Progress 03/15/23 to 03/14/24
Outputs
Target Audience:The target audience for this reporting period includes graduate and undergraduate students, extension agents, farmers, participants in the 2023 APS (Denver, Co) and ICPP (Lyon, France) meetings interested in diagnostics, Leeward Community College students, and USDA APHIS. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Graduate students learned about metagenome diagnostics and validation. Undergraduate students were exposed to nanopore sequencing and data analysis for detection. Undergraduates were introduced to database development. A Junior Researcher (postdoc) received training in metagenome detection. How have the results been disseminated to communities of interest?The results were disseminated through presentations at scientific meetings and workshops. Classroom teaching was also used to share the outcomes. What do you plan to do during the next reporting period to accomplish the goals?Year 2 Plan: Genome Sequencing and Database Expansion: We will sequence approximately 100 more genomes using both Nanopore and Illumina technologies, and these will be added to the database. Additionally, more genomes from NCBI will be screened for accurate metadata and genome quality and will also be added to the database. BacPath Pipeline Validation: The pipeline validation is still underway and will be completed by next year. We obtained sequencing data from infected plant tissue (artificially and naturally infected), but not all data has been analyzed yet. Most of the data will be analyzed and compared in the next few months. NextStrain/tracking Analyses: The NextStrain pipeline is running smoothly, but we will analyze the genomes once all bacterial strain sequencing is completed. We expect to start working on tracking in the next few months. Publications: One review article on metagenome-based detection is under preparation and should be completed in the next few months. Another article on pipeline validation and database development will be prepared and submitted in Year 2. Presentations: The outcomes of the results will also be disseminated through presentations at scientific meetings and workshops.
Impacts
What was accomplished under these goals?
Strains of all Pectobacterium and Dickeya species: Representative strains from our collection were selected based on accurate metadata information. Strains of other Pectobacterium and Dickeya species were obtained from international culture collections. So far, we have obtained representative strains of almost each species of Pectobacterium and Dickeya. Approximately 100 strains were acquired to develop resources for diagnostic protocol validation and genome sequencing. Genome Sequencing and Database Development: High-quality DNA was isolated from purified bacterial strains, and only genomes with accurate metadata were selected for sequencing. More than 100 complete genomes have been sequenced using Nanopore and Illumina technologies, assembled (hybrid), and added to our customized database. All sequenced genomes will later be submitted to the NCBI GenBank database. So far, the database contains about 400 complete genomes with accurate metadata. The database includes not only genomes of Pectobacterium and Dickeya species but also genomes from other bacterial species infecting plants and other environmental pathogens. ?BacPath Pipeline Validation: The pipeline has been developed and is now undergoing rigorous validation. The in-silico validation for specificity and sensitivity is completed. We mixed 12 chromosomes of the host (potato) with different concentrations and reads of pathogens P. carotovorum and D. dianthicola. Mock metagenomes were created using MetaSim and CAMISim. Initially, we mixed the complete genome of the pathogen with the complete genome of the host in concentrations of 10, 100, and 1,000. Next, we mixed the complete host genome with different concentrations. For specificity, P. carotovorum was mixed with D. dianthicola, and P. versatile with D. solani. The read length was 250 bp. All tests were repeated three times to ensure reproducibility. The pipeline accurately detected the pathogens at a concentration of 10, with no false matches detected. In in-vitro validation, pipeline validation with infected plant tissues is underway. For specificity, plants were inoculated with known strains individually or with multiple bacterial species. DNA was isolated directly from infected tissues and sequenced using Nanopore Flongle and, in some cases, both Nanopore and Illumina sequencers. All tests were repeated three times. When the sequence data was processed through BacPath, all outcomes were accurate with no false matches. In sensitivity assays, 12 ng of host DNA was mixed with different concentrations (1 fg to 1 ng) of P. parmentieri and D. dianthicola. The samples were sequenced using both Nanopore (long reads) and Illumina (short reads) sequencers. So far, no false results have been obtained. The data is still under analyses, and we expect to have all final results available in the next few months. Experiments with naturally infected samples and their comparative analyses are underway.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Montesinos S, D, Dobhal S, Arif M (2023). BacPath: Diagnostics pipeline for highly reliable detection of bacterial plant pathogens. Presented at APS Annual Meeting - Plant Health 2023, Denver, Co.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Arif M, Stack JP (2023). Advancement in plant pathogen diagnostics in high-throughput sequencing era. Presented in ICPP 2023, Lyon, France.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Arif M (2023). Opportunities in Biosecurity, at Plant Biosecurity in Theory and Practice workshop, Biosecurity Research Institute, Kansas State University, Manhattan, KS, USA
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Arif M (2023). Plant Diagnostics in Genomic Era, at Plant Biosecurity in Theory and Practice workshop, Biosecurity Research Institute, Kansas State University, Manhattan, KS, USA
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Arif M (2023). Biosecurity alert: Coffee leaf rust in Hawaii, at Plant Biosecurity in Theory and Practice workshop, Biosecurity Research Institute, Kansas State University, Manhattan, KS, USA
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