臺灣博碩士論文加值系統

蘭花產業為我國觀賞花卉最具產值者，其中蝴蝶蘭更被農委會選為四大外銷旗艦作物之一。近年來我國蝴蝶蘭外銷之品目逐漸轉以種苗為主，因此病毒之感染與否即成為界定種苗品質優劣標準之一。蘭花病毒中，齒舌蘭輪斑病毒 (Odontoglossum ringspot virus, ORSV) 與蕙蘭嵌紋病毒 (Cymbidium mosaic virus, CymMV) 是感染蘭花之近五十九種病毒中分佈最普遍影響最嚴重的二種。在田間此二病毒並不藉由媒介昆蟲傳播，而是以污染栽培環境與工作器具，再經由傷口入侵而造成感染。本研究乃針對過去本實驗室所篩選具有分解蘭花病毒鞘蛋白能力的放線菌菌株 (CA5) 之培養流程及實際應用方式進行研究，期能提升 CA5 所分泌鞘蛋白分解酵素之生產效率，並能掌握 CA5 鞘蛋白分解酵素之有效應用方式，以降低此二種病毒在生產健康蘭花種苗的過程中之危害。
試驗中首先針對培養之三角瓶結構進行研究，比較一般與二種具溝槽或檔板設計之三角瓶對培養 CA5 菌數量及所生產酵素活性之影響，結果發現以具有溝槽之三角瓶由於可以在震盪培養時產生較高的溶氧量，因而可在較短的時間內獲得較高之菌量與酵素相對活性 (relative activity unit, RU)。此外本研究特別針對大量培養前之接種源之培養方式進行探討，確定將菌種先於大豆粉液態培養基於 32 ℃ 下培養至第 5 天可作為最佳之接種源。將其以 1 % (v/v) 比例接種於相同培養基中，於30 ℃ 與150 rpm震盪下培養 6 天所獲得之濾液，即可達到4149 RU/ml之最高分解蘭花病毒鞘蛋白之相對活性，顯著高於過去以菌絲孢子懸浮液做為接種源，於一般三角瓶內培養14天所獲之3029 RU/ml相對活性。此一培養模式之確立估計可使 CA5 酵素之總相對活性比過去所建立之培養程序提升 4 倍，而時程可以縮短 3 天。
利用CA5 培養濾液再經 60 % 飽和硫酸銨濃縮沉澱之酵素材料 (CA5P) 進行實際應用方式與效果之評估。測試結果發現以CA5酵素處理確定污染ORSV或CymMV病毒之蘭花種子超過一分鐘後，即無法再以 ELISA 或 RT-PCR 偵測到病毒存在。但以相同方式處理含有蘭花絨毛組織之種子時，則仍可以偵測到病毒訊號，顯示蘭花種子僅外表上污染病毒，因此可以藉由CA5鞘蛋白分解酵素加以清除。另以酵素材料處理污染 ORSV 或 CymMV 之固體表面試驗中，亦確定稀釋4 倍之酵素濃縮液處理30秒後，已明顯無法利用 ELISA 再偵測到病毒的殘留，且於接種試驗中亦確定已完全摧毀此二病毒之感染能力。若改以稀釋 8 及16 倍之酵素濃縮液但延長處理時間，發現延長時間助益不大，此證明 30秒內即可達到主要分解病毒之效果。對於蘭園經常使用之可能造成病毒污染與傳播的材料用具，以固體表面試驗之方法實際以 CA5 酵素材料處理 30 秒，不論材質如刀片、魔繩、塑膠夾、塑膠名牌、手套及蘭花組培苗，甚至指甲，其表面汙染之二種蘭花病毒也都能被分解，且利用 ELISA 已偵測不到有病毒訊號。
本研究亦測試CA5 培養濾液與酵素濃縮液之保存方式，將其冷凍保存於 - 20 ℃ 下，每隔 30 天取出回溫，取樣測試其分解蘭花病毒鞘蛋白之活性變化，歷經150 天發現 CA5 培養濾液經反覆冰凍和解凍，仍可維持原保存前之 83 % 之相對活性 (RU)。相較於保存在 4 ℃ 或 25 ℃ 之兩種方式，分別僅剩餘 53 % 及 6 % 之相對活性，可見 CA5 酵素活性之保存應以冷凍方式較佳。
總之，針對 CA5 菌株之培養與病毒鞘蛋白分解酵素之生產，本研究已經建立一個較過去培養方式更為有效與高產之培養模式。並且證實應用所生產之酵素濃縮液可以在 30 秒的處理時間下，可以將污染於各種蘭園中常見之工具材料表面上之蘭花病毒，清除到無法以接種方式偵測到病毒感染力及以ELISA無法偵測到病毒訊號之程度。另外也證實可以應用此酵素濃縮液在一分鐘內完全清除污染於蘭花種子表面之 ORSV 與 CymMV。

Among the various ornamentals produced in Taiwan, orchids are the one with the highest annual output value. Phalaenopsis orchids, especially, are selected by the Council of Agriculture as one of the four major representative agricultural products in Taiwan. In recent years, tissue culture plantlets have become the major exportation item for Taiwan’s Phalaenopsis industry. Therefore, the cleanness of the plantlets free from virus infection has become one of the major concerns for quality assessment of the exporting Phalaenopsis plantlets. At present, Odontoglossum ringspot virus (ORSV) and Cymbidium mosaic virus (CymMV) are recognized as the most widely spread and economically important viral agents to orchid industry among the 30 known viruses infecting orchids. These two viruses are not disseminated by any known insect vectors, but they can be transmitted easily through mechanical wounds on the host plants by tools contaminated with virus particles. In the previous studies, our laboratory has successfully screened and obtained a pure culture of an isolate of actinomycetes, namely CA5, which can secrete a protease-like substance in its culture filtrate that can degrade coat proteins of ORSV and CymMV and abolish their infectivities. This research attempts to establish a standard cultivation protocol that can produce higher yield of CA5 protease and also to test the feasible application scheme for this novel protease to sanitize the tools and growing environment of orchids.
In the first part of this research, we tested the cultivation effects on the total cell output and the relative activity unit (RU) of CA5 protease activity by conventional flasks comparing with two special designed flasks that could stimulate higher oxygen input into the culture media. Of the three types of flasks tested, the baffled shake flask (BSF) showed the highest cell output and RU value within a shorter period of cultivation. Using BSF as cultivation flasks, we further found that a best CA5 inoculum for subsequent large scaled production could be prepared by culturing CA5 in soybean meal liquid media (SMLM) at 32 C, 150 rpm, for 5 days. With this standardized inoculum, we were able to establish an optimum standard cultivation protocol for CA5 and its secreted protease by starting with the addition of 2 ml ( 1 % (v/v) ) inoculum into a BSF containing 200 ml of SMLM (pH 8.0) and cultured at 30 C, 150 rpm, for 6 days. The greatest cell output and RU of CA5 protease activity could be obtained from this 6 days culture filtrate. With this new protocol, about 5.2 times increase of the RU value of CA5 protease activity was harvested from the culture filtrate comparing to that obtained from our previous protocol. To test the application scheme of CA5 protease, we generally prepared the materials, subsequently named as CA5P, by concentrating the CA5 culture filtrate with 60 % of ammonium sulfate and followed by dialyzing CA5P overnight. Treating orchid seeds contaminated by ORSV or CymMV with 8x H2O-diluted CA5P for 1 min, it was found that virus signals could not be detected from the treated seeds by ELISA or RT-PCR. In another treatment with virus-contaminated orchid seeds and fiber tissues, virus signals could still be detected by RT-PCR after treating similarly with CA5P. This result indicated that ORSV and CymMV were only contaminated on the surface of orchid seeds instead of residing inside the cells while the fiber tissues were actually infected by viruses which were not accessible by CA5P. In another experiment, 4x H2O-diluted CA5P was used to treat 30 seconds on the materials commonly used in orchid nurseries including razor blade, scissors, plastic clip, plastic labels, plastic gloves, orchid plantlets and nail pieces coating previously with ORSV and CymMV particles and the result confirmed that no virus signals could be detected by ELISA. In the last part of our study, we tested the effect of storage temperature on the maintenance of the relative activities of CA5 protease. Three different storage temperature (-20, 4, and 25 C) were tested to store the CA5P for 180 days and every one month the storage materials were allowed to revert to room temperature and part of samples were taken to check their RU of protease activity. The result showed that storing CA5P at -20 C was the best treatment that could preserve at least 83% of RU value comparing with the original one before storage. Instead, preserving at 4 and 25 C could only maintain 53 and 6 % of the original RU value, respectively.
Altogether, we have succeeded in establishing an optimum cultivation protocol for the production of CA5 protease. It takes only 11 days to reach a production peak of CA5 protease comparing to our old protocol taking at least 14 days to reach its maximum. Furthermore, we are confident that CA5P could be practically applied in routine orchid cultivation for sanitizing the growing environment to free from possible viral contamination. In addition, CA5P can also be used to disinfect orchid seeds to free from possible ORSV and CymMV contamination before seeding in the cultivation medium.

目 錄
書名頁
碩士論文授權書
論文口試委員會審定書 (中文版)
論文口試委員會審定書 (英文版)
中文摘要 ------------------------------------------------- I
英文摘要 ------------------------------------------------- IV
誌謝 ----------------------------------------------------- VII
目錄 ----------------------------------------------------- IX
表目錄 --------------------------------------------------- XII
圖目錄 --------------------------------------------------- XIII
壹、前言 ------------------------------------------------- 1
貳、前人研究 --------------------------------------------- 7
一、常見之蘭花病毒介紹 ------------------------------- 7
二、蘭花病毒之檢測方法 ------------------------------- 11
三、生物防治策略在病毒上的應用 ----------------------- 12
四、放線菌之介紹 ------------------------------------- 15
五、鏈黴菌之研究與應用 ------------------------------- 16
六、鏈黴菌大量培養之影響因子 ------------------------- 17
參、材料與方法 ------------------------------------------- 20
一、菌種培養保存與病毒接種保存 ----------------------- 20
二、分解蘭花病毒鞘蛋白成份之蛋白分解酵素之分析 ------- 21
三、影響 CA5 菌株生產蛋白質分解酵素之因素探討 ------- 24
四、固體表面污染病毒之清除試驗 ----------------------- 28
五、不同材料表面污染蘭花病毒之清除試驗 --------------- 30
六、蘭花種子表面污染病毒之清除試驗 ------------------- 30
七、酵素濃縮液處理蘭花種子之組織培養實生苗試驗 ------- 33
八、CA5培養濾液與酵素濃縮液之保存穩定性分析 -------- 34
肆、結果 ------------------------------------------------ 35
一、CA5 鏈黴菌培養型態之觀察 ------------------------ 35
二、CA5 蛋白分解酵素之相對活性測定 ------------------ 35
三、培養濾液於濃縮處理後其分解鞘蛋白能力之相對活性單位
(relative activity unit) 變化 -------------------------- 36
四、影響 CA5 菌株蛋白分解酵素產生因素之探討 --------- 37
五、以 CA5 酵素濃縮液清除污染之蘭花病毒之效果評估 --- 40
六、不同材料表面污染蘭花病毒之清除試驗 --------------- 42
七、蘭花種子表面污染病毒之清除試驗 ------------------- 43
八、不同溫度保存下之 CA5 菌株培養濾液與酵素濃縮液之分
解相對活性分析 ------------------------------------ 44
伍、討論 ------------------------------------------------- 46
陸、表 --------------------------------------------------- 55
柒、圖 --------------------------------------------------- 57
參考文獻 ------------------------------------------------- 83
附錄 ----------------------------------------------------- 89


表目錄
表一、CA5 菌株之培養濾液經不同方式製備後之分解病毒鞘蛋白
之相對活性與比活性 --------------------------------- 54
表二、影響蘭花病毒傳播之各式栽培蘭花工具與材料之Indirect
ELISA試驗 ----------------------------------------- 55


圖目錄
圖一、本研究所測試之三種不同結構之三角瓶 ----------------- 56
圖二、CA5 菌株於 ISP medium 4 中之不同大小菌落生長形態 -- 57
圖三、CA5 菌株培養於含有大豆粉液態培養基之不同構造 250 ml
三角瓶生長情形 ------------------------------------- 58
圖四、CA5 培養濾液與酵素濃縮液中所含分解蘭花病毒鞘蛋白相對
活性單位 (relative activity unit, RU) 之比較 ------------- 59
圖五、Bovine serum albumin (BSA) 蛋白質定量標準曲線 -------- 60
圖六、CA5 菌株分別培養於含有大豆粉液態培養基之檔板三角瓶、
溝槽三角瓶及一般三角瓶中，經過 6 天與 8 天測定其分解
蘭花病毒鞘蛋白之相對活性累積情形 ------------------- 61
圖七、CA5 菌株培養於二種含有大豆粉液態培養基之 500 ml 三
角瓶中其分解蘭花病毒鞘蛋白之相對活性累積之情形 ----- 62
圖八、以 ISP medium 4 平板培養 CA5 菌株於不同溫度下 3-11
天所量測之菌落大小 --------------------------------- 63
圖九、以大豆粉液態培養基於 30 及 32 ℃下培養 CA5 菌株 14
天內之活菌數變化 ----------------------------------- 64
圖十、以大豆粉液態培養基於 30 及 32 ℃ 下培養 CA5 菌株 14
天內之分解蘭花病毒鞘蛋白之相對活性變化 ------------- 65
圖十ㄧ、不同培養條件下之 CA5 分解蘭花病毒鞘蛋白之相對活性
比較 ----------------------------------------------- 66
圖十二、以不同 pH 值大豆粉液態培養基培養 CA5 菌株其累積之
相對活性與 pH 值之變化 ---------------------------- 68
圖十三、以不同稀釋倍數之病汁液接種奎藜葉片後所呈現之病斑數
目 ------------------------------------------------- 69
圖十四、不同稀釋之 CA5 酵素濃縮液分解固體表面黏附 ORSV
病毒之效果 ----------------------------------------- 70
圖十五、不同稀釋之 CA5 酵素濃縮液分解固體表面黏附 CymMV
病毒之效果 ----------------------------------------- 72
圖十六、利用 CA5 酵素濃縮液於不同時間處理下測試其清除固體
表面黏附 ORSV 病毒之效果 -------------------------- 74
圖十七、利用 CA5 酵素濃縮液於不同時間處理下測試其清除固體
表面黏附 CymMV 病毒之效果 ------------------------ 76
圖十八、一般蘭園中常用可能污染病毒顆粒之材料與工具 ------- 78
圖十九、利用 CA5 酵素濃縮液清除污染病毒之蘭花種子之效果 -- 79
圖二十、應用 RT-PCR 分析 CA5 酵素濃縮液處理污染 ORSV 之
蘭花種子之效果 ------------------------------------- 80
圖二十一、不同溫度下保存之 CA5 培養濾液與酵素濃縮液其分解
蘭花病毒鞘蛋白相對活性之變化 ---------------------- 82

李哖，文心蘭栽培原理，p. 2-19，文心蘭專刊，財團法人台灣區花卉發展協
會，台北，269 pp (2002)。

吳錦勳、李盈穎，一間公司幹掉一個王國，p. 107-134，商業周刊出版社，
台北，1026期 (2007)。

吳容儀、戴廷恩、莊耿彰、謝廷芳，文心蘭亞族之介紹及未來展望，p. 48-53，
財團法人台灣區花卉發展協會出版，台北，243 期，75 pp (2007)。

吳惠琦，探討溫度及剪切力對 Actinoplanes spp. 生產 acarbose 之發酵影
響，國立中央大學化學工程與材料工程研究所，碩士論文 (2004)。

林良平，土壤微生物學，南山堂出版社，台北，911 pp (1997)。

林維明，花市洋蘭圖鑑，天下遠見股份有限公司出版，台北，192 pp (2006)。

林讚標，台灣蘭科植物，南天書局有限公司出版，台北，355 pp (1988)。

林益德，可同時偵測齒舌蘭輪班及蕙蘭嵌紋病毒之多株抗體之研發及應
用，碩士論文，私立朝陽科技大學生物技術研究所 (2006)。

周廷光，台灣蘭花毒素病害之研究，中國園藝，台北，26:127-142 (1980)。

周武吉，養蘭技藝，p. 10，台灣英文出版社，台北，152 pp (1992)。

邱智湟，可分解齒舌蘭輪斑及蕙蘭嵌紋病毒鞘蛋白並摧毀其感染力之放線
菌蛋白酶之篩選與應用，私立朝陽科技大學生物技術研究所，碩士論
文 (2006)。

張清安。植物病毒鑑定及診斷新技術。「植物保護新科技研討會」專刊，
台灣省農業試驗所特刊，台中，57:35-45 (1996)。

張清安，本省應用無病毒種苗之回顧與展用，p. 63-73，植物保護學會會刊
(1997)。

張清安、曾雅詩、陳金枝、鄧汀欽，應用聚合酶連鎖反應及核酸探針雜配
法偵測齒舌蘭輪斑病毒，植物病理學會刊 8:29-36 (1999)。

張清安、陳加忠，蘭花母本保存園及附介質輸美蘭花溫室之設施與管理，
行政院農業委員會動植物防疫檢疫局出版，台北，63 pp (2004)。

張清安，植物保護技術專刊1－蘭花病毒病，行政院農業委員會動植物防疫
檢疫局出版，台北，62 pp (2005)。

張清安，蘭花病毒之診斷、鑑定與偵測，p. 103-118，植物重要防疫檢疫病
害診斷鑑定技術研習會專刊(五) (2006)。

張清安，台灣蘭花病毒病害防制之策略、歷程與未來展望(上)，p. 16-23，
財團法人台灣區花卉發展協會出版，台北，248 期，80 pp (2008)。

張清安，台灣蘭花病毒病害防制之策略、歷程與未來展望(下)，p. 16-22，
財團法人台灣區花卉發展協會出版，台北，249 期，80 pp (2008)。

陳加忠，省思與檢討—對商業周刊報導的回應之一，中興大學生物系統工
程研究室 (2007)。

黃敏展，蘭花栽培藝術，p. 253-257，自然科學文化事業公司，台北，301 pp
(1993)。

齊藤龜三，世界原生蘭圖鑑，p. 19-20，晨星出版有限公司，194 pp (1997)。

賴本智、楊秀蘭、張清安、陳任芳、魏國慶，蘭花經濟栽培技術，p. 2，行
政院青年輔導委員會，台北，148 pp (1994)。

謝式坢鈺、韓又新，台灣發生之蕙蘭嵌紋病毒，中國園藝，台北，21(6):292-298
(1975) 。

Baker, K. F. . Evolving concepts of biological control of plant pathogens. Annu.
Rev. Phytopathology. 25: 67-85 (1987).

Baranwal, V. K., Lodha, M. L., Varma, A., and Kapoor,
H. C. Purification and properties of growth stage-dependent antiviral
proteins from the leaves of Celosia cristata. Plant science 154:13-21
(2000).

Baranwal, V. K., and Verma, H. N. Characteristics of a virus inhibitor from the
leaf extract of Celosia cristata. Plant Pathology. 46:523–529 (1997).

Beunink, J.; Schedel, M.; Steiner, U. Osmotically controlled fermentation
process for the preparation of acarbose. U.S.Patent 6130072 (2000).

Brain, B. M. G., and Deborah, R. F.. Biological control of plant Pathogens :
research, commercialization, and application in the USA (2002).

Brandes, J., and Bercks, R. Gross morphology and serology as a basis for
classification of elongated plant viruses. Adv. Virus Reseach.
11:1-24 (1965).

Choi, B. T. ; Shin C. S., Reduced formation of byproduct component c in
acarbose fermentation by Actinoplanes spp. CKD485-16.Biotechnol. Prog ,
19:1677-1682 (2003).

Chng, C. G., Wong, S. M., Mahtani, P. H., Loh, C. S., Goh, C. J., Kao, M. C. C.,
Chung, M. C. M., and Watanabe, Y.. The complete sequence of a
Singapore isolate of odontoglossum ringspot virus and comparison with
other tobamoviruses. Gene 171: 155-161 (1996).

Chang, C. A., Purcifull, D. E., and Hiebert, E. Purification, characterization, and
immunological analysis of nuclear inclusions induced by bean yellow
mosaic and clover yellow vein potyviruses. Phytopathology 78:1266-1275
(1988).

Compton, S. J., and Jones, C. G. Mechanism of dye response and interference in
bradford protein assay. Anal. Biochemistry. 151:369-374 (1985).

Emmert, E. A. B., and Handelsman, J. Biocontrol of plant disease: a (Gram-)
positive perspective. FEMS Microbiology. Letters. 171:1-9 (1999).

Hegde, V. R., Pu, H., Patel, M., Das, P. R., Butkiewicz, N., Arreaza, G., Gullo,
V. P., Chan, T. M. Two antiviral compounds from the plant stylogne
cauliflora as inhibitors of HCV NS3 protease. Bioorganic & Medicinal
Chemistry Letters. 13:2925-2928 (2003).

Hellen, C. U., and Wimmer, E. Viral proteases as targets for chemotherapeutic
intervention. Curr. Opin. Biotechnology. 3:643–649 (1992).

Hollings, M., and Stone, O. M.. Cymbidium ringspot virus. CMI/AAB.
Description of Plant Viruses, pp. 178 (1977).

Horan, A. C.. Secondary metabolite production Actinomycetes other than
Streptomyces. In Encyclopedia of Bioprocess Technology: Fermentation,
Biocatalysis and Bioseparation ed. Flickinger, M. C. and S. W. Drew,
2333-2348. New York: Wiley and Sons. (1999)

Hsieh, R. M., Chen, C. C., Lin, Y. S., Chen W. H., Chang, C. A. Phalaenopsis
virus elimination by tissue culture. Taiwan Sugar Res. Inst. 180:39-47
(2003).

Iwai, Y. and S. Omura. Cultural conditions for screening of new antibiotics, J.
Antibiotics, 34, 123-141 (1992).

Jensen, D. D., and Gold, A. H. A virus ring spot of odontoglossum orchid:
symptoms, transmission and electron microscopy. Phytopathology 41:
648–653 (1951).

Kramer, K. J., and Subbaratnam, M. Insect chitinases: molecular biology and
potential use as biopesticides. Insect Biochemistry. Molecule Biology.
27:887-900 (1997).

Lawson, R.H.,and Hsu, H.T. Orchid. p.409-420. In:Virus and virus-like diseases
of bulb and flower crops. Loebenstein et.al.(eds), John Wiley & Sons,West
Sussex, United Kingdom, 543 pp.(1995)

Lazzarini, A., L. Caveletti, G. Toppo and F. Marinelli. Rare genera of
actinomycetes as potential producers of new antibiotics. Antonie van
Leeuwenhoek, 78, 399-405 (2000).

Lechevalier, L. F., and Lechevalier, H. A. The chemotaxonomy of
actinomycetes. P227-291 in : Actinomycete Taxonomy, SIM special
Publication in No. 6. A. Dietz, and D. W. Thayer eds., VA, USA (1980).

Mabrouk, M. M., and Sabry, S. A. Degradation of poly (3-hydroxybutyrate) and its copolymer poly (3-hydroxybutyrate-co- 3- hydroxyvalerate) by a marine Streptomyces spp. SNG9. Microbiology Research. 156:323-335 (2001).

Manna, A., Giri, P., and Paul, A. K. Degradation of poly (3- hydroxybutyrate) by soil streptomyces. World Journal of Microbiology & Biotechnology 15, 705–709 (1999).

Manners, D. and Wilson, G. Studies on β-glucanases: some properties of a bacterial endo-β-1,3-glucanase system. Biochem. J. 135:11-18 (1973).

McCarthy, A. J. and S. T. Williams. Actinomycetes as agents of biodegradation
in the environment – a review. Gene, 115, 189-192 (1992).

Miyadoh, S. Atlas of Actinomycetes. Tokyo, Asakura Publishing Co., Ltd
(1997).

Namba, R. and Ishii, M.. Failure of aphids to transmit the odontoglossum
ringspot and cymbidium mosaic viruses to orchid plantlets derived from
meristem cultures. Phytopathology. 61:582-583 (1971).

Paul, H. L. Odotonglossum ringspot virus. Description of Plant Viruses, pp. 155
(1975).

Piret, J. M. and A. L. Demain. Actinomycetes in Biotechnology. An overwier. In:
Goodfellow, M., S. T. Williams and M. Mordarski editors. Actinomycetes in Biotechnology. London: Academic Press, 461-482 (1988).

Prinham, T. G., Lindenfelser, L. A., Shotowell, O. L., Stodola, F. H., Benedict,
R. G., Foley, G., Jackson, R. W., Zaumeyer, W. J., Preston, W. H., and
Mitchell, J. W. Antibiotics against plant disease in laboratory and
greenhouse survey. Phytopathology. 46:568-575 (1956).

Sambrook, J. and Russell, D.W.. Molecular Cloning. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New York (2001).

Sanglier, J.J., H. Haag, J.A. Huck and T. Fehr. Review of Actinomycetes
compounds 1990-1995. Expert Opinion and Investigational Drugs,
5,207-223 (1996).

Volff, J. N., and Altenbuchner, J. Genetic instability of the Streptomyces
chromosome. Molecule Microbiol. 27:239-246 (1998).

Wang, Q. M., Protease inhibitors as potential antiviral agents for the treatment of picornaviral infections. Prog. Drug Res. Spec. p. 229-253 (2001).

Weinberg, E. D.. Secondary metabolism: Control by temperature and inorganic
phosphate. Dev. Ind. Microbiol, 15, 70-81 (1973).

Yoshida, N., Y. Tani and K. Ogata. Cryomycin, a new peptide antibiotic
produced only at low temperature. J. Antibiotics, 25, 653-659 (1972).

Yoshida, N., S. Hayashi, Y. Tani and K. Ogata. M-81, a new peptide antibiotic
produced by Streptomyces griseus subsp. Psychrophilus at moderate
temperature. J. Antibiotics, 27, 128-132 (1974).

Zettler, F. W., N. J. Ko, G. C. Wisler, M. S. Elliott, and S. M. Wong. Viruses of orchids and their control. Plant Dis. 74: 621-626 (1990).