Syntactic bootstrapping in children with mental retardation

Thesis (B.Sc)--University of Hong Kong, 2004."A dissertation submitted in partial fulfilment of the requirements for the Bachelor of Science (Speech and Hearing Sciences), The University of Hong Kong, December 31, 2004."Also available in print.published_or_final_versionSpeech and Hearing SciencesBachelorBachelor of Science in Speech and Hearing Science

Occurrence, Pathogen Characterization and Control of Phomopsis destruens Causing Foot Rot of Sweet Potato

由 Phomopsis destruens (Harter) Boerema 引起之甘藷基腐病為近年來嚴重影響國內甘藷生產之重要限制因子，本病害不僅危害鮮藷用之「台農57 號」及「台農66 號」，亦危害葉菜用甘藷「台農71 號」，顯示台灣主要栽培的甘藷品種皆不具抗病性。其病徵為在甘藷主藤蔓靠土表的莖基部造成黑褐色乾枯，使受害植株之地上部生長勢衰弱、葉片黃化枯萎、新葉變小，同時由於乾枯部位緊鄰塊根生長處，因此塊根發育亦會受阻，造成塊根受害部位表面呈淡褐色濕腐，進而導致產量嚴重損失及儲藏時之病害問題。本病原菌在甘藷病株藤蔓表面佈滿大小不一、黑色、凸起狀之柄子器，內含兩型分生孢子。本病害之主要感染源為罹病種苗，因此清潔種苗來源為防治本病害之重要策略；於曾發病之甘藷田進行淹水處理2 週以上或與水稻輪作，可減少病藷及病殘體上之菌體，避免作為下一期作之感染源；另外藥劑防治試驗結果顯示，貝芬菲克利及腐絕均具有顯著防治本病害發生之效果。綜合上述本病害之防治策略為1.選用經認證之健康種苗進行栽植。2.田間栽培時期及採收前若發現發病植株應整株含地下部進行清除，並移出田間，以減少田間感染源。3.發病區建議於種植前進行淹水或水稻及非寄主作物輪作，以降低田間感染源密度。 Foot rot disease is a major limiting factor of sweet potato production in recent years. The disease was harmful to commercial root-used varieties of sweet potato, TN57 and TN66, as well as vegetable-used variety, TN71. It suggested that the majority of sweet potato cultivars in Taiwan were not resistant to this disease. The symptoms of this disease included yellowing of leaves, necrotic lesions on vine, and wilting of the sweet potato plants. The pathogen might grow from cankers on the vines down into storage roots, having brown wet rot lesions on the skin of storage root, and caused to severe yield loss and storage disease problems. The fungus generated two types of conidia from stromataic pycnidia on infected vines. The disease seedling was a major inoculum source. Therefore, good quality of seedlings would be an important factor to control the foot rot of sweet potato in the field. It was recommended that the field with the foot root disease should be rotated with rice annually or being flooded for more than 2 wk after harvesting season, and it could eradicate the pathogen as well as avoid the infection of foot rot disease next season. In the chemical control, the result showed that Carbendazim + Hexaconazole and Thiabendazole were effectively inhibited mycelial growth. From all the experimental data, possible integrated control strategies to the disease include (1) cultivating certified healthy seedling, (2) eliminating all disease-infected plants in the field, and (3) adopting flooding treatment or rotation of rice and non-host crops prior to planting season to reduce the pathogen density of the field

(67(4):387-400)Physiological Characteristics, Pathogenicity and Fungicide Screening of Citrus Alternaria Brown Spot Disease Caused by Alternaria alternata

柑橘褐斑病在2012–2013 年間於台灣之「茂谷柑」、「佛利蒙柑」及「帝王柑」等柑橘園區之春梢期陸續發生。本研究除針對該病原菌進行生理特性調查外，亦對不同柑橘品種之病原性及其防治藥劑進行篩選評估。柑橘褐斑病菌分離株 (Alternaria alternata Alt-001) 可在「茂谷柑」、「明尼橘柚」、「桶柑」、「佛利蒙柑」、「晚崙西亞」、「豔陽柑」及「台農天王柑」等柑橘品種之離葉嫩葉傷口接種時，造成褐色病斑，但在粗皮檸檬之嫩葉上則不造成病斑。本病原最適菌絲生長溫度為25℃，而25–30℃為孢子發芽最適溫度。測試殺菌劑對其菌絲生長及孢子發芽影響時，結果顯示快得寧在10 mg a.i. L-1 有效濃度下可有效抑制菌絲生長，抑制率達99.2%；而快得寧、氫氧化銅、鋅錳乃浦及扶吉胺等4 種藥劑可顯著降低病原菌之孢子發芽，其中植株噴布快得寧、氫氧化銅及鋅錳乃浦藥劑處理21 d 後，A. alternata Alt-001分離株在其葉片上之孢子發芽率仍顯著低於噴布水之對照組。快得寧、氫氧化銅、鋅錳乃浦及扶吉胺為目前官方推薦柑橘真菌性病害之防治藥劑，從藥效評估結果，上述藥劑可一併作為防治柑橘褐斑病之用藥。 Alternaria brown spot of citrus (Citrus reticulata) was found in orchards of ‘Murcott’, ‘Fremont’, and ‘Gonggan’ citrus in Taiwan during 2012–2013. This disease seriously affected the growth of citrus in spring shoot period. The objective of this study is to investigate the physiological characteristics and pathogenicity of Alternaria alternata, the causal agent of the brown spot disease, and further screen the effectiveness of fungicides. Seven cultivars included ‘Murcott’, ‘Minneola’, ‘Tankan’, ‘Fremont’, ‘Valencia’, ‘Sunburst’, and ‘Tainung Giant’. Results showed that brown-to-black lesions appeared on the detached young leaves inoculated through wounds, but no symptom appeared on rough lemon after wound inoculation. The optimal temperature for mycelial growth and spore germination of A. alternata strain Alt-001 and Alt-008 was 25℃ and 25–30℃, respectively. Mycelial growth was effectively inhibited by 10 mg a.i. L-1 oxine-copper, the inhibition rate of 99.2% was measured by screening. Spore germination of A. alternata was inhibited by oxine-copper, copper(II) hydroxide, mancozeb, and fluazinam in vitro. Futhermore, spore germination of A. alternata was continuously inhibited by oxine-copper, copper(II) hydroxide, and mancozeb in greenhouse 21 d after fungicide treatment. These fungicides have been recommended to control other fungal diseases of citrus. Therefore, it is suggested that they could also be used to control Alternaria brown spot of citrus in term of efficacy

(68(4): 333-340)Fruit Rot of Passion Fruit Caused by Lasiodiplodia theobromae in Taiwan

2017 年7 月中旬於南投縣埔里鎮大坪頂地區之百香果果園中發現一種新病害，受害果實出現墨綠色至黑色塊斑而外圍有黃暈，病斑易擴大聯結成大範圍灰黑色並呈現凹陷的塊斑，又於塊斑上出現黑色小點 (為病原菌之柄子器)。剖開果實可見果皮內側黑化且易產生黑灰色菌絲，果肉失去原有的風味。本研究自罹病果實穩定分離得到一種真菌，培養於馬鈴薯葡萄糖培養基 (potato dextrose agar; PDA) 培養基上，該菌絲生長快速，於24℃下培養4 d 後，其菌落呈現灰黑色毛絨狀，具有濃密之氣生菌絲。本菌之分生孢子初期呈卵圓形、透明、單室且細胞壁厚，逐漸轉變成褐色，中間有一隔膜且具有明顯縱紋。依據本病害之病徵表現、病原菌形態特徵及分子序列鑑定結果，將此菌鑑定為Lasiodiplodia theobromae。經人工接種孢子懸浮液於「台農1 號」百香果果實後，發現L. theobromae 對百香果具有病原性，並能再分離得原接種的真菌，完成柯霍氏法測，證明此菌為引起百香果果腐病之病原菌。另外，無傷口者，則不易造成果實發生果腐病。本文由L. theobromae 引起百香果果腐病，為台灣百香果病害新紀錄。 A new disease caused by fruit rot of passion fruit (Passiflora edulis) was found in major passion fruit production areas at Puli Township, Nantou County of Taiwan in 2017. The infected fruits showed dark green to black patches with yellow halo. The lesions usually enlarged and joined into a large black and sunken patch with the appearance of black dots (the pycnidia of the pathogen). The flesh inside was blackened with soar flavor and easy to produce black gray hyphae. A kind of fungus was consistently isolated from the diseased tissue. The mycelium of the fungus grew rapidly on potato dextrose agar and formed a grayish-black fluffy colony with dense aerial hyphae at 24℃ after being cultured for 4 d. The conidia of the fungus were initially oval, hyaline, unicellular and had thick cell wall, but days later showed dark-brown color and developed one septum with longitudinal lines. The causative fungus was identified as Lasiodiplodia theobromae based on morphological characteristics and internal transcribed spacer (ITS) rDNA sequence blasting on National Center for Biotechnology Information (NCBI) GenBank database. The identified L. theobromae could cause similar symptoms on the fruits on trees and the detached fruits by inoculating the tested fruits with conidia suspension through wound treatment, and the same pathogen could be re-isolated from the diseased fruit tissue to fulfill the Koch’s rules. Immature and mature passion fruit could show disease symptoms when inoculated with L. theobromae through wound treatment but no symptom was found to those without wound treatment. This study is the first report about fruit rot of passion fruit caused by L. theobromae in Taiwan

(66(1):66-73) Screening of Fungicides for Foot Rot of Sweet Potato Caused by Phomopsis destruens

甘藷基腐病為近年來甘藷之重要病害之一，本研究針對該病原菌 (Phomopsis destruens) 進行其防治藥劑篩選評估，測試殺菌劑對本菌之菌絲生長及分生孢子發芽影響。結果顯示，撲克拉錳、貝芬依滅列、克熱淨、菲克利腐絕、貝芬撲克拉、滅特座、貝芬菲克利及腐絕等8 種藥劑處理皆可有效抑制菌絲生長，而在10 mg a.i. L-1 有效成分濃度下，賽普護汰寧、撲克拉錳、貝芬依滅列、克熱淨、菲克利腐絕、貝芬撲克拉、亞托敏、依普同、滅特座、扶吉胺、貝芬菲克利及腐絕等12 種藥劑皆可顯著降低病原菌之分生孢子發芽。另於溫室實際應用克熱淨、滅特座、扶吉胺、貝芬菲克利、腐絕及菲克利腐絕等6 種化學藥劑進行甘藷基腐病之防治測試，結果顯示腐絕、菲克利腐絕及貝芬菲克利等3 種藥劑施用時可顯著抑制此本病害之發生。進一步瞭解腐絕在藷塊上農藥殘留情形，結果顯示於施用後30 d 及60 d，藷塊之藥劑殘留量皆為0.1 mg L-1 以下，為低殘留藥劑，建議未來可考量優先以腐絕為供試藥劑進行甘藷基腐病之田間防治試驗。 Foot rot is one of emerging diseases for sweet potato in recent years. The objective of this study was to screen fungicides for foot rot diseases caused by Phomopsis destruens. The results showed that mycelial growth was effectively inhibited by prochloraz-manganese, carbendazim + imazalil, iminoctadine triacetate, hexaconazole + thiabendazole, carbendazim + prochloraz, metconazole, carbendazim + hexaconazole and thiabendazole. Spore germination of P. destruens was inhibited by cyprodinil + fludioxonil, prochloraz-manganese, carbendazim + imazalil, iminoctadine triacetate, hexaconazole + thiabendazole, carbendazim + prochloraz, azoxystrobin, iprodione, thiabendazole, metconazole, fluazinam and carbendazim + hexaconazole in 10 mg a.i. L-1. In greenhouse experiments, the results indicated that thiabendazole, hexaconazole + thiabendazole and carbendazim + hexaconazole were effective fungicides for controlling foot rot. Fungicide residues in the case of tubers showed that remains of pesticide in tubers were approximately 0.1 ppm after drenching Thiabendazole after 30 days and 60 days. The results revealed that thiabendazole was low remains of fungicide. Therefore, it is recommended that thiabendazole is the priority chemical for controlling foot rot disease over other fungicides in the future-field experiment of sweet potato

(66(2):158-166)Circular Spot of Sweet Potato Caused by Sclerotium rolfsii in Taiwan

甘藷白絹病為2015 年陸續在彰化、雲林及屏東甘藷栽培田出現之嚴重病害。其主要病徵為藷塊上產生黃褐色圓型病斑，大多數病斑在甘藷採收後雖不會繼續擴大，但嚴重影響品質。由病組織所分離之病原菌，培養時產生白色菌絲，具有扣子體，並會產生褐色菌核 (brown sclerotia)，依據形態及分子鑑定為Sclerotium rolfsii Sacc.。本菌於15–35℃培養時菌絲均會生長，30℃為最適生長溫度。病原菌不僅在甘藷藷塊造成病斑，亦對葉用甘藷苗具有病原性，造成幼苗莖基部褐化，植株倒伏，枯死。測試9 種殺真菌劑對本菌菌絲之生長抑制情形，結果顯示50.0% 福多寧 (flutolanil)、75.0% 滅普寧 (mepronil)、7.5% 依普座(epoxiconazole)、23.0% 菲克利 (hexaconazole) 及25.0% 賓得克利 (pencycuron + btebuconazole) 在10 mg a.i. L-1 有效濃度下對其菌絲生長抑制率均可達89.2% 以上。本文為國內第一篇闡明由S. rolfsii 引起之甘藷白絹病並提供防治藥劑篩選結果之報告，可供農業研究人員參考。 In 2015, a severe disease outbreak on sweet potato storage root with the symptoms of circular yellowish brown to brown lesions occurred in Chunghua, Yunlin, and Pingtung areas. The pathogen isolated from the diseased tissue had white mycelium, clamp connection, and it produced brown sclerotia, which was identified as Sclerotium rolfsii Sacc. based on these morphological characters and ITS sequence. The growth temperature of pathogen isolates ranged from 15 to 35℃, and the optimum growth temperature was 30 ℃. The pathogen isolate, SCL-003, showed pathogenic ability on sweet potato storage roots and seedlings. Nine fungicides were selected for evaluating the inhibition effect on mycelial growth of two pathogen isolates (SCL-001 and SCL-003). The results showed that the effective fungicides with more than 89% inhibition rate were 50% flutolanil, 75.0% mepronil, 7.5% epoxiconazole, 23.0% hexaconazole, and 25.0% pencycuron + tebuconazole with 10 mg a.i. L-1 dilution concentration. This is the first report of circular spot disease on sweet potato caused by Sclerotium rolfsii in Taiwan

(64(1):21-31)Survey and Fungicides Screening for Mango Postharvest Disease Caused by Phomopsis mangiferae

Phomopsis mangiferae 係引起台灣檬果果實果腐病及蒂腐病病原菌之一，本研究針對該病原菌進行其在田間引起病害之發生率調查及其防治藥劑篩選評估。由2009–2011 年間在台南市玉井及官田地區之愛文檬果果實採後病害調查資料顯示，官田地區檬果果實發生採後果腐或蒂腐病之平均罹病率有51.5%，皆由P. mangiferae 所引起，明顯高於玉井地區之11.9%。由此得知，官田地區的果腐病或蒂腐病之主要病原菌為P. mangiferae。續將3 株P. mangiferae 分離株之分生孢子無傷口接種於田間檬果樹上之未成熟果實，結果顯示大部分果實均可於果實採收後於接種部位出現果腐病斑，顯示本菌於無傷口接種時亦具有感染能力。測試賽普護汰寧、貝芬依滅列、依普同、腐絕、貝芬菲克利、免得爛、甲基多保淨、亞托待克利、亞托敏及得克利等10 種藥劑對P. mangiferae 之菌絲生長及孢子發芽影響，結果顯示得克利、亞托待克利及貝芬菲克利在10–100 mg a.i. L-1 有效成分濃度下，對所測試之3 株P. mangiferae 菌株具有97% 以上之菌絲生長抑制率；而賽普護汰寧、亞托敏及亞托待克利處理下，則可顯著降低病原菌之分生孢子發芽率。以上藥劑皆為目前檬果炭疽病之推薦防治藥劑，於田間防治時可參考作為防治由P. mangiferae 引起之採收後病害之用。 Phomopsis mangiferae is one of pathogens causing stem-end rot or fruit rot disease of mango in Taiwan. The objectives of this study were to survey and screen fungicides for mango postharvest diseases caused by P. mangiferae. A field survey was performed at Yujing and Guntian Townships in Tainan City during 2009–2011. The results showed this disease incidence was 51.5% in Guntian, greater than 11.9% in Yujing. The results showed that P. mangiferae was the major pathogen of stemend rot or fruit rot disease of mango in Guntian Township. Moreover, three isolates of P. mangiferae, Ph-453, Ph-546 and Ph-550, were inoculated on immature mango fruits, and symptom of fruit rot appeared on the fruits after harvest and ripening. Pathogenicity tests indicated that all of three isolates were pathogenic to mango fruit after harvested. The ten fungicides (cyprodinil + fludioxonil, carbendazim + imazalil, iprodione, thiabendazole, carbendazim + hexaconazole, metiram, thiophanate methyl, azoxystrobin + difenoconazole, azoxystrobin and tebuconazole) were tested theirs efficacy on inhibiting mycelial growth and spore germination of P. mangiferae Ph-453, Ph-546 and Ph-550 isolates in vitro, respectively. The results showed that tebuconazole, azoxystrobin + difenoconazole, and carbendazim + hexaconazole had more than 97% of inhibition when their effective concentrations were from 10 mg a.i. L-1 to 100 mg a.i. L-1. cyprodinil + fludioxonil, azoxystrobin, and azoxystrobin + difenoconazole were effectively inhibited spore germination. These fungicides are currently recommended to the control of anthracnose for mango, and they could also be used to control the postharvest diseases of mango fruit caused by P. mangiferae in the field

(65(1):45-53)The Study of Physiological Characteristics and Control of Phomopsis destruens Causing Foot Rot of Sweet Potato

甘藷基腐病 (foot rot) 由Phomopsis destruens 所引起，為近年來限制甘藷生產之重要病害。本研究於甘藷田區進行甘藷基腐病發生調查，得知此病源菌不僅危害鮮藷用之「台農57 號」及「台農66 號」，亦危害葉菜用甘藷「台農71 號」，顯示台灣主要栽培的甘藷品種皆不具抗病性。本病原菌絲生長之最適溫度為20℃，於25–30℃時為分生孢子發芽適溫，而15–30℃範圍本病害均會發生。於健康種苗、罹病種苗及病土對甘藷基腐病發生之影響試驗中，發現罹病種苗種植於健康土中具有100% 發病率，顯示罹病種苗為本病害重要感染源之一，因此清潔種苗來源為防治本病害之重要策略。另外，本研究進行淹水處理對病害發生之影響評估，結果發現病藷淹水1 wk 後即失去作為感染源之能力，而罹病藤蔓於淹水2 wk 以上亦可喪失作為田間感染源之能力。因此，建議發病田區應儘量與水稻輪作，若未能種植水稻仍應執行淹水2 wk 以上，以消滅病藷及罹病殘體上之菌體，避免成為下一期作之感染源。 Foot rot disease is a major limiting factor for sweet potato production in recent years. In this study, foot rot of sweet potato was investigated in different growing regions of Taiwan. Results showed that the disease was harmful to commercial root-used varieties of sweet potato, ‘TN57’ and ‘TN66’, as well as vegetable-used variety, ‘TN71’, suggesting that the major cultivars of sweet potato in Taiwan were not resistant to the disease. The optimal temperature of mycelial growth and spore germination of Phomopsis destruens were 20℃ and 25–30℃, respectively. The maximum incidence of foot rot disease occurred in range of 15–30℃. The incidence of foot rot was 100% when the diseased seedlings were grown in uninoculated soil for 12 wk, suggesting that the disease seedling was a major inoculum source. Therefore, good quality of seedlings would be an important factor to control the foot rot of sweet potato in the field. However, when the field grown with diseased tuber and stem were treated with flooding for 1 wk and 2 wk, respectively, the disease incidence of foot rot was reduced to zero. As a result, the field with the foot root disease should be rotated with paddy rice annually or flooded for more than 2 wk after the previous harvest to eradicate the pathogen avoiding the infection in the next season

Potato common scab caused by Streptomyces scabies in Taiwan - biological characteristics of the pathogen and an attempted biocontrol by antagonistic Bacillus subtilis var. amyloliquefaciens WG6-14

民國 95 年冬季在中台灣的台中縣潭子鄉與雲林縣斗南鎮地區所收成之馬鈴薯突見瘡痂病之大發生，由於類似的大規模發生前所未見，且在過去兩年間發病率與嚴重程度均有增加情形，一時成為倍受栽培業者關注之課題。本研究旨在探討最近在中南部地區普遍發生之馬鈴薯瘡痂病病原菌之生物特性，及其可能解決方法。本研究計利用由罹病薯塊樣品所分離到 7 個具典型鏈黴菌菌落形態之分離菌株做為供試病原菌，其中 T1、T2、T3、T4、T5 與 T6 等為本研究由採集自潭子地區罹病薯塊分離獲得，D1 菌株則由行政院農業委員會台南區農業改良場 鄭安秀博士提供，分離自斗南地區所採集樣本。7 個供試病原菌株經於 ISP4 (International Streptomyces Project ) 培養基上培養，所長成菌落俱呈灰色，顯微鏡檢視下可見，其均可產生氣生菌絲並於其上著生螺旋狀之孢子鏈；且均可於 ISP 單一碳素源供給下生長良好；另由tyrosine agar 上黑色素 (melanin) 產生特性及膜脂肪酸含量檢測等，所獲結果顯示此 7 個供試菌株之生理、生物特性均與Streptomyces scabies 相當一致。繼而由選殖、解序各供試菌株之 16S rDNA 序列結果，經GenBank分析比對，進而證實其與同屬於 S. scabies 之已知 4 個菌株相同度 (identity) 高達99％，綜合上述型態特徵、生理與生化特性及16S rDNA 序列分析檢測結果，證實本研究中所應用 7 個供試病原菌株均歸屬於 Streptomyces scabies。由於鏈黴菌屬 (Streptomyces) 成員為土壤中腐生性、並被廣泛應用於對抗土傳性病原真菌的有益微生物，本研究進而比較此些病原菌株與本實驗室經一系列實驗證實對植物性病害具有防治效果的鏈黴菌 S1、S3、S4、B4 與 M6 等 5 個菌株間之差異性，次釐清供生物殺菌劑發展應用鏈黴菌株於作物栽培上廣為應用後可能存在之潛在風險。上述 7 個病原菌株於人工接種下，均可造成馬鈴薯薄片組織之褐化與蘿蔔新生幼苗生長之嚴重受抑制反應，另以 1×108 cfu/ml 濃度孢子懸浮液澆灌盆缽種植馬鈴薯則 7 個菌株皆可在所結成薯塊上造成表皮破裂、龜裂狀、隆起之典型瘡痂病病徵；而相對的 5 個供生物殺菌劑發展應用之鏈黴菌株，於馬鈴薯組織上與薯塊上均未見有典型瘡痂病徵表現。有鑑於 thaxtomin A 產生為本病病原菌感染攸關重要因子，將所自 12 個供試鏈黴菌株經培養，並萃取其毒質成分，所含 thaxtomin A 成分除經內加標品高效液相層析 (HPLC) cochromagraphy 證實存在，並利用串聯式液相層析質譜儀 (LC/MS/MS) 以 MRM 監測模式系統，由 m/z＝437 母離子及m/z＝155、140 與 107 等 3 個子離子斷片之檢出進一步加以證實。綜合檢出結果，已證實 7 個可導致典型嚴重瘡痂病徵的菌株其培養液中 thaxtomin A 產量均可達 mg/ml 以上濃度，相對的 5 個供生物製劑發展應用之鏈黴菌株則培養液中均未檢測到有此毒質之產生。另值得一提的是，7 個供試病原菌株中以 T3 菌株產率最高，其次為 T5、T6、D1、T4、T1，而以 T2 菌株為最低者。，經檢視此 7 個供試菌株於蘿蔔幼苗系統所呈現發病狀況可以明白看出各供試菌株之 thaxtomin A 毒質產生能力與其致病毒力表現有密切關係；另將 7 個供試菌株之基因體 DNA 作為模版，以 txtA 基因之專一性引子對添加後，進而證實均可如預期增幅出大小約為 398bp 之片段，經選殖、解序比對後，發現其與已知 S. acidiscabies 之 txtA 基因相同度 (identity) 高達 99.7％，繼之利用所選殖 txtA 片段所製備核酸探針進行南方雜合反應，証實於上述 5 個供生物性殺菌劑發展應用之鏈黴菌菌株之基因體中均未測到 txtA 基因之存在，此亦顯示其余成為瘡痂病病原菌之風險性應極低。為瞭解本病生物防治應用之可行性，本研究以對峙培養檢測 7 株已知對多數重要病原真菌與細菌具優異抗生活性且產孢性狀良好之枯草桿菌群 (Bacillus subtilis group; BSG) 菌株對 S. scabies T1 菌株之拮抗性，經實驗結果證實以 Bacillus subtilis var. amyloliquefaciens WG6-14 拮抗性最為優異，其次為 TKS1-1、SP4-17與WP8-12。在溫室試驗中，分別以 D1 與 T1 兩供試病原菌株之孢子懸浮液 (菌量約為 1×108 cfu/ml) 澆灌行人工接種，處理六週後採收所產生薯塊，其發病率 (infection rate) 與罹病度 (disease severity) 分別 71％ 與 66.7％ 及 43.8％ 與 44.5％ 左右，相對的接種同時以 WG6-14 澆灌處理組則可使D1 與 T1 接種病害發病率降到只有 35％ 與 28％，罹病度降到只有 11.7％ 與 10.5％，另外接種後一個禮拜以 WG6-14 澆灌處理組則 D1 與 T1 接種下發病率分別降到只有 34％ 與 45％、罹病度分別降到只有 11.2％ 與 16.7％，除了防治效果卓著，另值得注意的是，WG6-14 之澆灌處理對馬鈴薯根系與薯塊生長發育均有顯著促進效果，與未處理之對照組比較，D1 與 T1 菌株所接種植株經 WG6-14 處理後，其植株結薯總數分別多 1.4-2.0 倍與 1.7-2.8 倍。此一結果顯示，WG6-14 之施用可以兼具促進植株生長與保護薯塊使不被病原菌感染之双重效果，其確為馬鈴薯栽培與瘡痂病病害管理上極值得被推薦應用之生物製劑。An outbreak of tuber infection showing symptoms closely resembling that of common scab disease was observed during the winter of 2006 on field harvested potatoes in central Taiwan at both Tantzu Taichung and Dounan Yunlin. The disease appeared to be new for potato cultivation in Taiwan, and the increasing incidence and severity of the disease have drawn great attention among growers during the past 2 years. The main objectives of this investigation were to explore the biological and pathological characteristics of the causal agent and the possible resolution to solve the problem. A total of 7 Streptomyces strains isolated from diseased tubers were used for the performed tests. Among them, T1, T2, T3, T4, T5 and T6 were from Tantzu; and D1 (kindly given by Dr. Ann-show Cheng of Tainan DAIS) was from Dounan. Comparative study by use of ISP4 (International Streptomyces Project) medium on the morphological and physiological characteristics of these 7 tested isolates indicated that all of them grew up into grayish colony typical of Streptomyces morphology and produced spores in spiral chains on the aerial mycelia. They all appeared to grow well with the provision of each individual ISP sugar as sole carbon source, produced melanin pigment on tyrosine agar, and consisted in their membrane lipid fatty acid profile similar to that known for Streptomyces scabies. The 16S rDNA of these tested isolates were cloned and sequenced, pair-wise comparison of the sequences with the data provided by GenBank indicated all of them shared a 99% identity with that known for Streptomyces scabies. A phylogenetic tree constructed based on 16S rDNA sequences known for Streptomyces further concluded the 7 tested isolates in the same group as that of S. scabies. The morphological, physiological and molecular characteristics observed indicated that all the 7 tested isolates belong to members of S. scabies. As the majority of Streptomyces spp. are known to be saprophytic and some are important beneficial soil microbial resources which have been widely used as biological control agents, 5 Streptomyces strains S1, S3, S4, B4 and M6 known with potential of biofungicide application were included as compared strains for the pathogenicity study to learn if they may pose potential threat on crop production. Upon artificial inoculation, all the 7 diseased potato tuber derived strains were shown to cause necrotic response on potato tuber slices, reduced stem height and leaf thickening on radish seedlings. An artificial inoculation by drenching application of 1x108 cfu/ml spore suspension on pot grown potato plants (cv. Kennebec) confirmed that all 7 tested isolates were pathogenic and led to development of typical necrotic common scab symptoms. As a comparison, none of the 5 strains with potential of biofungicide application caused observable symptoms on potato tuber slices and pot grown potato plant; although slight inhibition of radish seedling growth was detected. Thaxtomin A is known to be a major determinant of Streptomyces spp. that causing the development of common scab symptoms on potato. The production of thaxtomin A of the tested strains was examined by thin layer chromatography, high performance liquid chromatography (HPLC), and a triple-quad mass detector equipped HPLC system (LC/MS/MS). The identity of thaxtomin A detected was demonstrated by use of internal standard co-chromatography in HPLC, and by LC/MS/MS at multiple reaction monitoring mode the detection of precursor ion at m/z=437 and the major product ions at m/z=150, 140 and 107, respectively. All the 7 diseased tuber-isolated strains were shown to produce中文摘要--------------------------------------------------I 英文摘要--------------------------------------------------IV 目錄----------------------------------------------------VIII 壹、前 言--------------------------------------------------1 貳、前人研究-----------------------------------------------4 一、馬鈴薯之經濟重要性-----------------------------------4 二、馬鈴薯瘡痂病之發生與危害概況-------------------------5 三、植物毒質thaxtomin 研究概況---------------------------7 四、馬鈴薯瘡痂病之防治策略-------------------------------9 五、生物防治 (Biological control)-----------------------10 六、枯草桿菌群 (Bacillus subtilis group) 特性及應用概況-10 七、參考文獻--------------------------------------------13 參、材料與方法--------------------------------------------21 一、供試藥品來源----------------------------------------21 二、供試培養基------------------------------------------21 (一) 幾丁質培養基 (chitin medium)---------------------21 (二) 馬鈴薯蔗糖瓊脂培養基 (potato sucrose agar, PSA)--22 (三) International Streptomyces Project (ISP4) 培養基-22 (四) 燕麥粉頭培養基 (oatmeal medium)------------------22 三、供試菌株來源與培養----------------------------------23 (一) 供試馬鈴薯瘡痂病菌菌株之來源、保存---------------23 (二) 供生物殺菌劑發展應用拮抗性鏈黴菌菌株-------------23 四、接種源之製備----------------------------------------23 五、供試菌形態之光學顯微鏡與掃瞄式電子顯微鏡檢視--------24 六、供試菌株生理生化特性測定----------------------------24 (一) 碳素源利用性-------------------------------------24 (二) 脂肪酸分析 (Fatty acid analysis)-----------------25 七、供試病原菌株 16S rDNA 及 txtA 基因序列之聚合酶連鎖反應增幅、解序---------------26 八、txtA基因片段選殖、質體DNA抽取及南方雜合檢測---------27 (一) txtA基因片段選殖與質體DNA抽取--------------------27 (二) 南方雜合檢測2-1. txt A探針(probe)之製備----------------------28 2-2. 核酸之轉漬-----------------------------------28 2-3. 雜合反應-------------------------------------29 2-4. 洗片與壓片-----------------------------------29 九、所分離病原菌株病原性之生物測定 (Bioassay)-----------30 (一) 蘿蔔幼苗系統-------------------------------------30 (二) 馬鈴薯薄片圓盤法生物測定 (tuber slice assay)-----30 (三) 馬鈴薯植株接種試驗-------------------------------31 十、thaxtomin A 萃取、純化與分析------------------------31 (一) thaxtomin A 萃取流程-----------------------------31 (二) 薄層層析 (TLC；thin-layer chromatography) 分離純-------32 (三) 高效液相層析 (High-Performance Liquid Chromatography, HPLC)---32 (四) 串聯式液相層析質譜儀 ( Liquid Chromatograph/Mass Spectrometer/ Mass Spectrometer)------------------32 十一、供試枯草桿菌群菌株之來源及拮抗性篩選測試----------33 十二、拮抗性枯草桿菌發酵液澆灌處理對瘡痂病菌感染之影響--34 肆、結果-------------------------------------------------35 一、供試病原菌株菌落形態與生理、生化特性----------------35 二、供試病原菌菌株 16S rDNA 序列增幅與同源性分析--------36 三、thaxtomin A synthetase (txtA) 基因片段之偵測--------37 四、供試菌株之病原性測試--------------------------------37 五、thaxtomin A 萃取、純化與分析------------------------39 六、馬鈴薯瘡痂病菌拮抗性枯草桿菌群菌株篩選--------------40 七、Bacillus subtilis var. amyloliquefaciens WG6-14 菌株於 馬鈴薯瘡痂病防治之應用性評估------------------------40 伍、討論-------------------------------------------------43 陸、參考文獻-------------------------------------------50 柒、圖表說明----------------------------------------------56 捌、附錄--------------------------------------------------7

Study of New Business Model for the Medical Tourism in Taiwan: A Health Examining Business Orientation

　　觀光醫療已經是亞洲各國極力發展的重點產業，也是台灣近來積極發展的重點產業之一，而在亞洲觀光醫療產業發展上，各國也各自發展特色醫療，如治療目的的重症醫療（關節置換或器官移植等）、美容目的（醫美整形、變性手術）或鎖定預防醫學族群（健康檢查或spa等），而在台灣醫療服務業於健保發展多年下，已具備足夠醫療實力及服務品質，然台灣醫療業者在面對健保赤字及多項管制下，多年前已開始發展自費高單價健檢服務，且已有既定鎖定台灣內需市場之商業模式。 本研究目的為探討在觀光醫療商機中，以健檢產業為發展主軸之發展策略及商業模式。主要以台灣已發展健檢產業良好之業者為個案，來探討其現行商業模式作為觀光醫療方向之延伸可行性，本研究透過文獻分析及個案業者訪談之研究方法，來了解觀光醫療產業中健檢產業之商業模式，並由業者角度提出期待政策需求，並進一步提出對業者及政府之建議。 　　本研究從全球觀光醫療發展狀況及台灣健檢醫療個案以商業模式分析，並業者訪談，研究結果顯示：一、觀光醫療是亞洲各國發展重點，但台灣政策仍缺乏有力行銷策略來拓展國際知名度。二、大陸高階市場具成長潛力，鎖定高階服務，台灣優質健檢醫療有利基。三、業者以因應健保困境而建立的商業模式，延伸至觀光醫療市場，但舊有商業模式似乎對新市場吸引力道不足。四、台灣觀光醫療品牌之國際形象，仍有待建立，政府應快速指派專責單位整合政策配套與民間資源，作聚焦性行銷。 　　依據個案研究及訪談結果，針對政府及業者分別提出未來建議方向：建議政府需盡速成立專責單位針對觀光醫療政策作更好的整合，透過資源整合將台灣醫療打造國際品牌形象，而在發展政策上，政府不可僅仰賴自由經濟示範區中的國際醫療專區規畫來發展觀光醫療產業，現階段健康檢查是目前台灣業者在觀光醫療相關服務項目中建構最完整的領域，政府應該針對業者提出中短期的配套政策來鼓勵及支援，讓業者更能於現有基礎下，有更有的發揮空間。而對於業者則建議應該重新檢視目前的商業模式，重新了解目標消費者需求，將服務流程再作創新後，聚焦目標大陸市場後，掌握商機。　　Medical Tourism is one of the key developing industries in Asia countries, also the major developing industry in Taiwan these years. Asia countries focus on specific fields of medical tourism, such as the treatment of severe medical symptom (joint replacement or organ transplant etc.), cosmetic purposes (plastic surgery, sex reassignment surgery) or preventive medicine (health examining, or spa, etc.). Taiwan has established a complete medical environment to provide sufficient service and capability due to the experience of the National Health Insurance. However, the financial defect of National Health Insurance system results in financial deficit of National Health Insurance. The operators of hospital and clinics have been developing own expense health examination in Taiwan’s local medical market to face the situation. 　　This study aimed to explore business opportunities in medical tourism, especially in the health examining orientation. Cases in this study not only developed outstanding health examining industry in Taiwan, but also owned the positive attitude to develop medical tourism. This study aimed to investigate extending feasibility of the current business model from the medical tourism perspective. This research investigates through the literature analysis and case study as the research methods to understand the business model of the medical tourism industry in health examining orientation. Besides, this research collects the opinions from operators’ point of views to present to industry and government. 　　In this study, we investigated from the overview of the development of global medical tourism to the case study of Taiwan health examination. The preliminary results show: Firstly, medical tourism is the Asian countries development priorities, but Taiwan's policy still lacks a strong marketing strategy to expand our international reputation. Secondly, China market with high growth potential, locked high service, high-quality health examination of Taiwan has niches. Thirdly, the established business model for Taiwan domestic market is not attracted to extending to the medical tourism market. Fourthly, the international image of Taiwan Medical Tourism brand is still needed to be established, Taiwan government should appoint a dedicated unit to integrate all resources to develop a focused strategy. Based on case studies and interviews, we provide suggestions to both industry and government. We suggest that Taiwan government should immediately appoint a dedicated unit to integrate all resources to develop a better strategy and establish the international brand of Taiwan medical service. About the policy of medical tourism, government shouldn’t only focus on the project of medical tourism zone of “Free Economic Zone” for future development. Till now, the best one of the services related to medical tourism in Taiwan is the service of health examination. Taiwan government should make a proposal for industry to encourage and support them to persistently invest in medical tourism. In addition, we suggest that industry should re-investigate the original business model. They could try to investigate the needs of target customer and re-structure the service process, then focus on target market-China to get the future opportunities