Biological Characteristics and Control of the Causal Agent of Cruciferous Vegetable Anthracnose

西元1997年夏天，在台灣主要蔬菜栽培區如雲林縣、苗栗縣、新竹、花蓮、高雄與台中等地，發現許多白菜葉片出現淡黃色至灰褐色圓形斑，中間白色透明發亮，有時會有穿孔現象，多數病斑會癒合成大形壞疽斑；病斑主要發生於下位葉，嚴重時受害葉片會出現乾枯下垂的病徵；且此病害多發生於有機蔬菜栽培區。將罹病組織分離到的炭疽病菌回接於白菜，四天後，植株葉片即會產生如自然感染的病徵。進一步，將具病原性之菌株接種於甘藍、蘿蔔、芥菜、芥藍、不結球白菜、結球白菜、莧菜、茼蒿、萵苣、芹菜、甕菜及菠菜等蔬菜，結果發現甘藍、蘿蔔、芥菜、芥藍、不結球白菜會出現病徵。在馬鈴薯葡萄糖瓊脂平板培養此病原菌，其菌絲平鋪生長於培養基表面，呈白色或墨綠色至黑色，會產生鮭紅色分生孢子堆；分生孢子單胞，圓筒形或紡錘形，透明無色，內有大型油滴，大小平均為16.65×4.68mm ; 附著器呈圓形至不規則形; 分生孢子盤 (acervuli) 於葉表皮下埋生，孢子盤內散生剛毛，剛毛深褐色。綜合上述病原菌的形態、產孢方式及病原性等特性，進而與國內外相關文獻比較，並參照Sutton, B. C.出版The Coelomycetes, Fungi Imperfecti with Pycnidia, Acervuli and Stromata一書的分類系統，筆者將本病原菌鑑定為Colletotrichum higginsianum Sacc. apud Higgins。C. higginsanum的菌絲生長、分生孢子發芽及附著器形成的最適溫度皆為24-28 oC。病害之發生會隨溫度上升而提高。白菜接種病原菌後濕度維持20小時以上，植株的罹病度即可達80%以上。本病原菌可感染定經草 (lindernia antipoda (L.) Alston) 外，亦可在土壤中以寄主殘體存活35天以上。篩選8種藥用植材對C. higginsianum之分生孢子發芽與菌絲生長的抑制效果，發現丁香具有完全抑菌的效果。以不同濃度之丁香油處理病原菌之分生孢子，發現750 ppm丁香油可以完全抑制分生孢子發芽。利用光學顯微鏡和掃描式電子顯微鏡觀察處理過丁香油的病原菌，發現丁香油會使病原菌菌絲膨大變形外，亦會造成附著器之原生質外漏。比較丁香水溶性浸出液、丁香油及丁香酚防治白菜炭疽病的效果，結果顯示丁香油與丁香酚具有相等的防治功效，且較丁香浸出液的防治率高50% 以上。利用不同濃度之丁香油溶液防治白菜炭疽病，發現濃度高於1500 ppm 時，防治效果可達70% 以上；惟再提高施用濃度，其防治效果並無顯著提昇。在丁香油溶液中分別添加植物生長的基本元素N、P、K、Ca及Mg元素等鹽類，然後噴佈於植齡25天的白菜植體上，結果發現KNO3、H3PO3、K2SO4、Ca(NO3)2·4H2O及MgSO4等可顯著增進丁香油防治白菜炭疽病的效果。Since 1997, Pak-choi (Brassica rapa L. Chinese Group) anthracnose has become severe in organic farms at Yunlin, Miaoli, Hisnchu, Hualien, Kaohsiung and Taichung Counties in Taiwan. The symptoms consisted of small, circular to irregular, pale gray to straw-colored lesions occurring in lower leaves. The lesions may become perforated with splits through the dried necrotic area. Under favorable conditions, the numerous lesions often coalesce and form large irregular spots, and infected leaves become yellowing and wilting. Symptoms on mid-ribs, petioles and stems appeared as sunken and elongated lesions with gray to dark, brown or black border. The fungus was consistently isolated from diseased Pak-choi leaves and its pathogenicity was confirmed by spraying the conidial suspension onto the leaves of cruciferous and other families vegetables. The symptoms developed only on inoculated plants of cruciferous vegetables. Other inoculated plants remained symptomless. The colonies of the pathogen on PDA plates usually had little aerial mycelia, but occasionally produced fluffy patches of white aerial mycelia or produced the dark mycelial bodies onto the agar. Conidia were produced in dense, continuous, cinnamon-colored and setae were not developed in culture, but were produced on the host. The pathogen produced rod-shaped, rod-ends, hyaline, one-celled conidia in acervuli. Average of conidial size was 16.65×4.68μm. According to the morphology and the pathogenicity on cruciferous plants, the pathogen was identified as Colletotrichum higginsianum Sacc. apud Higgins. The optimum temperatures for mycelial growth, conidial germination, and appressorial formation of C. higginsianum isolates PA-01 and PA-19 were at 24-28 ℃. The disease severity increased with increment of temperatures from 20 to 32 ℃ in the growth chamber. The disease severity of Pak-choi anthracnose was more than 80% when the plants were inoculated near 98-100% relative humidity at 28℃for 20 hr. C. higginsianum showed the pathogenicity on the weeds (lindernia antipoda (L.) Aslton) and was able to survive better in soil with pak-choi plant debris. Eight medicinal plant materials were evaluated for their effects on conidial germination and mycelial growth of the pathogen isolates PA-01 and PA-19 on PDA plates. Among those, clove (Eugenia caryophyllata Thunb.) inhibited completely the conidial germination and mycelial growth at the rate of 1% (W/V) whether it was autoclaved or not. In advance, it was proved that clove oil, the major component of clove, was completely effective in inhibiting conidial germination at 750 ppm. Observations under light and scanning electron microscopes indicated that the hyphae swelled and cytoplasma of appressoria leaked out when the fungus was treated with clove oil for 12 hours. The effects of clove water-soluble extract, clove oil, and eugenol were respectively used to control Pak-choi anthracnose. The results showed that clove oil and eugenol were equally effective in reducing more than 50% disease severity compared to clove water-soluble extract. Clove oil was significantly able to reduce more than 75% disease severity of Pak-choi anthracnose at 1500 ppm. In greenhouse tests, clove oil could reduce disease severity of Pak-choi anthracnose when it was sprayed one day in advance of the pathogen inoculation or at the same time as the pathogen was inoculated, but couldn't when it was sprayed one day after the pathogen was inoculated. The effects of clove oil on control of Pak-choi anthracnose were markedly affected when it was mixed with plant nutrients. It was found that KNO3, H3PO3, K2SO4, Ca(NO3)2˙4H2O, and MgSO4 were able to enhance the effect of clove oil on control of the disease.前言…………………………………………………………………1 材料與方法…………………………………………………………5 供試菌之培養與保存 …………………………………………… 5 供試植株 ………………………………………………………… 5 接種源之製備 …………………………………………………… 6 噴霧接種法…………………………………………………………6 病害調查法…………………………………………………………6 十字花科蔬菜炭疽病菌之鑑定 ………………………………… 7 病原菌之形態……………………………………………………7 寄主範圍之測定…………………………………………………7 病原菌之生理特性…………………………………………………7 溫度對菌絲生長的影響…………………………………………7 溫度對分生孢子發芽率與附著器形成的影響…………………8 病害的發生條件……………………………………………………8 植株位葉與病害發生之關係……………………………………8 病原菌接種濃度與病害發生之關係……………………………8 溫度對植株發病的影響…………………………………………9 保濕時間對白菜炭疽病發生的影響……………………………9 白菜炭疽病菌於土中存活的證據…………………………………10 白菜炭疽病之防治…………………………………………………10 不同藥用植材對炭疽病菌分生孢子發芽與菌絲生長的影響…10 丁香油對分生孢子發芽的影響…………………………………11 丁香油處理對病原菌形態的影響………………………………11 丁香浸出液、丁香油及丁香酚對白菜炭疽病罹病度之影響…12 不同濃度丁香油對白菜炭疽病罹病度之影響…………………12 溫室中丁香油水溶液防治白菜炭疽病的效果…………………12 植物營養對丁香油防治白菜炭疽病效果的影響………………13 氮肥對丁香油防治白菜炭疽病效果的影響…………………13 磷肥對丁香油防治白菜炭疽病效果的影響…………………13 鉀肥對丁香油防治白菜炭疽病效果的影響…………………13 鈣、鎂肥對丁香油防治白菜炭疽病效果的影響……………14 丁香油配合氮、磷、鉀、鈣和鎂等鹽類混合物防治白菜炭 疽病的效果……………………………………………………14 結果…………………………………………………………………15 白菜炭疽病之發生與病徵…………………………………………15 病原菌之鑑定………………………………………………………15 病原菌之形態……………………………………………………15 病原菌之寄主範圍………………………………………………16 病原菌之生理特性…………………………………………………16 溫度對菌絲生長之影響…………………………………………16 溫度對分生孢子發芽率與附著器形成之影響…………………16 病害的發生條件……………………………………………………17 植株位葉與病害發生之關係……………………………………17 接種濃度與病害發生之關係……………………………………17 溫度對植株發病的影響…………………………………………18 接種後保濕時間對白菜炭疽病發生的影響……………………18 白菜炭疽病菌於土中存活的證據…………………………………18 白菜炭疽病之防治…………………………………………………19 不同藥用植材對分生孢子發芽與菌絲生長的影響……………19 丁香油對分生孢子發芽的影響…………………………………19 丁香油處理對病原菌形態的影響………………………………20 丁香浸出液、丁香油及丁香酚對白菜炭疽病罹病度之影響…20 丁香油防治白菜炭疽病之效果…………………………………20 溫室中丁香油水溶液防治白菜炭疽病的效果…………………20 植物營養對丁香油防治白菜炭疽病效果的影響………………21 氮肥對丁香油防治白菜炭疽病效果的影響……………………21 磷肥對丁香油防治白菜炭疽病效果的影響……………………21 鉀肥對丁香油防治白菜炭疽病效果的影響……………………21 鈣、鎂肥對丁香油防治白菜炭疽病效果的影響………………22 丁香油配合氮、磷、鉀、鈣和鎂等鹽類混合物防治白菜 炭疽病的效果……………………………………………………22 討論…………………………………………………………………23 引用文獻……………………………………………………………27 中文摘要……………………………………………………………31 英文摘要……………………………………………………………33 圖表…………………………………………………………………36 附錄一………………………………………………………………6

[[alternative]]The effects of inspiratory muscle training on performance and antioxidant capacity

[[abstract]]本研究的主要目的為在探討吸氣肌訓練對男性運動員的運動表現及抗氧化能力的影響。研究方法以18歲以上大專男性優秀運動選手16名為受試對象(平均年齡：18 ± 2.3歲，平均身高：176.6 ± 5.8 公分，平均體重：65.2 ± 6.0 公斤)，以隨機分配方式，分為實驗組(n = 8)與安慰組(n = 8)，均使用POWERbreathe呼吸肌訓練器，進行為期4週吸氣肌訓練，全部的受試者於訓練介入前後接受肺功能( FVC, MVV, PImax,PEmax )、最大攝氧量及抗氧化能力( MDA and SOD )之測試。統計方法以重覆量數二因子變異數分析，評估吸氣肌訓練介入的成效，顯著水準設定為α =. 05。結果發現，MVV及 PImax在實驗組及安慰組組內達顯著差異(P .05)。在抗氧化能力部分，SOD 與MDA在組內均達顯著差異(P .05) In parts of antioxidant capacity, SOD and MDA in the group reached a significant difference (P <.05). Conclusion: 4 weeks IMT could improve maximal inspiratory muscle strength and blood concentrations of SOD. Recommendations for future needs with physical training limb movement, perhaps in order to effectively enhance athletic performance

Technical Development and Research Improvement in Biological Control of Plant Pests (II)

環保意識的抬頭與人類對食品安全的關注,使得農作物病蟲害的生物防治工作受到社會大眾的重視,因此,本計畫目的在於研發作物病蟲害生物防治策略,以便能有效在農業生態體系中推展應用.本計畫涵蓋有九個子計畫,它們個別的工作目標如下:即 (1) 由夏威夷引進銀葉粉蝨的天敵Encarsia pergandiella, E. hispida及E. mineoi,探討它們防蟲之功效與飼養、檢疫等工作; (2) 探討斑潛蠅發生生態,調查本土現有斑潛蠅之天敵種類及引進有效的寄生性天敵,作為生物防治之用; (3) 評估黑殭菌各菌株致病力與抗環境逆境;利用黑殭菌DNA多元性特定條帶作出DNA-probe,供田間生態及施用存活調查; (4) 探討大量飼育捕植蹣類天敵,建立捕植對卵孢子發芽、感染與傳播的影響;施用增產菌FG-1菌株與滅達樂,以期延緩病害發生與促進薑株生長; (6) 利用本地產蟲生線蟲(Steinernema abbasi),研發出適合台灣蔬菜害蟲之安全有效的生物防治技術; (7) 觀察飼育紅點唇瓢蟲之基本需求,評估其生物防治梨圓介殼蟲之潛力; (8) 利用生物分析法篩選具有抑制病原菌的作物殘渣與植體,並輔以HPLC或GC分析它們所含有的硫配糖體種類及分解產物,藉以瞭解各分解產物的殺菌與抑菌功效; (9) 針對本地種蟲生線蟲Steinernema abbasi對蔬菜主要害蟲如小菜蛾、白粉蝶、大菜螟及菜心螟蟲的致病力進行生物檢定與施用技術.Biological control of plant pests has received considerable attention throughout the world, drawing momentum from the growing public concern regarding the widespread use of hazardous chemicals in pest control. In order to develop and apply biocontrol strategies in agriecosystem, the objectives of this project including nine sub-projects are (1) to import a whitefly parasitoids, Encarsia pergandiella, E. hispida and E. mineoi from Hawaii and study their efficacy on controlling the silver leaf whitefly; (2) to study the distribution, occurrence, seasonal abundance, population dynamics and damage of leafminers in southern area and test the basic biology, parasitic rates of introduced parasitoids; (3) to screen Metarhizium anisopliae strains having high entomopathogenicity, more resistant against UV light and the fungicides; to analyze the genetic diversity and relationship of the tested green mascardine fungi by using electrophoretic fingerprinting; (4) to develop biocontrol technologies of the spider mites with their predaceous mites, which depends upon the availble mass rearing skills and their lowering costs determined by the design of artificial rearing arena and supplemental foods; (5) to study the ecology of ginger soft rotdisease including the transmission of the disease through seed ginger; the temperature effect on the oospore germination, infection and spreading. Also, to study the use of both metalaxyl and YIB (yield increasing bacteria) isolate Fg-1 to delay the disease development and increase the plant growth; (6) to investigate biological control of vegetable pests using the indigenous species, Steinernema abbasi, in safe and effective control measures; (7) to study the basic rearing requirement for Chilocorus kuwanae and to provide a preliminary assessment of its potential as a biological agent of the San Jose Scale, Quadraspidiotus perniciosus; (8) to determine the effect of volatile compound released from leaf and seed of Brassica species on the natural isothiocynate fumigant for integrated control of crop soilborne diseases; (9) to develop a vermiculite, formulation of Steinernema abbasi to control four vegetable pests, such as diamondback moth, imported cabbageworm, cabbage pyralid and cabbage webworm