臺灣博碩士論文加值系統

紅龍果為原產於南美之仙人掌科果樹，因其高產、營養豐富、貯藏期長及容易產調之特性，近年於世界各地廣泛栽培。 5℃冷藏可抑制紅龍果大多數採後病害，惟Alternaria果腐病仍會發生，並成為長期貯運的限制因子，本研究之目的在於探討病原菌之種類、田間感染時機及防治方法。經蒐集台灣9縣市之紅龍果果實及枝條共53株Alternaria分離株，利用gapdh基因進行建構親緣演化樹，分離株可區分為3個支序群，其中11株屬於A 支序群、13株屬B 支序群、29株屬C 支序群。再選擇28株以Alt a 1、gapdh、endoPG、ITS、rpb2及tef1等基因建構多基因親緣演化樹，4株A 支序群菌株屬於A. alternata，4株B 支序群菌株屬於A. gaisen， 20株C 支序群菌株分群接近A. burnsii及A. tomato，尚無法完全區分親緣關係，暫定為複合種(A. burnsii species complex；ABSC)，又可分為ABSC 1-3共3個支序群。為探討各親緣種的病原性，利用菌絲塊接種果實及肉質莖評估病原性，3個親緣種皆有菌株對果實有明顯之病原性，也有弱病原性之菌株；3個親緣種皆有菌株能對肉質莖產生外圍木質化的橘色或黑色的病斑。選擇A. alternata (HLF6102)、A. gaisen (F211144)、ABSC 1 (YPH103及H16-4)及ABSC 3 (F218004)等5株菌株作為代表菌株，調查溫度對菌絲生長及分生孢子發芽的影響，發現此5株菌株均可在5-30℃生長，5℃培養35天時，A. alternata (HLF6102)的菌落直徑最大。此5株分離株分生孢子在5-30℃時均可發芽，而5℃培養24小時發芽率最高之分離株為ABSC 1的YPH103及H16-4。接種分生孢子於果實上再以掃描式電子顯微鏡觀察侵入途徑，發現可產生類似附著器的膨大構造固著於果皮，再經由傷口及氣孔侵入感染。田間果實發育期感病時期分析顯示，‘大紅’及‘蜜寶’二品種果實在夏季於盛花後7-21天為較感病時期，冬季發病率低，無可區分之感病時期。為防治田間潛伏感染。調查A. alternata (HLF6102)及ABSC (YPH103)對紅龍果已登記之化學殺菌劑感受性，培養基試驗顯示二菌株對賽普護汰寧、得克利及克熱淨較敏感，遂進行‘大紅’及‘蜜寶’二品種的田間噴佈試驗，於果實採收貯藏後及模擬櫥架後調查罹病等級，顯示施用賽普護汰寧二次或三次較其他二種殺菌劑之處理能穩定降低罹病等級。非農藥防治方面，篩選出肉桂、丁香、檸檬香茅及百里香等精油能5℃揮發及接觸抑制HLF6102及YPH103菌絲生長，肉桂及百里香混合劑較單劑更能抑制HLF6102及YPH103菌絲生長。6.4%碳酸鉀及碳酸鈉能抑制HLF6102及YPH103菌絲生長，而6.4%碳酸鉀亦能抑制HLF6102分生孢子發芽。採後之非農藥處理仍需進行配方優化，方可整合田間防治，達到生產高品質之安全果品。

Pitayas or dragon fruits are native cacti of South America. They are commercially planted worldwide due to their high productivity, rich nutrients, and long shelf-life. Alternaria fruit rot often occur during storage at 5℃. The disease become the major limiting factor of long-term storage and transportation. This study was focused on identification of the pathogens, determination of disease susceptibility during fruit development, and control methods. We collected 53 Alternaria isolates from pitaya stems and fruits. The 53 isolates were selected to construct a phylogenetic tree based on the sequence gapdh, and clustered in 3 clades, 11 isolates in clade A, 13 isolates in clade B, and 29 isolates in clade C. Among them, 28 isolates were selected for a phylogenetic analysis based on partial sequences of ITS, Alt a 1, gapdh, rpb2, tef1, and endoPG genes. The result displayed that 4 isolates of A clade were A. alternata, 4 isolates of B clade were A. gaisen, and 20 isolates of C clade were closely related A. burnsii and A. tomato. A. burnsii species complex (ABSC). Five isolates, A. alternata (HLF6102), A. gaisen (F211144), ABSC 1 (YPH103 and H16-4), and ABSC 3 (F218004), were selected to determinate temperature effects on mycelium growth and condial germination. All the selected isolates grew mycelium and germinated condia in 5-30℃. Under 5℃, A. alternata HLF6102 has the biggest colony diameter, and ABSC 1 (YPH103 and H16-4) has the highest condial germination rate under 5℃. Scanning electron micrographs of pitaya peel inoculated with YPH103 condia indicate that Alternaria could form appressoria-like structure and attach firmly on the peel surface, and hyphae could penetrate fruit through stomata and wounds. To investigate disease susceptibility of developing fruit, conidia of HLF6102 and YPH103 were inoculated on ‘Da-Hong’ and ‘Mi-Pao’ pitayas during fruit developing period in different trials. Both cultivars were highly susceptible at 7-21 days after full bloom in summer but no distinguishable highly susceptible stage in winter. To develop control methods of Alternaria fruit rot, chemical fungicide sensitivity assays were conducted by using isolate HLF6102 and YPH103. Iminoctadine triacetate, tebuconazole, and cyprodinil + fludioxonil effectively inhibiting Alternaria mycelial growth in vitro, and were applied on field trials. Field trials demonstrated that the application of cyprodinil + fludioxonil two or three times stably decreased the disease severity scale of Alternaria fruit rot on ‘Da-Hong’ and ‘Mi-Pao’ pitaya fruits compared to treatments of the other fungicides. Cinnamon, clove, lemon grass, and thyme essential oil could inhibit HLF6102 and YPH103 mycelial growth by directly contact and vapour. The combination of cinnamon and thyme essential oils show better efficiency than single essential oil. The 6.4% of potassium carbonate and sodium carbonate could inhibit HLF6102 and YPH103 mycelial growth, and 6.4% of potassium carbonate could inhibit HLF6102 conidial germination. Non-chemical control need additional recipe optimization and may be integrated with field control to produce high quality pitayas in the future.

目次
中文摘要 i
Abstract iii
表次索引 vii
圖次索引 viii
前言 1
前人研究 3
材料方法 9
一、試驗菌株來源、保存及接種源製備 9
二、DNA萃取、基因片段增幅及定序 10
三、親緣演化樹之建構 11
四、形態特徵 11
五、紅龍果果實接種試驗 12
六、紅龍果肉質莖接種試驗 12
七、溫度對菌絲生長及分生孢子發芽的影響 13
八、紅龍果Alternaria之侵入途徑 14
九、田間果實感病時期分析 14
十、化學殺菌劑防治紅龍果褐斑病 15
十一、無機鹽類對紅龍果褐斑病之防治 17
十二、精油對紅龍果褐斑病之防治 18
結果 22
一、分子鑑定 22
二、形態特徵 24
三、紅龍果果實接種試驗 26
四、紅龍果肉質莖接種試驗 26
五、 溫度對菌絲生長及分生孢子發芽的影響 27
六、紅龍果Alternaria之侵入途徑 28
七、田間果實感病時期分析 29
八、化學殺菌劑對褐斑病菌絲之抑制 29
九、化學殺菌劑防治紅龍果褐斑病 30
十、無機鹽類對紅龍果褐斑病之防治 32
十一、精油對紅龍果褐斑病之防治 33
討論 36
一、分子鑑定 36
二、形態特徵 39
三、紅龍果果實接種試驗 40
四、紅龍果肉質莖接種試驗 41
五、 不同菌株菌絲生長及分生孢子發芽速率之溫度效應 41
六、紅龍果Alternaria之侵入途徑 42
七、田間果實感病時期分析 43
八、化學殺菌劑對褐斑病菌絲之抑制 44
九、化學殺菌劑防治紅龍果褐斑病之田間試驗 45
十、無機鹽類對紅龍果褐斑病之防治 47
十一、精油對紅龍果褐斑病之防治 48
結語 51
參考文獻 52
附錄 111

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