, M., and İ. Özkoç (2010) Molecular characterization of Rhizoctonia solani AG4 using PCR-RFLP of the rDNA-ITS region. Turk. J. Biol. 34: 261-269.
Chen, Y., A. F. Zhang, W. X. Wang, Y. Zhang, and T. C. Gao (2012) Baseline sensitivity and efficacy of thifluzamide in Rhizoctonia solani. Ann. Appl. Biol. 161: 247-254.
Dixit, C. K., S. K. Vashist, F. T. O’Neill, B. O’Reilly, B. D. MacCraith, and R. O’Kennedy (2010) Development of a high sensitivity rapid sandwich ELISA procedure and its comparison with the conventional approach. Anal. Chem. 82: 7049-7052.
Dubey, S. C., A. Tripathi, and B. K. Upadhyay (2012) Molecular diversity analysis of Rhizoctonia solani isolates infecting various pulse crops in different agro-ecological regions of India. Folia Microbiol. 57: 513-524.
Dubey, S. C., A. Tripathi, B. K. Upadhyay, and A. Kumar (2016) Development of conventional and real time PCR assay for detection and quantification of Rhizoctonia solani infecting pulse crops. Biologia 71: 133-138.
Groth, D. E. (2005) Azoxystrobin rate and timing effects on rice sheath blight incidence and severity and rice grain and milling yields. Plant Dis. 89: 1171-1174.
Handiseni, M., Y.-K. Jo, and X.-G. Zhou (2015) Integration of Brassica cover crop with host resistance and azoxystrobin for management of rice sheath blight. Plant Dis. 99: 883-885.
IRRI (1996) Injuries caused by diseases. p. 25 In: Standard evaluation system for rice. Int. Rice Res. Inst. 4th (ed.) Manila, Philippines. 52 pp.
James, T., Y. J. M. Moncalvo, S. Li, and R. Vilgalys (2001) Polymorphism at the ribosomal DNA spacers and its relation to breeding structure of the widespread mushroom Schizophyllum commune. Genetics 157: 149-161.
Johanson, A., H. C. Turner, G. J. McKay, and A. E. Brown (1998) A PCR-based method to distinguish fungi of the rice sheath-blight complex, Rhizoctonia solani, R. oryzae and R. oryzae-sativae. FEMS Microbiol. Lett. 162: 289-294.
Joshi, M., P. K. Singh, S. A. Waza, V. Singh, S. Goswami, Pallavi, S. Kumar, A. K. Singh, N. W. Zaidi, and U. S. Singh (2016) Establishing an association between molecular markers and sheath blight (Rhizoctonia solani Kuhn) resistance in rice. Plant Omics 9: 281-288.
Khan, I. A., F. S. Awan, A. Ahmad, and A. A. Khan (2004) A modified mini-prep method for economical and rapid extraction of genomic DNA in plants. Plant Mol. Biol. Rep. 22: 89a-89e.
Lievens, B., M. Brouwer, A. C. R. C. Vanachter, B. P. A. Cammue, and B. P. H. J. Thomma (2006) Real-time PCR for detection and quantification of fungal and oomycete tomato pathogens in plant and soil samples. Plant Sci. 171: 155-165.
Lin, Y. H., J. Y. Chang, E. T. Liu, C. P. Chao, J. W. Huang, and P. F. L. Chang (2009) Development of a molecular marker for specific detection of Fusarium oxysporum f. sp. cubense race 4. Eur. J. Plant Pathol. 123: 353-365.
Liu, D., S. Coloe, R. Baird, and J. Pedersen (2000) Rapid mini-preparation of fungal DNA for PCR. J. Clin. Microbiol. 38: 471.
Lu, W., L. Pan, H. Zhao, Y. Jia, Y. Wang, X. Yu, and X. Wang (2014) Molecular detection of Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzicola, and Burkholderia glumae in infected rice seeds and leaves. Crop J. 2: 398-406.
Martinelli, F., R. Scalenghe, S. Davino, S. Panno, G. Scuderi, P. Ruisi, P. Villa, D. Stroppiana, M. Boschetti, L. R. Goulart, C. E. Davis, and A. M. Dandekar (2015) Advanced methods of plant disease detection. A review. Agron. Sustain. Dev. 35: 1-25.
Murray, M. G., and W. F. Thompson (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 8: 4321-4325.
Okubara, P. A., K. L. Schroeder, and T. C. Paulitz (2005) Real-time polymerase chain reaction: applications to studies on soilborne pathogens. Can. J. Plant Pathol. 27: 300-313.
Ou, S. H. (1985) Sheath blight. p. 272-286 In: Rice diseases 2nd (ed.) Commonwealth agricultural bureau, Great Britain, UK. 380 pp.
Porebski, S., L. Grant Bailey, and B. R. Baum (1997) Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Mol. Biol. Rep. 15: 8-15.
Prasad, B., and G. C. Eizenga (2008) Rice sheath blight disease resistance identified in Oryza spp. accessions. Plant Dis. 92: 1503-1509.
Puchooa, D (2004) A simple, rapid and efficient method for the extraction of genomic DNA from lychee (Litchi chinensis Sonn.). Afr. J. Biotechnol. 3: 253-255.
Rodrigues, F. Á., F. X. R. Vale, G. H. Korndörfer, A. S. Prabhu, L. E. Datnoff, A. M. A. Oliveira, and L. Zambolim (2003a) Influence of silicon on sheath blight of rice in Brazil. Crop Prot. 22: 23-29.
Rodrigues, F. Á., F. X. R. Vale, L. E. Datnoff, A. S. Prabhu, and G. H. Korndörfer (2003b) Effect of rice growth stages and silicon on sheath blight development. Phytopathology 93: 256-261.
Sayler, R. J., and Y. Yang (2007) Detection and quantification of Rhizoctonia solani AG-1 IA, the rice sheath blight pathogen, in rice using real-time PCR. Plant Dis. 91: 1663-1668.
Skottrup, P., H. Frøkiær, S. Hearty, R. O’Kennedy, J. Hejgaard, M. Nicolaisen, and A. F. Justesen (2007) Monoclonal antibodies for the detection of Puccinia striiformis urediniospores. Mycol. Res. 111: 332-338.
Štorchová, H., R. Hrdličková, J. Jr. Chrtek, M. Tetera, D. Fitze, and J. Fehrer (2000) An improved method of DNA isolation from plants collected in the field and conserved in saturated NaCI/CTAB solution. Taxon 49: 79-84.
Su’udi, M., J. M. Park, W. R. Kang, D. J. Hwang, S. Kim, and I. P. Ahn (2013) Quantification of rice sheath blight progression caused by Rhizoctonia solani. J. Microbiol. 51: 380-388.
Susheela, K., and C.S. Reddy (2013) Variability in Rhizoctonia solani (AG-1 IA) isolates causing sheath blight of rice in India. Indian Phytopathol. 66: 341-350.
White, T. J., T. Bruns, S. Lee, and J. W. Taylor (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. p. 315-322. Innis, M. A., D. H. Gelfand, J. J. Sninsky, and T. J. White (eds.) In PCR protocols: A guide to methods and applications. Academic Press, San Diego, CA. 482 pp.
Yuan, Z., Y. Zhang, G. Xu, D. Bi, H. Qu, X. Zou, X. Gao, H. Yang, H. He, X. Wang, J. Bao, S. Zuo, X. Pan, B. Zhou, G. L. Wang, S. Qu (2018) Comparative transcriptome analysis of Rhizoctonia solani-resistant and -susceptible rice cultivars reveals the importance of pathogen recognition and active immune responses in host resistance. J. Plant Biol. 61: 143-158.
Zhang, C. Q., Y. H. Liu, X. Y. Ma, Z. Feng, and Z. H. Ma (2009) Characterization of sensitivity of Rhizoctonia solani, causing rice sheath blight, to mepronil and boscalid. Crop Prot. 28: 381-386.
Zhang, J., and J. McD. Stewart (2000) Economical and rapid method for extracting cotton genomic DNA. J. Cotton Sci. 4: 193-201.
Zheng, A., R. Lin, D. Zhang, P. Qin, L. Xu, P. Ai, L. Ding, Y. Wang, Y. Chen, Y. Liu, Z. Sun, H. Feng, X. Liang, R. Fu, C. Tang, Q. Li, J. Zhang, Z. Xie, Q. Deng, S. Li, S. Wang, J. Zhu, L. Wang, H. Liu, and P. Li (2013) The evolution and pathogenic mechanisms of the rice sheath blight pathogen. Nat. Commun. 4: 1424.