Specific and sensitive detection of Phytophthora nicotianae by simple and nested-PCR
Phytophthora nicotianae Breda de Haan is one of the most important soil-borne plant pathogens. The identification of this pathogen based on morphological or physiological characters is time-consuming and labour-intensive and requires comprehensive knowledge of fungi. Molecular analysis of the intern...
| Autores principales: | , , , , , |
|---|---|
| Formato: | article |
| Lenguaje: | Inglés |
| Publicado: |
2017
|
| Acceso en línea: | http://hdl.handle.net/20.500.11939/5337 |
| _version_ | 1855032263372177408 |
|---|---|
| author | Grote, D. Olmos, Antonio Kofoet, A. Tuset, Juan J. Bertolini, Edson Cambra, Mariano |
| author_browse | Bertolini, Edson Cambra, Mariano Grote, D. Kofoet, A. Olmos, Antonio Tuset, Juan J. |
| author_facet | Grote, D. Olmos, Antonio Kofoet, A. Tuset, Juan J. Bertolini, Edson Cambra, Mariano |
| author_sort | Grote, D. |
| collection | ReDivia |
| description | Phytophthora nicotianae Breda de Haan is one of the most important soil-borne plant pathogens. The identification of this pathogen based on morphological or physiological characters is time-consuming and labour-intensive and requires comprehensive knowledge of fungi. Molecular analysis of the internal transcribed spacer (ITS) regions of rDNA is a novel and very effective method of species determination. Based on this concept, conventional and single closed tube nested-PCRs were developed for the specific and sensitive detection of P. nicotianae. Two new specific primers, designed from the spacer regions ITS1 and ITS2, internal to the nucleotide sequence flanked by universal primers ITS4 and ITS6, were used. To evaluate the specificity of the method, 36 morphologically characterized isolates were tested. A positive reaction, characterized by an amplification product of 737 bp, was shown by all P. nicotianae isolates and two P. nicotianae/cactorum hybrids. No amplification product was observed when other Phytophthora species and genera were assayed. The sensitivity of this method was analysed by serial dilutions of a defined amount of fungal DNA in a healthy root extract. Nested-PCR was at least 1000 times more sensitive than conventional PCR. In addition, samples from different infection sites, origins and crops, samples from nutrient solution, water and the rockwool used in hydroponic cultures, were analysed to validate this method. |
| format | article |
| id | ReDivia5337 |
| institution | Instituto Valenciano de Investigaciones Agrarias (IVIA) |
| language | Inglés |
| publishDate | 2017 |
| publishDateRange | 2017 |
| publishDateSort | 2017 |
| record_format | dspace |
| spelling | ReDivia53372025-04-25T14:42:07Z Specific and sensitive detection of Phytophthora nicotianae by simple and nested-PCR Grote, D. Olmos, Antonio Kofoet, A. Tuset, Juan J. Bertolini, Edson Cambra, Mariano Phytophthora nicotianae Breda de Haan is one of the most important soil-borne plant pathogens. The identification of this pathogen based on morphological or physiological characters is time-consuming and labour-intensive and requires comprehensive knowledge of fungi. Molecular analysis of the internal transcribed spacer (ITS) regions of rDNA is a novel and very effective method of species determination. Based on this concept, conventional and single closed tube nested-PCRs were developed for the specific and sensitive detection of P. nicotianae. Two new specific primers, designed from the spacer regions ITS1 and ITS2, internal to the nucleotide sequence flanked by universal primers ITS4 and ITS6, were used. To evaluate the specificity of the method, 36 morphologically characterized isolates were tested. A positive reaction, characterized by an amplification product of 737 bp, was shown by all P. nicotianae isolates and two P. nicotianae/cactorum hybrids. No amplification product was observed when other Phytophthora species and genera were assayed. The sensitivity of this method was analysed by serial dilutions of a defined amount of fungal DNA in a healthy root extract. Nested-PCR was at least 1000 times more sensitive than conventional PCR. In addition, samples from different infection sites, origins and crops, samples from nutrient solution, water and the rockwool used in hydroponic cultures, were analysed to validate this method. 2017-06-01T10:12:10Z 2017-06-01T10:12:10Z 2002 MAR 2002 article Grote, D., Olmos, A., Kofoet, A., Tuset, J.J., Bertolini, E., Cambra, M. (2002). Specific and sensitive detection of Phytophthora nicotianae by simple and nested-PCR. European Journal of Plant Pathology, 108(3), 197-207. 0929-1873 http://hdl.handle.net/20.500.11939/5337 10.1023/A:1015139410793 en openAccess Impreso |
| spellingShingle | Grote, D. Olmos, Antonio Kofoet, A. Tuset, Juan J. Bertolini, Edson Cambra, Mariano Specific and sensitive detection of Phytophthora nicotianae by simple and nested-PCR |
| title | Specific and sensitive detection of Phytophthora nicotianae by simple and nested-PCR |
| title_full | Specific and sensitive detection of Phytophthora nicotianae by simple and nested-PCR |
| title_fullStr | Specific and sensitive detection of Phytophthora nicotianae by simple and nested-PCR |
| title_full_unstemmed | Specific and sensitive detection of Phytophthora nicotianae by simple and nested-PCR |
| title_short | Specific and sensitive detection of Phytophthora nicotianae by simple and nested-PCR |
| title_sort | specific and sensitive detection of phytophthora nicotianae by simple and nested pcr |
| url | http://hdl.handle.net/20.500.11939/5337 |
| work_keys_str_mv | AT groted specificandsensitivedetectionofphytophthoranicotianaebysimpleandnestedpcr AT olmosantonio specificandsensitivedetectionofphytophthoranicotianaebysimpleandnestedpcr AT kofoeta specificandsensitivedetectionofphytophthoranicotianaebysimpleandnestedpcr AT tusetjuanj specificandsensitivedetectionofphytophthoranicotianaebysimpleandnestedpcr AT bertoliniedson specificandsensitivedetectionofphytophthoranicotianaebysimpleandnestedpcr AT cambramariano specificandsensitivedetectionofphytophthoranicotianaebysimpleandnestedpcr |