Engineered pine endophytic Bacillus toyonensis with nematocidal and colonization abilities for pine wilt disease control
IntroductionThe pinewood nematode (PWN) is responsible for causing pine wilt disease (PWD), which has led to the significant decline of conifer species in Eurasian forests and has become a globally invasive quarantine pest. Manipulating plant-associated microbes to control nematodes is an important...
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Frontiers Media S.A.
2023-12-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2023.1240984/full |
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| author | Dongzhen Li Dongzhen Li Yongxia Li Yongxia Li Xuan Wang Xuan Wang Wei Zhang Wei Zhang Xiaojian Wen Xiaojian Wen Zhenkai Liu Zhenkai Liu Yuqian Feng Yuqian Feng Xingyao Zhang Xingyao Zhang |
| author_facet | Dongzhen Li Dongzhen Li Yongxia Li Yongxia Li Xuan Wang Xuan Wang Wei Zhang Wei Zhang Xiaojian Wen Xiaojian Wen Zhenkai Liu Zhenkai Liu Yuqian Feng Yuqian Feng Xingyao Zhang Xingyao Zhang |
| author_sort | Dongzhen Li |
| collection | DOAJ |
| description | IntroductionThe pinewood nematode (PWN) is responsible for causing pine wilt disease (PWD), which has led to the significant decline of conifer species in Eurasian forests and has become a globally invasive quarantine pest. Manipulating plant-associated microbes to control nematodes is an important strategy for sustainable pest management. However, it has proven difficult to find pine-associated bacteria that possess both nematocidal activity and the ability to colonize pine tissues.MethodsThe stress experiments with turpentine and pine tissue extract were carried out to screen for the desired target strain that could adapt to the internal environment of pine trees. This strain was used to construct an engineered nematocidal strain. Additionally, a fluorescent strain was constructed to determine its dispersal ability in Pinus massoniana seedlings through plate separation, PCR detection, and fluorescence microscopy observations. The engineered nematocidal strain was tested in the greenhouse experiment to assess its ability to effectively protect P. massoniana seedlings from nematode infection.ResultsThis study isolated a Bacillus toyonensis strain Bxy19 from the healthy pine stem, which showed exceptional tolerance in stress experiments. An engineered nematocidal strain Bxy19P3C6 was constructed, which expressed the Cry6Aa crystal protein and exhibited nematocidal activity. The fluorescent strain Bxy19GFP was also constructed and used to test its dispersal ability. It was observed to enter the needles of the seedlings through the stomata and colonize the vascular bundle after being sprayed on the seedlings. The strain was observed to colonize and spread in the tracheid after being injected into the stems. The strain could colonize the seedlings and persist for at least 50 days. Furthermore, the greenhouse experiments indicated that both spraying and injecting the engineered strain Bxy19P3C6 had considerable efficacy against nematode infection.DiscussionThe evidence of the colonization ability and persistence of the strain in pine advances our understanding of the control and prediction of the colonization of exogenously delivered bacteria in pines. This study provides a promising approach for manipulating plant-associated bacteria and using Bt protein to control nematodes. |
| first_indexed | 2024-03-09T02:37:47Z |
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| id | doaj.art-b841f6e0cc0b475ebbef485d5582ba46 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-03-09T02:37:47Z |
| publishDate | 2023-12-01 |
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| series | Frontiers in Microbiology |
| spelling | doaj.art-b841f6e0cc0b475ebbef485d5582ba462023-12-06T08:26:01ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2023-12-011410.3389/fmicb.2023.12409841240984Engineered pine endophytic Bacillus toyonensis with nematocidal and colonization abilities for pine wilt disease controlDongzhen Li0Dongzhen Li1Yongxia Li2Yongxia Li3Xuan Wang4Xuan Wang5Wei Zhang6Wei Zhang7Xiaojian Wen8Xiaojian Wen9Zhenkai Liu10Zhenkai Liu11Yuqian Feng12Yuqian Feng13Xingyao Zhang14Xingyao Zhang15Key Laboratory of Forest Protection of National Forestry and Grassland Administration, Ecology and Nature Conservation Institute, Chinese Academy of Forestry, Beijing, ChinaCo-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, ChinaKey Laboratory of Forest Protection of National Forestry and Grassland Administration, Ecology and Nature Conservation Institute, Chinese Academy of Forestry, Beijing, ChinaCo-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, ChinaKey Laboratory of Forest Protection of National Forestry and Grassland Administration, Ecology and Nature Conservation Institute, Chinese Academy of Forestry, Beijing, ChinaCo-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, ChinaKey Laboratory of Forest Protection of National Forestry and Grassland Administration, Ecology and Nature Conservation Institute, Chinese Academy of Forestry, Beijing, ChinaCo-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, ChinaKey Laboratory of Forest Protection of National Forestry and Grassland Administration, Ecology and Nature Conservation Institute, Chinese Academy of Forestry, Beijing, ChinaCo-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, ChinaKey Laboratory of Forest Protection of National Forestry and Grassland Administration, Ecology and Nature Conservation Institute, Chinese Academy of Forestry, Beijing, ChinaCo-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, ChinaKey Laboratory of Forest Protection of National Forestry and Grassland Administration, Ecology and Nature Conservation Institute, Chinese Academy of Forestry, Beijing, ChinaCo-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, ChinaKey Laboratory of Forest Protection of National Forestry and Grassland Administration, Ecology and Nature Conservation Institute, Chinese Academy of Forestry, Beijing, ChinaCo-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, ChinaIntroductionThe pinewood nematode (PWN) is responsible for causing pine wilt disease (PWD), which has led to the significant decline of conifer species in Eurasian forests and has become a globally invasive quarantine pest. Manipulating plant-associated microbes to control nematodes is an important strategy for sustainable pest management. However, it has proven difficult to find pine-associated bacteria that possess both nematocidal activity and the ability to colonize pine tissues.MethodsThe stress experiments with turpentine and pine tissue extract were carried out to screen for the desired target strain that could adapt to the internal environment of pine trees. This strain was used to construct an engineered nematocidal strain. Additionally, a fluorescent strain was constructed to determine its dispersal ability in Pinus massoniana seedlings through plate separation, PCR detection, and fluorescence microscopy observations. The engineered nematocidal strain was tested in the greenhouse experiment to assess its ability to effectively protect P. massoniana seedlings from nematode infection.ResultsThis study isolated a Bacillus toyonensis strain Bxy19 from the healthy pine stem, which showed exceptional tolerance in stress experiments. An engineered nematocidal strain Bxy19P3C6 was constructed, which expressed the Cry6Aa crystal protein and exhibited nematocidal activity. The fluorescent strain Bxy19GFP was also constructed and used to test its dispersal ability. It was observed to enter the needles of the seedlings through the stomata and colonize the vascular bundle after being sprayed on the seedlings. The strain was observed to colonize and spread in the tracheid after being injected into the stems. The strain could colonize the seedlings and persist for at least 50 days. Furthermore, the greenhouse experiments indicated that both spraying and injecting the engineered strain Bxy19P3C6 had considerable efficacy against nematode infection.DiscussionThe evidence of the colonization ability and persistence of the strain in pine advances our understanding of the control and prediction of the colonization of exogenously delivered bacteria in pines. This study provides a promising approach for manipulating plant-associated bacteria and using Bt protein to control nematodes.https://www.frontiersin.org/articles/10.3389/fmicb.2023.1240984/fullpinewood nematodeBtbacterial colonizationplant-associated microbesengineering bacteriaBacillus |
| spellingShingle | Dongzhen Li Dongzhen Li Yongxia Li Yongxia Li Xuan Wang Xuan Wang Wei Zhang Wei Zhang Xiaojian Wen Xiaojian Wen Zhenkai Liu Zhenkai Liu Yuqian Feng Yuqian Feng Xingyao Zhang Xingyao Zhang Engineered pine endophytic Bacillus toyonensis with nematocidal and colonization abilities for pine wilt disease control Frontiers in Microbiology pinewood nematode Bt bacterial colonization plant-associated microbes engineering bacteria Bacillus |
| title | Engineered pine endophytic Bacillus toyonensis with nematocidal and colonization abilities for pine wilt disease control |
| title_full | Engineered pine endophytic Bacillus toyonensis with nematocidal and colonization abilities for pine wilt disease control |
| title_fullStr | Engineered pine endophytic Bacillus toyonensis with nematocidal and colonization abilities for pine wilt disease control |
| title_full_unstemmed | Engineered pine endophytic Bacillus toyonensis with nematocidal and colonization abilities for pine wilt disease control |
| title_short | Engineered pine endophytic Bacillus toyonensis with nematocidal and colonization abilities for pine wilt disease control |
| title_sort | engineered pine endophytic bacillus toyonensis with nematocidal and colonization abilities for pine wilt disease control |
| topic | pinewood nematode Bt bacterial colonization plant-associated microbes engineering bacteria Bacillus |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2023.1240984/full |
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