Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/30924
標題: 唐菖蒲萎凋病菌之鑑定與田間病害傳播
Methods for Identification and Detection of Fusarium oxysporum f. sp. gladioli , the Causal Agent of Gladiolus Fusarium Wilt and It's Dissemination
作者: 陳昱初
Chen, Yu-Chu
關鍵字: Gladiolus Fusarium wilt;唐菖蒲萎凋病;selective medium;specific primer;Lloyd,s index;spatial aggregation;infected gladiolus corm;病原菌鑑定;病原性測定;選擇性培養基;病害之田間分佈
出版社: 植物病理學系所
引用: 引用文獻 1. 王毓華. 1993. 唐菖蒲採粉結實及其種子發芽之研究. 國立中興大學園藝學研究所碩士論文。 2. 林向. 1991. 季節、地區及品種對唐菖蒲開花品質與結球之影響. 國立臺灣大學園藝學研究所碩士論文。 3. 高衛婷. 1999. 百合萎凋病菌Fusarium oxysporum f. sp. lilii生物學及病原性測定. 國立中興大學植物病理學系碩士論文. 台中市. 58pp。 4. 孫守恭. 1975. Fusarium 屬病原菌在土壤中的生態. 植保會刊17:216-232。 5. 孫守恭、黃振文. 1996. 台灣植物鐮孢菌病害. 世維出版社出版. 台中市. 170pp。 6. 黃振文、孫守恭。1997。臺灣產鐮孢菌。世維出版社。116頁。 7. 徐世典等編. 2002. 台灣植物病害名彙(四版). 中華植病學會出版. 台中. 386頁。 8. 陳昱初、黃一修、謝文瑞. 2004. 高屏地區唐菖蒲萎凋病之傳播途徑與田間分佈分析. 植病會刊13:177-182。 9. 蔡素蕙. 1993. 唐菖蒲之切花栽培(上). 臺灣花卉園藝74:42-45。 10. 蔡素蕙. 1993. 唐菖蒲之切花栽培(下). 臺灣花卉園藝75:33-36。 11. 謝式坢鈺. 1985. 唐菖蒲萎凋病之生態及防治. 植保會刊27:247-256。 12. Awuah, R. T., and Lorbeer, J. W. 1986. A sorbose-based selective medium for enumerating propagules of Fusarium oxysporum f. sp. apii race 2 in organic soil. Phytopathology 76:1202-1205. 13. Bald, J.G., Suzuki,T., and Doyle, A. 1971 Pathogenicity of Fusarium oxysporum to Easter lily, Narcissus and Gladiolus. Ann. Appl. Biol. 67:331-342. 14. Bower, J. H., and Locke, J. C. 2000. Effect of botanical extracts on the population density of Fusarium oxysporum in soil and control of Fusarium wilt in the greenhouse. Plant Dis. 84:300-305. 15. Buxton, E. W. 1955. The taxonomy and variation in culture of Fusarium oxysporum from gladiolus. Trans. Br. Mycol. Soc. 38:202-212. 16. Campbell, C. L., and Madden, L. V. 1990. Analysis of Spatial in Epidemics. Pages 295-311 in: Introduction to Plant Disease Epidemiology. John Wiley & Sons, New York , 532 pp. 17. Cook, R. J., and Schroth, M. N. 1965. Carbon and nitrogen compounds and germination of chlamydospores of Fusarium solani f. sp. phaseoli. Phytopathology 55:254-256. 18. Edel, V., Steinberg, C., Avelange, I., Laguerre, G., and Alabouvette, C. 1995. Comparison of three molecular methods for the characterization of Fusarium oxysporum strains. Phytopathology 85:579-585. 19. Klotz, L. V., Nelson, P. E., and Toussoun, T. A. 1988. A medium for enhancement of chlamydospore formation in Fusarium species. Mycologia 80:108-109. 20. Komada, H. 1975. Development of a selective medium for quantitative isolation of Fusarium oxysporum from natural soil. Rev. Plant Prot. Res. 8:114-125. 21. Linderman, R. G. 1981. Fusarium disease of flowering bulbcrops. The Pennsylvania State University Press. University Park and London. Pp.129-141. 22. Magie, R. O. 1966.Gladiolus Fusarium disease development and control.Gladiolus 41:106-110. 23. Manulis, S., Kogan, N., Reuvenm, M., and Ben-Yephet, Y. 1994. Use of the RAPD technique for identification of Fusarium oxysporum f. sp.dianthi from carnation. Phytopathology 84:98-101. 24. Massey, L. M. 1926. Fusarium rot of gladiolus corms. Phytopathology 16:509-523. 25. Mes, J. J., van Doorn, J., Roebroeck, E. J. A., and Boonekamp, P. M. 1994. Detection and identification of Fusarium oxysporum f. sp. gladioli by RFLP and RAPD analysis. P 63-68. Mordern Assays for Plant Pathogenic Fungi. CAB International, Oxford, 1994. 26. Mes, J. J., van Doorn, J., Roebroeck, E. J. A., van Egmond, E., van Aratrijk, J., and Boonekamp, P. M. 1994. Restriction fragment length polymorohisms, races and vegetative compatibility groups within a worldwide collection of Fusarium oxysporum f. sp. gladioli. Plant Pathol. 43:362-370. 27. Mullis, K. B., and Faloonan, F. A. 1987. Specific synthesis of DNA in vitro via a polymerase catalysed chain reaction. Method Enzymol. 155:335-350. 28. Nash, S. M., and Snyder, W. C. 1962. Quantitative estimation by plate counts of propagules of the bean root rot Fusarium in field soil. Phytopathology 52:567-572. 29. Hartman, G. L., Kull, L., and Huang, Y. H. 1998. Occurrence of Sclerotinia sclerotiorum in Soybean Fields in East-Central Illinois and Enumeration of Inocula in Soybean Seed Lots. Plant Dis. 82:560-564. 30. Roebroeck, E. J. A., Groen, N. P. A., and Mes, J. J.1990. Detection of laten Fusarium oxysporum in Gladiolus corms.Acta.Hortic. 266:469-476. 31. Snyder, W. C., and Hansen, H. N. 1945. The species concept in Fusarium with reference to discolor and other sections. Am. J. Bot. 32:657-666. 32. Straathof, Th. P., Jansen, J., and Loffler, H. J. M. 1993. Determination of Fusarium oxysporum in Lilium. Phytopathology 83:568-572. 33. Sun, E. J., Su, H. J., and Ko, W. H. 1978. Identification of Fusarium oxysporum f. sp. cubense race 4 from soil or host tissue by cultural characters. Phytopathology 68:1672-1673. 34. Wilfret, G.J. 1980. Gladiolus pp.166-181.In: Introduction to Floriculture. R.A. Larson, ed. Academic Press, New York, 35. Wong, W. C. 1988. A differential medium for the identification of races 1 and 4 of Fusarium oxysporum f. sp. cubense. Letters Appl. Microbiol. 6:51-54.
摘要: 
本文主要在研發選擇性培養基以供分離、鑑定與偵測土壤及種球之唐菖蒲萎凋病菌(Fusarium oxysporum f. sp. gladioli),並探討病害接種原來源及病害田間之傳播。調查發現臺灣唐菖蒲種植區,包括宜蘭、台中后里、彰化、雲林、高雄及屏東等地均有病害發生。從各地採集之罹病種球、根、莖、葉片,及發病田土壤經分離培養後所得之病原菌,根據Snyder 與Hansen之分類系統,以形態特性為鑑定依據,並經病原性測定後,獲得82個唐菖蒲萎凋病尖鐮孢菌菌株,從中選取Fog051等5個菌株做為研發選擇性培養基的供試菌株。選擇性培養基每公升含L-asparagine, 2 g; D-galactose, 20 g; MgSO4‧7H2O, 0.5 g; NaB4O7‧10H2O, 1 g; K2HPO4, 1 g; KCl, 0.5 g; Fe(EDTA), 5 mg; oxgall, 0.5 g; PCNB(pentachloronitrobenzene), 1 g; Benomyl(50% WP), 1 g; streptomycin sulfate, 0.3 g; Agar, 20 g,以10%磷酸(phosphoric acid , H3PO4)溶液調整酸鹼值,不同酸鹼值之培養基具不同之功用,pH 4.0時唐菖蒲萎凋病菌孢子可正常發芽,但可抑制所有其它供試菌株孢子發芽,同時發現只有唐菖蒲與百合萎凋病菌(F. oxysporum f.sp. lilii)之菌絲在此酸鹼值培養基上可正常生長,但兩者之菌落形態明顯不同。當培養基酸鹼值調為 2.0時,僅唐菖蒲萎凋病菌菌絲可以生長,其它供試菌株之菌絲生長皆被抑制。以pH 4.0之培養基分離人工製作之病土,其回收率可達96%以上,最低密度可偵測到每克土壤中含有50個繁殖體。以pH 2.0之培養基分離唐菖蒲罹病植株及球莖組織可於三天內偵測出病原菌。本研究亦利用聚合酶連鎖反應偵測唐菖蒲萎凋病菌,透過電腦軟體Prime程式分析選取之專一性核酸引子FOG-W6L-001F (序列為5’-TGAAAGCAGATGGAAGAAACAG-3’) 與FOG-W6L-006R (序列為5’-ACGACTCTTCCTCGGACAAA- 3’),測試唐菖蒲病原性菌株(Fog051、Fog052),百合病原性菌株(F016、FOL001),及非病原性菌株(F032),經PCR增幅及電泳分析結果顯示Fog051、Fog052皆會在721bps 位置產生專一性之去氧核醣核酸片段,其他菌株則無增幅產物出現。調查屏東縣東港、萬丹等地之唐菖蒲採種田種球帶菌率,發現唐菖蒲植株罹病度與種球的帶菌率間具有顯著的相關性(r2=0.738),證明帶菌種球是傳播本病害的途徑之一。此外,在高屏地區的唐菖蒲栽培田區,以羅依得氏指數(Lloyd,s index of patchiness)統計分析萎凋病在田間的傳播,發現病害分佈呈聚集狀,且其病害指數分佈圖亦呈現病害群集(aggregation)發生的現象,由此可知帶菌土壤也是本病害傳播的另一途徑。

Gladiolus Fusarium wilt , caused by Fusarium oxysporum f. sp. gladioli, is the most of destructive soil-borne disease during it's growing period. Isolation and the identification of the pathogen was not only laborious but also time-consuming. It is necessary to develope an efficient technique to estimate the population of F. oxysporum f.sp. gladioli ,the gladiolus Fusarium wilt pathogen, in natural soil and diseased plants, which is applicable to research on ecology of the pathogen. The objective of this study was to develop a selective medium for isolation and detection of F. oxysporum f.sp. gladioli. The selective medium (pH2.0) consisted K2HPO41g/L, KCL 0.5g/L, MgSO4·7H2O 0.5g/L, Fe-Na-EDTA 0.05g/L, L-Asperagine 2g/L, D-galactose 20 g /L, PCNB 1g/L, Oxgall 0.5g/L, Streptomycin sulfate 300ppm, Benomyl 1000 ppm, agar 20g, distilled water 1L. Test result proved that F. oxysporum f.sp. lilii , F. oxysporum f.sp. luffae , F. oxysporum f.sp. cucumerinum , F. oxysporum f.sp. tracheiphilum , F. oxysporum f.sp. monodricae , F. oxysporum f.sp. melonis , F. oxysporum f.sp. cubensei and non-pathogenic F. oxysporum were not able to grow on the selective medium plate. This medium was more sensitive than those of Nash-PCNB and Komada for distinguishing F. oxysporum f.sp. gladioli from the other Fusarium species. The recovery rates of F. oxysporum f.sp. gladioli (FOG051) from the artificially infested soil was 96%. RAPD was used first to screen the total DNA of pathogenic and non- pathogenic Fusarium oxysporum isolates using 100 random primers. The primer OPAW-06 (5''-TTTGGGCCCC-3'') was selected, and the specific DNA segment (890bp) was amplified in the total DNA of pathogenic isolates present. The specific DNA segment amplified was recovered, cloned, and sequenced. The specific primer set FOG-W6L-001F/FOG-W6L-006R was designed and assembled according to the nucleotide sequence of the specific DNA segment. The detection of F. oxysporum f.sp. gladioli from mycelium can be conducted using the FOG-W6L-001F/FOG-W6L-006R specific primer. The results showed that the specific primer set developed can successfully detect F. oxysporum f.sp. gladioli in pure culture. In Kaohsiung-Pingtung area disease occurrence and distribution in monoculture fields had been studied from March 2002 to March 2003. According to Lloyd,s index analysis, field distribution of the disease has a spatial aggregation that matched a grid pattern plotted by disease severity. The results showed that the disease was soil-borne. In addition to being soil-borne, infected gladiolus corms causing disease in field were also found in this study and were correlated to disease severity based on the data from Dunggang and Wandan in Pingtung County. This evidence proved that F. oxysporum f. sp. gladioli was transmitted through either infested soil or infected gladiolus corms.
URI: http://hdl.handle.net/11455/30924
其他識別: U0005-2505200615225800
Appears in Collections:植物病理學系

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