CAJ | 학술논문

【目的】分离并纯化出一株裂解性青枯雷尔氏菌(Ralstonia solanacearum)噬菌体，并测定其各项生物学特性，为开发新的抗烟草青枯病制剂提供依据。【方法】取烟草青枯病重病田中健康烟株的根际土壤制成土壤悬浮液，并通过在青枯雷尔氏菌菌液中加入过滤后的土壤悬浮液富集噬菌体，用双层平板法验证噬菌体的存在后挑取单个最大噬菌斑进行反复纯化，直到得到单一清晰的噬菌斑。纯化后的单个噬菌斑加入对数早期的青枯雷尔氏菌菌液中进行增殖培养，将增殖液按常规方法进行噬菌体颗粒浓缩后，取20μL 浓缩液用磷钨酸染色并通过电子显微镜观察噬菌体的形态特征；同时将浓缩液进行 SDS-PAGE 电泳，观察蛋白条带大小和数量；用λ噬菌体 DNA 提取试剂盒提取噬菌体增殖液中的噬菌体核酸进行琼脂糖凝胶电泳，确定其基因组片段大小；最后用常规方法测定噬菌体的滴度、最佳感染复数、一步生长曲线，并通过比较加入噬菌体液前后青枯雷尔氏菌菌液的 OD600值变化测定其对温度、pH、紫外线、氯仿的敏感性。【结果】分离并纯化出了一株裂解性青枯雷尔氏菌噬菌体，命名为∈RS-1,噬菌斑为圆形，清晰透明，边缘光滑，直径1—2 mm，经电镜观察其形态为蝌蚪状，头部为二十面体的立体对称，直径约为94 nm，并有一带伸缩尾鞘的长尾大约为27 nm×100 nm，按照国际病毒分类委员会分类标准，其属于有尾噬菌体目（Caudovirales），肌尾噬菌体科(Myoviridae)的裂解性噬菌体，核酸性质为 dsDNA；噬菌体浓缩液经 SDS-PAGE 分析至少可以观察到25条蛋白条带，相对分子质量在10—100 kD，说明其蛋白外壳至少含有25个结构蛋白；将提取的 DNA 进行琼脂糖凝胶电泳显示其条带大于48 kb，符合肌尾噬菌体科基因组大小范围（31—317 kb）；生物学特性的测定显示该噬菌体对青枯雷尔氏菌的最佳感染复数为0.01；其吸附和感染青枯雷尔氏菌时的潜伏期约为30 min，爆发期约为80 min，裂解量约为156；该噬菌体的裂解活性在28℃时最高，在28—50℃均较强，但在温度超过60℃后活性基本丧失；其对酸碱的耐受力较强，在 pH 3—8的范围内均有较强的裂解活性，当 pH 值超过9后活性开始降低；其对紫外线有一定的耐受能力，经紫外线照射0—9 min 后裂解活性依然较强，12 min 后活性开始下降，21 min 后活性基本丧失；其对氯仿不敏感，5%浓度的氯仿对其活性基本没有影响。【结论】分离到了裂解性的青枯雷尔氏菌噬菌体，属于有尾噬菌体目，肌尾噬菌体科，经过测定其各项生物学特性可知其潜伏期较短，裂解能力较强，具有很好的杀菌效果，且其裂解活性持续时间长，并能在不同温度、不同酸碱性的环境内有较强的适应能力，具有开发为抗青枯雷尔氏菌菌剂的潜力。【目的】分離併純化齣一株裂解性青枯雷爾氏菌(Ralstonia solanacearum)噬菌體，併測定其各項生物學特性，為開髮新的抗煙草青枯病製劑提供依據。【方法】取煙草青枯病重病田中健康煙株的根際土壤製成土壤懸浮液，併通過在青枯雷爾氏菌菌液中加入過濾後的土壤懸浮液富集噬菌體，用雙層平闆法驗證噬菌體的存在後挑取單箇最大噬菌斑進行反複純化，直到得到單一清晰的噬菌斑。純化後的單箇噬菌斑加入對數早期的青枯雷爾氏菌菌液中進行增殖培養，將增殖液按常規方法進行噬菌體顆粒濃縮後，取20μL 濃縮液用燐鎢痠染色併通過電子顯微鏡觀察噬菌體的形態特徵；同時將濃縮液進行 SDS-PAGE 電泳，觀察蛋白條帶大小和數量；用λ噬菌體 DNA 提取試劑盒提取噬菌體增殖液中的噬菌體覈痠進行瓊脂糖凝膠電泳，確定其基因組片段大小；最後用常規方法測定噬菌體的滴度、最佳感染複數、一步生長麯線，併通過比較加入噬菌體液前後青枯雷爾氏菌菌液的 OD600值變化測定其對溫度、pH、紫外線、氯倣的敏感性。【結果】分離併純化齣瞭一株裂解性青枯雷爾氏菌噬菌體，命名為∈RS-1,噬菌斑為圓形，清晰透明，邊緣光滑，直徑1—2 mm，經電鏡觀察其形態為蝌蚪狀，頭部為二十麵體的立體對稱，直徑約為94 nm，併有一帶伸縮尾鞘的長尾大約為27 nm×100 nm，按照國際病毒分類委員會分類標準，其屬于有尾噬菌體目（Caudovirales），肌尾噬菌體科(Myoviridae)的裂解性噬菌體，覈痠性質為 dsDNA；噬菌體濃縮液經 SDS-PAGE 分析至少可以觀察到25條蛋白條帶，相對分子質量在10—100 kD，說明其蛋白外殼至少含有25箇結構蛋白；將提取的 DNA 進行瓊脂糖凝膠電泳顯示其條帶大于48 kb，符閤肌尾噬菌體科基因組大小範圍（31—317 kb）；生物學特性的測定顯示該噬菌體對青枯雷爾氏菌的最佳感染複數為0.01；其吸附和感染青枯雷爾氏菌時的潛伏期約為30 min，爆髮期約為80 min，裂解量約為156；該噬菌體的裂解活性在28℃時最高，在28—50℃均較彊，但在溫度超過60℃後活性基本喪失；其對痠堿的耐受力較彊，在 pH 3—8的範圍內均有較彊的裂解活性，噹 pH 值超過9後活性開始降低；其對紫外線有一定的耐受能力，經紫外線照射0—9 min 後裂解活性依然較彊，12 min 後活性開始下降，21 min 後活性基本喪失；其對氯倣不敏感，5%濃度的氯倣對其活性基本沒有影響。【結論】分離到瞭裂解性的青枯雷爾氏菌噬菌體，屬于有尾噬菌體目，肌尾噬菌體科，經過測定其各項生物學特性可知其潛伏期較短，裂解能力較彊，具有很好的殺菌效果，且其裂解活性持續時間長，併能在不同溫度、不同痠堿性的環境內有較彊的適應能力，具有開髮為抗青枯雷爾氏菌菌劑的潛力。
【목적】분리병순화출일주렬해성청고뢰이씨균(Ralstonia solanacearum)서균체，병측정기각항생물학특성，위개발신적항연초청고병제제제공의거。【방법】취연초청고병중병전중건강연주적근제토양제성토양현부액，병통과재청고뢰이씨균균액중가입과려후적토양현부액부집서균체，용쌍층평판법험증서균체적존재후도취단개최대서균반진행반복순화，직도득도단일청석적서균반。순화후적단개서균반가입대수조기적청고뢰이씨균균액중진행증식배양，장증식액안상규방법진행서균체과립농축후，취20μL 농축액용린오산염색병통과전자현미경관찰서균체적형태특정；동시장농축액진행 SDS-PAGE 전영，관찰단백조대대소화수량；용λ서균체 DNA 제취시제합제취서균체증식액중적서균체핵산진행경지당응효전영，학정기기인조편단대소；최후용상규방법측정서균체적적도、최가감염복수、일보생장곡선，병통과비교가입서균체액전후청고뢰이씨균균액적 OD600치변화측정기대온도、pH、자외선、록방적민감성。【결과】분리병순화출료일주렬해성청고뢰이씨균서균체，명명위∈RS-1,서균반위원형，청석투명，변연광활，직경1—2 mm，경전경관찰기형태위과두상，두부위이십면체적입체대칭，직경약위94 nm，병유일대신축미초적장미대약위27 nm×100 nm，안조국제병독분류위원회분류표준，기속우유미서균체목（Caudovirales），기미서균체과(Myoviridae)적렬해성서균체，핵산성질위 dsDNA；서균체농축액경 SDS-PAGE 분석지소가이관찰도25조단백조대，상대분자질량재10—100 kD，설명기단백외각지소함유25개결구단백；장제취적 DNA 진행경지당응효전영현시기조대대우48 kb，부합기미서균체과기인조대소범위（31—317 kb）；생물학특성적측정현시해서균체대청고뢰이씨균적최가감염복수위0.01；기흡부화감염청고뢰이씨균시적잠복기약위30 min，폭발기약위80 min，렬해량약위156；해서균체적렬해활성재28℃시최고，재28—50℃균교강，단재온도초과60℃후활성기본상실；기대산감적내수력교강，재 pH 3—8적범위내균유교강적렬해활성，당 pH 치초과9후활성개시강저；기대자외선유일정적내수능력，경자외선조사0—9 min 후렬해활성의연교강，12 min 후활성개시하강，21 min 후활성기본상실；기대록방불민감，5%농도적록방대기활성기본몰유영향。【결론】분리도료렬해성적청고뢰이씨균서균체，속우유미서균체목，기미서균체과，경과측정기각항생물학특성가지기잠복기교단，렬해능력교강，구유흔호적살균효과，차기렬해활성지속시간장，병능재불동온도、불동산감성적배경내유교강적괄응능력，구유개발위항청고뢰이씨균균제적잠력。
Objective]In order to provide a new formulation of anti tobacco bacterial wilt, a lytic phage of Ralstonia solanacearum was isolated and purified from natural environments, and its biological characteristics were determined.[Method]Rhizosphere soil samples were taken from healthy tobacco plants growing in the disease field of tobacco bacterial wilt. Soil suspension after filtration was added into the R. solanacearum bacterial liquid to enrich the phage by co-cultivating. The existence of phage was detected using the way of two-layer plating method and a single clear of plague was obtained through purifying the single maximum phage repeatedly. The purified single phage was added into R. solanacearum bacterial liquid at logarithmic phase to propagate the phage culture. 20 μL concentrate of the phage particles by the conventional method was stained with phosphotungstic acid to observe their morphological characteristics by electron microscope. Meanwhile, the concentrate of phage’s fluid was used to detect the number and size of phage’s structure protein by SDS-PAGE electrophoresis, and confirm the size of genome fragment by agarose gel electrophoresis after extracting the total nucleic acid of the phage using the phage DNA extraction kit. The other biological properties include titer, MOI, one-step growth curve were measured by the conventional method, and the sensitivity to temperature, pH, ultraviolet ray and chloroform was tested by comparing the change in the OD600 of the R. solanacearum bacterial liquid before and after adding the phage.[Result]A strain of lytic R. solanacearum phage named as ∈RS-1 was isolated and purified. Its plaques are circular, clear and transparent with smooth edge, showing 1-2 mm of diameter. The electron microscope result showed that the phage particles like tadpoles with an icosahedra head of 94 nm diameter, and a long flexible tail about 27 nm×100 nm. According to the classification standards of the international commission on virus classification, it is a lytic phage of Myoviridae in Caudovirales, and its nucleic acid is dsDNA. There were 25 protein bands showing relative molecular mass of 10-100 kD in the result of SDS-PAGE, meaning the phage had at least 25 structural proteins. The agarose gel electrophoresis showed that its genome size was greater than 48 kb, fitting to the characteristics of Myoviridae (31-317 kb). Measurement of biological properties showed that the multiplicity of infection (MOI) of the R. solanacearum phage was 0.01. Its incubation period was 30 min, burst phase time was 80 min, and the burst size was 156 when infecting and locating on the R. solanacearum. The activity of phage ∈RS-1 was strong from 28℃ to 50℃, reaching a supreme at 28℃, however showing inactivation at 60℃. And it had wide susceptibility to pH changes, but its activity was decreased when the pH was above 9. The phage ∈RS-1 was also insensitive to ultraviolet ray, its activity was stable when exposed to ultraviolet ray for 0-9 min and began to decline after 21 min, 24 min later it was inactivated. It was insensitive to chloroform, 5%concentration of chloroform had no effect on its activity. [Conclusion]In this experiment, a lytic R. solanacearum’s phage was isolated belonging to Myoviridae in Caudovirales. With some good biological properties including short incubation period, strong sterilization ability and stable in wide temperatures and pH, it has a potential to be developed as a new preparation for control the R. solanacearum bacterial disease.