Nanopore-Sequencing Metabarcoding for Identification of Phytopathogenic and Endophytic Fungi in Olive (<i>Olea europaea</i>) Twigs
Metabarcoding approaches for the identification of plant disease pathogens and characterization of plant microbial populations constitute a rapidly evolving research field. Fungal plant diseases are of major phytopathological concern; thus, the development of metabarcoding approaches for the detecti...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-11-01
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| Series: | Journal of Fungi |
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| Online Access: | https://www.mdpi.com/2309-608X/9/11/1119 |
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| author | Ioannis Theologidis Timokratis Karamitros Aikaterini-Eleni Vichou Dimosthenis Kizis |
| author_facet | Ioannis Theologidis Timokratis Karamitros Aikaterini-Eleni Vichou Dimosthenis Kizis |
| author_sort | Ioannis Theologidis |
| collection | DOAJ |
| description | Metabarcoding approaches for the identification of plant disease pathogens and characterization of plant microbial populations constitute a rapidly evolving research field. Fungal plant diseases are of major phytopathological concern; thus, the development of metabarcoding approaches for the detection of phytopathogenic fungi is becoming increasingly imperative in the context of plant disease prognosis. We developed a multiplex metabarcoding method for the identification of fungal phytopathogens and endophytes in olive young shoots, using the MinION sequencing platform (Oxford Nanopore Technologies). Selected fungal-specific primers were used to amplify three different genomic DNA loci (ITS, beta-tubulin, and 28S LSU) originating from olive twigs. A multiplex metabarcoding approach was initially evaluated using healthy olive twigs, and further assessed with naturally infected olive twig samples. Bioinformatic analysis of basecalled reads was carried out using MinKNOW, BLAST+ and R programming, and results were also evaluated using the BugSeq cloud platform. Data analysis highlighted the approaches based on ITS and their combination with beta-tubulin as the most informative ones according to diversity estimations. Subsequent implementation of the method on symptomatic samples identified major olive pathogens and endophytes including genera such as <i>Cladosporium</i>, <i>Didymosphaeria</i>, <i>Paraconiothyrium</i>, <i>Penicillium, Phoma, Verticillium</i>, and others. |
| first_indexed | 2024-03-09T16:41:22Z |
| format | Article |
| id | doaj.art-ad34f18c82cc4023b24029fce2aa73c0 |
| institution | Directory Open Access Journal |
| issn | 2309-608X |
| language | English |
| last_indexed | 2024-03-09T16:41:22Z |
| publishDate | 2023-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Journal of Fungi |
| spelling | doaj.art-ad34f18c82cc4023b24029fce2aa73c02023-11-24T14:51:13ZengMDPI AGJournal of Fungi2309-608X2023-11-01911111910.3390/jof9111119Nanopore-Sequencing Metabarcoding for Identification of Phytopathogenic and Endophytic Fungi in Olive (<i>Olea europaea</i>) TwigsIoannis Theologidis0Timokratis Karamitros1Aikaterini-Eleni Vichou2Dimosthenis Kizis3Laboratory of Toxicological Control of Pesticides, Scientific Directorate of Pesticides’ Control & Phytopharmacy, Benaki Phytopathological Institute, 8 St. Delta Street, 14561 Athens, Attica, GreeceBioinformatics and Applied Genomics Unit, Department of Microbiology, Hellenic Pasteur Institute, 127 Vasilissis Sofias Avenue, 11521 Athens, Attica, GreeceLaboratory of Mycology, Scientific Directorate of Phytopathology, Benaki Phytopathological Institute, 8 St. Delta Street, 14561 Athens, Attica, GreeceLaboratory of Mycology, Scientific Directorate of Phytopathology, Benaki Phytopathological Institute, 8 St. Delta Street, 14561 Athens, Attica, GreeceMetabarcoding approaches for the identification of plant disease pathogens and characterization of plant microbial populations constitute a rapidly evolving research field. Fungal plant diseases are of major phytopathological concern; thus, the development of metabarcoding approaches for the detection of phytopathogenic fungi is becoming increasingly imperative in the context of plant disease prognosis. We developed a multiplex metabarcoding method for the identification of fungal phytopathogens and endophytes in olive young shoots, using the MinION sequencing platform (Oxford Nanopore Technologies). Selected fungal-specific primers were used to amplify three different genomic DNA loci (ITS, beta-tubulin, and 28S LSU) originating from olive twigs. A multiplex metabarcoding approach was initially evaluated using healthy olive twigs, and further assessed with naturally infected olive twig samples. Bioinformatic analysis of basecalled reads was carried out using MinKNOW, BLAST+ and R programming, and results were also evaluated using the BugSeq cloud platform. Data analysis highlighted the approaches based on ITS and their combination with beta-tubulin as the most informative ones according to diversity estimations. Subsequent implementation of the method on symptomatic samples identified major olive pathogens and endophytes including genera such as <i>Cladosporium</i>, <i>Didymosphaeria</i>, <i>Paraconiothyrium</i>, <i>Penicillium, Phoma, Verticillium</i>, and others.https://www.mdpi.com/2309-608X/9/11/1119metabarcodinghigh-throughput sequencingnanopore sequencingoliveplant pathogen<i>Verticillium dahliae</i> |
| spellingShingle | Ioannis Theologidis Timokratis Karamitros Aikaterini-Eleni Vichou Dimosthenis Kizis Nanopore-Sequencing Metabarcoding for Identification of Phytopathogenic and Endophytic Fungi in Olive (<i>Olea europaea</i>) Twigs Journal of Fungi metabarcoding high-throughput sequencing nanopore sequencing olive plant pathogen <i>Verticillium dahliae</i> |
| title | Nanopore-Sequencing Metabarcoding for Identification of Phytopathogenic and Endophytic Fungi in Olive (<i>Olea europaea</i>) Twigs |
| title_full | Nanopore-Sequencing Metabarcoding for Identification of Phytopathogenic and Endophytic Fungi in Olive (<i>Olea europaea</i>) Twigs |
| title_fullStr | Nanopore-Sequencing Metabarcoding for Identification of Phytopathogenic and Endophytic Fungi in Olive (<i>Olea europaea</i>) Twigs |
| title_full_unstemmed | Nanopore-Sequencing Metabarcoding for Identification of Phytopathogenic and Endophytic Fungi in Olive (<i>Olea europaea</i>) Twigs |
| title_short | Nanopore-Sequencing Metabarcoding for Identification of Phytopathogenic and Endophytic Fungi in Olive (<i>Olea europaea</i>) Twigs |
| title_sort | nanopore sequencing metabarcoding for identification of phytopathogenic and endophytic fungi in olive i olea europaea i twigs |
| topic | metabarcoding high-throughput sequencing nanopore sequencing olive plant pathogen <i>Verticillium dahliae</i> |
| url | https://www.mdpi.com/2309-608X/9/11/1119 |
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