Cargando…

Development of a nested PCR assay for detecting Colletotrichum siamense and Colletotrichum fructicola on symptomless strawberry plants

Anthracnose is a major disease of strawberry that seriously impacts the strawberry industry. To prevent the spread of anthracnose through symptomless plants, it is important to detect pathogenic Colletotrichum spp. at the latent infection stage in the nursery. Previous PCR-based methods developed fo...

Descripción completa

Detalles Bibliográficos
Autores principales: Chung, Pei-Che, Wu, Hung-Yi, Chen, Yi-Chia, Hung, Ting-Hsuan, Chung, Chia-Lin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9239453/
https://www.ncbi.nlm.nih.gov/pubmed/35763511
http://dx.doi.org/10.1371/journal.pone.0270687
_version_ 1784737297258774528
author Chung, Pei-Che
Wu, Hung-Yi
Chen, Yi-Chia
Hung, Ting-Hsuan
Chung, Chia-Lin
author_facet Chung, Pei-Che
Wu, Hung-Yi
Chen, Yi-Chia
Hung, Ting-Hsuan
Chung, Chia-Lin
author_sort Chung, Pei-Che
collection PubMed
description Anthracnose is a major disease of strawberry that seriously impacts the strawberry industry. To prevent the spread of anthracnose through symptomless plants, it is important to detect pathogenic Colletotrichum spp. at the latent infection stage in the nursery. Previous PCR-based methods developed for the diagnosis or detection of Colletotrichum acutatum and Colletotrichum gloeosporioides have used primers targeting the internal transcribed spacer region of ribosomal DNA, β-tubulin gene, or mating type gene. In this study, to specifically detect Colletotrichum siamense and Colletotrichum fructicola, the most predominant and virulent Colletotrichum species causing strawberry anthracnose in Taiwan, we conducted a comparative genomics analysis of 29 Colletotrichum spp. and identified a non-conserved 1157-bp intergenic region suitable for designing specific primers for a nested PCR assay. In silico analysis and actual tests suggested that the new nested PCR assay could detect pathogenic C. siamense and C. fructicola, but not other strawberry pathogens (Botrytis sp., Fusarium spp., Neopestalotiopsis rosae, and Phytophthora sp.) or ubiquitous saprophytes (Fusarium spp. and Trichoderma spp.). The inner to outer primer ratio was optimized to 1:10 to eliminate unexpected bands and enhance the signal. The assay could detect as little as 1 pg of C. siamense genomic DNA, which corresponds to ~15 cells. Application of the new detection assay on 747 leaf samples collected from 18 strawberry nurseries in 2019 and 2020 showed that an average of 20% of strawberry mother plants in Taiwan were latently infected by C. siamense or C. fructicola. The newly developed assay is being applied to facilitate the production of healthy strawberry runner plants in Taiwan.
format Online
Article
Text
id pubmed-9239453
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-92394532022-06-29 Development of a nested PCR assay for detecting Colletotrichum siamense and Colletotrichum fructicola on symptomless strawberry plants Chung, Pei-Che Wu, Hung-Yi Chen, Yi-Chia Hung, Ting-Hsuan Chung, Chia-Lin PLoS One Research Article Anthracnose is a major disease of strawberry that seriously impacts the strawberry industry. To prevent the spread of anthracnose through symptomless plants, it is important to detect pathogenic Colletotrichum spp. at the latent infection stage in the nursery. Previous PCR-based methods developed for the diagnosis or detection of Colletotrichum acutatum and Colletotrichum gloeosporioides have used primers targeting the internal transcribed spacer region of ribosomal DNA, β-tubulin gene, or mating type gene. In this study, to specifically detect Colletotrichum siamense and Colletotrichum fructicola, the most predominant and virulent Colletotrichum species causing strawberry anthracnose in Taiwan, we conducted a comparative genomics analysis of 29 Colletotrichum spp. and identified a non-conserved 1157-bp intergenic region suitable for designing specific primers for a nested PCR assay. In silico analysis and actual tests suggested that the new nested PCR assay could detect pathogenic C. siamense and C. fructicola, but not other strawberry pathogens (Botrytis sp., Fusarium spp., Neopestalotiopsis rosae, and Phytophthora sp.) or ubiquitous saprophytes (Fusarium spp. and Trichoderma spp.). The inner to outer primer ratio was optimized to 1:10 to eliminate unexpected bands and enhance the signal. The assay could detect as little as 1 pg of C. siamense genomic DNA, which corresponds to ~15 cells. Application of the new detection assay on 747 leaf samples collected from 18 strawberry nurseries in 2019 and 2020 showed that an average of 20% of strawberry mother plants in Taiwan were latently infected by C. siamense or C. fructicola. The newly developed assay is being applied to facilitate the production of healthy strawberry runner plants in Taiwan. Public Library of Science 2022-06-28 /pmc/articles/PMC9239453/ /pubmed/35763511 http://dx.doi.org/10.1371/journal.pone.0270687 Text en © 2022 Chung et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Chung, Pei-Che
Wu, Hung-Yi
Chen, Yi-Chia
Hung, Ting-Hsuan
Chung, Chia-Lin
Development of a nested PCR assay for detecting Colletotrichum siamense and Colletotrichum fructicola on symptomless strawberry plants
title Development of a nested PCR assay for detecting Colletotrichum siamense and Colletotrichum fructicola on symptomless strawberry plants
title_full Development of a nested PCR assay for detecting Colletotrichum siamense and Colletotrichum fructicola on symptomless strawberry plants
title_fullStr Development of a nested PCR assay for detecting Colletotrichum siamense and Colletotrichum fructicola on symptomless strawberry plants
title_full_unstemmed Development of a nested PCR assay for detecting Colletotrichum siamense and Colletotrichum fructicola on symptomless strawberry plants
title_short Development of a nested PCR assay for detecting Colletotrichum siamense and Colletotrichum fructicola on symptomless strawberry plants
title_sort development of a nested pcr assay for detecting colletotrichum siamense and colletotrichum fructicola on symptomless strawberry plants
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9239453/
https://www.ncbi.nlm.nih.gov/pubmed/35763511
http://dx.doi.org/10.1371/journal.pone.0270687
work_keys_str_mv AT chungpeiche developmentofanestedpcrassayfordetectingcolletotrichumsiamenseandcolletotrichumfructicolaonsymptomlessstrawberryplants
AT wuhungyi developmentofanestedpcrassayfordetectingcolletotrichumsiamenseandcolletotrichumfructicolaonsymptomlessstrawberryplants
AT chenyichia developmentofanestedpcrassayfordetectingcolletotrichumsiamenseandcolletotrichumfructicolaonsymptomlessstrawberryplants
AT hungtinghsuan developmentofanestedpcrassayfordetectingcolletotrichumsiamenseandcolletotrichumfructicolaonsymptomlessstrawberryplants
AT chungchialin developmentofanestedpcrassayfordetectingcolletotrichumsiamenseandcolletotrichumfructicolaonsymptomlessstrawberryplants