兩種台灣柑桔潰瘍病病原細菌的噬菌體的研究

Abstract

由柑桔潰瘍病病斑分離得兩種Xanthomonas citri (Hasse) Dowson的噬菌體及兩種病原細菌。噬菌體中有一種是絲狀，產生直徑約0.5mm，小而模糊的溶菌斑，命名?Cf；另外一種產生圓形透明直徑2-3mm的溶菌斑命名?XCl。兩種病原細菌依病葉收集地方之不同而命名?南投及中和。實驗中並用得自日本的X. citri的兩種噬菌體CP1，CP2及兩種菌株；X. oryzae 507菌株及Xf噬菌體做?比較。 本論文實驗以Cf噬菌體?主，研究其外表形態，寄主特異性，穩定性，對細菌生長的影響及純化方法。 於電子顯微鏡下觀察顯示出Cf噬菌體是一種絲狀噬菌體，約長12400?，寬70?。由寄主範圍的實驗知XCl噬菌體的寄主範圍和日本的CP1噬菌體相類似但Cf噬菌體則限於X. citri的南投、中和兩菌株。中和菌株亦可做?CP1及Xp12噬菌體的寄主，而南投菌株則只能做?CP1噬菌體的寄主。 Cf噬菌體對於氯仿很敏感。trypsin及lysozyme兩種酵素則對Cf噬菌體沒有影響。Cf噬菌體經紫外?照射60秒鐘；於70℃，10分鐘則幾乎100％不活性化。此噬菌體於pH7時最?穩定。 Cf噬菌體的感染不會使寄主細菌發生溶菌現象，而XCl噬菌體則對寄主細菌會發生溶菌現象。 Cf噬菌體的純化可用硫酸銨?析的方法加至40％飽和度後收集沉澱，用不同離心速度除去雜質，用超速離心機於100,000XG離心使Cf噬菌體沉降下來，再經過CsCl density gradient centrifugation spinco SW 50L rotor於40,000rpm離心24小時則於密度1.27處有一乳白螢光的噬菌體環帶收集此處即得純化的噬菌體。 用acridine orange染色，結果?火?紅色，顯示Cf噬菌體係含有單鏈DNA。被Cf噬菌體感染的細菌體內似無完整噬菌體之堆積，Cf噬菌體於細菌內造好後即釋放出來。

Two bacteriophages of Xanthomonas citri (Hasse) Dowson and two strains of X. citri were isolated from lesions of citrus canker in Taiwan. One of the phages, filamentous in shape and producing turbid small plaques of about 0.5 mm in diameter, Was named Cf while the other, producing clear circular plaques of 2-3 mm in diameter, was named XCl. The two strains of X. citri were named Nantou and Chungho, based on the locality from which the specimens were collected. Two strains of X. citri and their phages, CP1 and CP2, from Japan, a filamentous phage, Xf, and a hexagonal shaped phage, Xp12, of Xanthomonas oryzae, were included in comparative studies. Investigations were carried out primarily on the properties of the Cf phage including its morphology, host specificity and effect on the growth of the host bacteria. The phage was also purified. Under electron microscopy, purified Cf phage was observed to be a filamentous form of about 12400? length and 70 ? width. In host range tests, the XCl phage was found to be similar to the Japanese CP1 phage, but the Cf phage was limited to the Nantou and Chungho host strains of bacteria only. The Chungho bacterium was a host of both the CP1 and Xp12 phages. On the other hand, the Nantou bacterium was a host of CP1 but not Xp12 phage. Cf phage was very sensitive to chloroform but resistant to trypsin and lysozyme. More than 90% of the phages were inactivated by ultraviolet light treatment for 60 sec. and heating at 70℃ for 10 min. almost completely inactivated it. The phage was stable at pH 7. Cf phage infection did not lyse the host cells, but XCl phage infection did. Cf phage was purified by 40% (NH4)2SO4 saturation, differential centrifugation, and final precipitation by centrifugation at 100,000 x G for 3 hrs. For further purification, the phage precipitates were suspended in CsCl at a density of 1.27 and centrifuged to equilibrium in a Spinco SW50L at 40,000 rpm for 24 hrs. Treated by acridine orange staining Cf phage exhibited a flaming red color indicating it is a single-stranded DNA phage. Free Cf pheges were continuously released from the infected host bacterial cells, but no indication of phage accumulation within the cells.