Globalization and climate change are accelerating the spread of plant pathogens in new areas, leading to considerable economic losses and environmental changes. Pathogens often spread from infected material accidentally introduced by humans in a new area. Currently, there are several traditional techniques that are routinely employed in plant disease surveillance and management. However, they have limitations like the incapacity to detect new variants of the pathogen, the inability to detect all subspecies/pathovars in one step, and/or the impossibility to analyze multiple samples simultaneously. Moreover, these limitations lead to a significant time consumption. Therefore, it is imperative to develop fast, adaptable and easy-to-use protocols for surveilling plant material at the entry point. These protocols should allow identification at subspecies, pathovars (pvs) or sequence types (ST) level to guide surveillance strategies, as these provide insight on their host specificity and potential damage. Ideally, these protocols should have high sensitivity and specificity. To achieve rapid, reliable, cost-effective and point-of-care diagnostics, long-reads Nanopore sequencing could be a promising solution. Oxford Nanopore Technology provides portable devices (MinION) that can sequence several samples in real-time. Additionally, one of the key benefits of long reads is their ability to provide enhanced taxonomic resolution. However, compared to earlier technologies, there is a lack of bioinformatic tools specifically designed for Nanopore sequences’ analysis. To meet these requirements, we developed a sensitive and specific diagnostic system that can be quickly applied to different organisms at the country entry points. We focused on setting an amplicon-Nanopore sequencing workflow and developed a pipeline able to process long-noisy reads. To test and compare our pipeline with the existing ones, the 16S rDNA gene of several known bacteria, with different taxonomic similarity, was amplified and then sequenced using Nanopore technology. The produced sequences were then analyzed with “in-house” pipeline as well as available software. Our pipeline showed a precision and a recall close to 1 comparable to the well-established Emu software. Additionally, we applied our system to two EU-regulated quarantine bacterial pathogens, Xanthomonas citri (Xc) and Xylella fastidiosa (Xf) for which pvs/subspecies identification is required to implement appropriate countermeasures. Xf is divided into subspecies that infect different host plants. Xc pvs have distinct host ranges and distributions and require a diagnostic system able to specifically identify Xc pv. aurantifolii and differentiate it from pv. citri. For this purpose, an amplicon-Nanopore sequencing, based on selected housekeeping genes (e.g. MLST), has been developed. These selected genes allow the distinction of their pathovars, subspecies or ST. Several plant species have been spiked with different bacterial suspensions at known concentrations. Then, a multiplex- (for Xf) and duplex- (Xc) PCR amplification of housekeeping genes has been developed. The prepared samples were finally sequenced with Nanopore and analyzed with our pipeline to test the workflow: our approach showed a high flexibility and enables precise identification at both the genus-species level for broader metabarcoding studies and at a deeper taxonomic level, such as pathovar or sequence type. References Curry, K. D., Wang, Q., Nute, M. G., Tyshaieva, A., Reeves, E., Soriano, S., ... & Treangen, T. J. (2022). Emu: species-level microbial community profiling of full-length 16S rRNA Oxford Nanopore sequencing data. Nature methods, 19(7), 845-853. European and Mediterranean Plant Protection Organization. PM 7/44 (2) Xanthomonas citri pv. citri and Xanthomonas citri pv. aurantifolii. EPPO Bulletin, 2023, 53(1), 62-96. European and Mediterranean Plant Protection Organization. PM 7/24 (5) Xylella fastidiosa. EPPO Bull. 2023, 53, 205–276. Venbrux, M., Crauwels, S., & Rediers, H. (2023). Current and emerging trends in techniques for plant pathogen detection. Frontiers in Plant Science, 14, 1120968.
Nanopore amplicon sequencing: rapid, sensitive and specific diagnostic system to limit plant pathogenic bacteria spread / Crosara, V.; Tatulli, G.; Scala, V.; Reverberi, M.; Pucci, N.; Faino, L.; Loreti, S.. - (2024). (Intervento presentato al convegno The 8th Xanthomonas Genomics Conference (8XGC) tenutosi a Shanghai, China).
Nanopore amplicon sequencing: rapid, sensitive and specific diagnostic system to limit plant pathogenic bacteria spread
V. Crosara
;M. Reverberi;L. Faino;
2024
Abstract
Globalization and climate change are accelerating the spread of plant pathogens in new areas, leading to considerable economic losses and environmental changes. Pathogens often spread from infected material accidentally introduced by humans in a new area. Currently, there are several traditional techniques that are routinely employed in plant disease surveillance and management. However, they have limitations like the incapacity to detect new variants of the pathogen, the inability to detect all subspecies/pathovars in one step, and/or the impossibility to analyze multiple samples simultaneously. Moreover, these limitations lead to a significant time consumption. Therefore, it is imperative to develop fast, adaptable and easy-to-use protocols for surveilling plant material at the entry point. These protocols should allow identification at subspecies, pathovars (pvs) or sequence types (ST) level to guide surveillance strategies, as these provide insight on their host specificity and potential damage. Ideally, these protocols should have high sensitivity and specificity. To achieve rapid, reliable, cost-effective and point-of-care diagnostics, long-reads Nanopore sequencing could be a promising solution. Oxford Nanopore Technology provides portable devices (MinION) that can sequence several samples in real-time. Additionally, one of the key benefits of long reads is their ability to provide enhanced taxonomic resolution. However, compared to earlier technologies, there is a lack of bioinformatic tools specifically designed for Nanopore sequences’ analysis. To meet these requirements, we developed a sensitive and specific diagnostic system that can be quickly applied to different organisms at the country entry points. We focused on setting an amplicon-Nanopore sequencing workflow and developed a pipeline able to process long-noisy reads. To test and compare our pipeline with the existing ones, the 16S rDNA gene of several known bacteria, with different taxonomic similarity, was amplified and then sequenced using Nanopore technology. The produced sequences were then analyzed with “in-house” pipeline as well as available software. Our pipeline showed a precision and a recall close to 1 comparable to the well-established Emu software. Additionally, we applied our system to two EU-regulated quarantine bacterial pathogens, Xanthomonas citri (Xc) and Xylella fastidiosa (Xf) for which pvs/subspecies identification is required to implement appropriate countermeasures. Xf is divided into subspecies that infect different host plants. Xc pvs have distinct host ranges and distributions and require a diagnostic system able to specifically identify Xc pv. aurantifolii and differentiate it from pv. citri. For this purpose, an amplicon-Nanopore sequencing, based on selected housekeeping genes (e.g. MLST), has been developed. These selected genes allow the distinction of their pathovars, subspecies or ST. Several plant species have been spiked with different bacterial suspensions at known concentrations. Then, a multiplex- (for Xf) and duplex- (Xc) PCR amplification of housekeeping genes has been developed. The prepared samples were finally sequenced with Nanopore and analyzed with our pipeline to test the workflow: our approach showed a high flexibility and enables precise identification at both the genus-species level for broader metabarcoding studies and at a deeper taxonomic level, such as pathovar or sequence type. References Curry, K. D., Wang, Q., Nute, M. G., Tyshaieva, A., Reeves, E., Soriano, S., ... & Treangen, T. J. (2022). Emu: species-level microbial community profiling of full-length 16S rRNA Oxford Nanopore sequencing data. Nature methods, 19(7), 845-853. European and Mediterranean Plant Protection Organization. PM 7/44 (2) Xanthomonas citri pv. citri and Xanthomonas citri pv. aurantifolii. EPPO Bulletin, 2023, 53(1), 62-96. European and Mediterranean Plant Protection Organization. PM 7/24 (5) Xylella fastidiosa. EPPO Bull. 2023, 53, 205–276. Venbrux, M., Crauwels, S., & Rediers, H. (2023). Current and emerging trends in techniques for plant pathogen detection. Frontiers in Plant Science, 14, 1120968.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.