학술논문
감자 재배포장에서 Ralstonia solanacearum과 Pectobacterium 종을 감염시키는 박테리오파지의 분리와 특성 / Isolation and characterization of bacteriophages infecting Ralstonia or Pectobacterium species from potato fields
Document Type
Dissertation/ Thesis
Author
Source
Subject
Language
English
Abstract
Ralstonia solanacearum과 Pectobacterium 종에 의해 발생되는 풋마름병과 무름병은 감자를 포함한 주요 가지과 작물에 파괴적인 병해들 중 하나다. 이런 병해를 방제하는 것은 아주 어렵고, 지금까지 적합한 방제법이 개발되지 않았다. 그리고 이 병원균들은 쉽게 퍼지며 식물 재배환경 속에서 수 년 동안 생존한다. 비록 화학약품들이 이 병해들을 방제하는데 일부 적용되고 있지만, 그 효능은 한정적이며 여러 문제들을 일으킨다. 그러므로 세균성 병해를 방제하기 위한 생물학적 방제 방법으로 파지의 적용이 좋은 대안으로 제시되고 있다.본 연구에서는, 한국의 감자재배포장에서 얻은 식물체와 토양샘플에서 다양한 R. solanacearum과 Pectobacterium 종을 용균 시키는 박테리오파지들이 발견되었다. 3~4번의 반복적인 용균반 picking을 통해 각각 R. solanacearum과 Pectobacterium종을 감염시키며 다양한 범위의 기주 특이성을 가진 173개와 189개의 파지들을 얻었고, 최종적으로 각각 12개와 24개의 핵심 파지들이 선발되었으며, 균주특이성을 기반으로 dendrogram이 작성되었다. 선발된 파지들의 유전적 다양성은 AFLP fingerprints를 이용하여 phylogenetic tree를 작성하여 확인했다. 그리고 다양한 온도, pH 조건에서 파지의 안정성이 조사되었는데, R. solanacearum 을 감염시키는 파지들은 16oC-44oC와 pH 6-10에서 안정적이었지만, Pectobacterium 종들을 감염시키는 파지들은 16oC-40oC 와 pH 6-7에서 안정적이었다. 선발된 파지들의 형태적 분석에서는 R. solanacearum를 감염시키는 파지들은 다양한 머리크기와 꼬리길이를 가진 Podoviridae로 분류되는 것으로 나타났지만, Pectobacterium 종들을 감염시키는 파지들은 다양한 머리크기와 꼬리길이를 가진 Podoviridae, Myoviridae, Siphoviridae의 세 가지 그룹으로 분류되는 것으로 나타났다. 추가적으로 감자 괴경에 Pectobacterium종을 감염시키는 선발된 파지를 접종하여 실질적으로 파지를 이용한 병해 제어의 가능성을 확인했다. 본 연구의 결과들은 세균성병해를 방제하는데 기여할 것이며, 나아가 유전적, 분자적 측면에 관한 추가적 연구를 통해 파지와 병원성 세균과의 상호작용에 대한 이해를 도울 것이다.
Bacterial wilt and soft rot caused by Ralstonia solanacearum and Pectobacterium species are one of the most devastating diseases in major Solanaceae crops including potato, respectively. It is very difficult to control these diseases and no promising methods have been developed to date. Although chemicals are applied to control the diseases, the pathogens are easily disseminated and survive for many years in plant farming system and they are of limited efficacy and cause several problems. Therefore, the use of phage therapy has been suggested to control the diseases as a biological agent. In this study, we discovered bacteriophages lysing diverse Ralstonia isolates and Pectobacterium species from plant and soil samples obtained from the potato cultivated fields in Republic of Korea. Three or four times repeated pickings of plaques allowed us to obtain 173 and 189 single phages infecting R. solanacearum and Pectobacterium species, respectively, showing a diverse spectrum of host-specificity. With these results, 12 and 24 core phages were selected and dendrograms were generated. Genetic diversity of the selected phages was confirmed by AFLP fingerprints generating phylogenetic trees. The stability of the phages was investigated in various temperatures and various conditions of pH in vitro. The phages infecting R. solanacearum were stable at 16oC–44oC and pH 6–10 whereas the phages infecting Pectobacterium species were stable at 16oC-40oC and pH 6-7. Moreover, morphological characterization of the phages infecting R. solanacearum revealed they were all classified into the Podoviridae with diverse head sizes, but the phages infecting Pectobacterium species were classified three different groups, Podoviridae, Myoviridae, and Siphoviridae with diverse head sizes and tail lengths. Additionally, inoculation of Pectobacterium species with selected phages on potato tubers confirmed the possibility to apply the phage therapy. The results of this research will contribute to control the diseases and further researches regarding genetic and molecular aspects will facilitate understanding phage and bacteria interaction.
Bacterial wilt and soft rot caused by Ralstonia solanacearum and Pectobacterium species are one of the most devastating diseases in major Solanaceae crops including potato, respectively. It is very difficult to control these diseases and no promising methods have been developed to date. Although chemicals are applied to control the diseases, the pathogens are easily disseminated and survive for many years in plant farming system and they are of limited efficacy and cause several problems. Therefore, the use of phage therapy has been suggested to control the diseases as a biological agent. In this study, we discovered bacteriophages lysing diverse Ralstonia isolates and Pectobacterium species from plant and soil samples obtained from the potato cultivated fields in Republic of Korea. Three or four times repeated pickings of plaques allowed us to obtain 173 and 189 single phages infecting R. solanacearum and Pectobacterium species, respectively, showing a diverse spectrum of host-specificity. With these results, 12 and 24 core phages were selected and dendrograms were generated. Genetic diversity of the selected phages was confirmed by AFLP fingerprints generating phylogenetic trees. The stability of the phages was investigated in various temperatures and various conditions of pH in vitro. The phages infecting R. solanacearum were stable at 16oC–44oC and pH 6–10 whereas the phages infecting Pectobacterium species were stable at 16oC-40oC and pH 6-7. Moreover, morphological characterization of the phages infecting R. solanacearum revealed they were all classified into the Podoviridae with diverse head sizes, but the phages infecting Pectobacterium species were classified three different groups, Podoviridae, Myoviridae, and Siphoviridae with diverse head sizes and tail lengths. Additionally, inoculation of Pectobacterium species with selected phages on potato tubers confirmed the possibility to apply the phage therapy. The results of this research will contribute to control the diseases and further researches regarding genetic and molecular aspects will facilitate understanding phage and bacteria interaction.