Progress 03/15/24 to 03/14/25
Outputs
Target Audience:The target audience for this reporting period includes graduate and undergraduate students, research scientists, extension agents, farmers, participants of the 2024 APS meeting (Memphis, Tennessee), the 2024 IPPC meeting (Athens, Greece), and the 2025 IPS meeting (Nagpur, India), as well as students from Leeward Community College and personnel from USDA APHIS. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Graduate students learned about genome sequencing, assembly, annotation, metagenome diagnostics and validation. Also, learned about database development. Undergraduate students were exposed to nanopore sequencing and data analysis for detection. Learning about genome assembly, annotation and how to write a genome announcement. A Junior Researcher (postdoc) received training in genomics. 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. Also, through publications. What do you plan to do during the next reporting period to accomplish the goals?Year 3 Plan (NCE): 1. BacPathDB database expansion: We have recently sequenced 96 new genomes using the PacBio platform. Assembly and annotation are in progress, and these high-quality genomes will soon be integrated into our database. 2. BacPath pipeline validation: Validation of the BacPath pipeline is nearly complete, with only a few remaining experiments to further enhance its reliability. Multi-operator validation and comparative analysis with other pipelines (Phytopipe and Metagenomics/WIMP) are underway and expected to finish soon. 3. NextStrain/strain tracking analyses: With the majority of genomes now available, we are ready to begin strain tracking analyses using the NextStrain pipeline, which is already running smoothly in our lab. 4. Publications and presentations: Manuscripts detailing the BacPath pipeline and BacPathDB are in preparation and expected to be submitted in the coming months. Additional articles, including a review on metagenome-based detection, comparative genomics studies, and new species descriptions, are also underway. In addition to publications, we will share our findings through presentations at scientific conferences and workshops to ensure broad dissemination and active engagement with the research community. We will present our research in upcoming APS meeting in Honolulu HI.
Impacts
What was accomplished under these goals?
Genome Sequencing: A new set of 96 strains has been sequenced using the PacBio long-read sequencing platform. This includes multiple strains from all known species of Pectobacterium and Dickeya, selected to represent diverse geographical locations and host plants. Additionally, the panel includes strains from an exclusivity panel, which consists of closely related but non-target organisms, essential for validating the specificity of diagnostic tools. The genome sequencing of these 96 strains has been completed. Assembly and annotation are currently in progress to ensure high-quality, accurate genomic information. Once the quality of each genome is assessed and verified, the sequences will be integrated into a curated database. This comprehensive and high-quality database will be the most complete resource available for all known pectinolytic bacteria. It will significantly enhance the reliability of metagenome-based diagnostics, ensuring accurate detection and identification. Moreover, it will support biosecurity initiatives and facilitate detailed epidemiological studies by enabling rapid and precise pathogen tracking and characterization. BacPathDB Database: At present, the database contains a total of 413 complete genomes, primarily representing the largest number of strains from the Pectobacterium and Dickeya species. In addition, the database includes genomes from a diverse range of other genera for validation (exclusivity), such as Erwinia, Brenneria, Pantoea, Burkholderia, Pseudomonas, Xanthomonas, Stenotrophomonas, Escherichia, Salmonella, Ralstonia, and Clavibacter. Genomes sourced from the NCBI underwent rigorous verification to ensure accurate metadata and high-quality assembly. Genomes downloaded from the NCBI database were selected based on stringent criteria: greater than 98% completeness and less than 3% contamination. The genomes sequenced and assembled in our lab are high-quality, complete assemblies, often generated using hybrid sequencing approaches, and are accompanied by accurate and well-curated metadata. Quality assessment for genome completeness and contamination was performed using CheckM 2.0 on the server, as well as the BRVC (PATRIC) annotation tool, ensuring the reliability of the genomic data included in the database. BacPath Pipeline Validation: At this point, the BacPath pipeline is fully validated, with the exception of a multi-operator validation that is currently underway. In silico analyses demonstrate no cross-identification with non-target species, confirming the pipeline's high specificity. The limit of detection for mock-inoculated samples is extremely low, with as few as one read being sufficient for detection. For in vitro validation, the pipeline also proved to be highly sensitive. Notably, it exhibited greater sensitivity with long-read sequencing compared to short-read sequencing, likely due to the increased sequence length and information content provided by long reads. In experiments, potato slices and whole plants were artificially inoculated with Dickeya dianthicola, D. fangzhongdai, and Pectobacterium parmentieri, both individually and in various combinations. In all cases, the pipeline accurately identified the pathogens present, whether in single or mixed infections. Both sequencing platforms, Oxford Nanopore Flongle for long reads and Illumina NovaSeq for short reads, were used for validation of the pipeline. This dual-platform approach ensured comprehensive assessment of the pipeline's performance across different sequencing technologies. Overall, the Bacpath pipeline enables rapid and accurate detection of soft rot bacteria in infected plant samples. It is now ready for implementation in routine and regulatory diagnostics, offering a powerful tool to enhance our biosecurity measures and support effective disease management in agricultural settings. Other Findings and Accomplishments: During the course of our investigation, we identified multiple novel species within the genera Dickeya, Pectobacterium, Pantoea, and Clavibacter. To date, three of these new species have already been published (see list of publications; one accepted in IJSEM and other two as preprint). These discoveries underscore the utility of our comprehensive genome database. The identification of these novel species highlights the database's value as a robust resource for taxonomic classification, accurate identification, and the description of new plant-associated bacteria. Our comprehensive genome database will be a valuable resource for researchers, U.S. farmers, and agricultural stakeholders by enabling rapid and accurate identification of plant pathogenic soft rot causing bacteria. This will support early detection and effective management of crop diseases, helping to reduce losses, improve biosecurity, and develop better disease control strategies.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2025
Citation:
Arif M, Zeng Q, Dobhal S (2025). Editorial: Research in the identification and control methods of rot diseases in plants. Frontiers in Microbiology, doi: 10.3389/fmicb.2025.1601422
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2025
Citation:
Komal, Dobhal S, Arizala D, Boluk G, Alvarez AM, Arif M (2025). Complete genome sequence of Dickeya oryzae strain A5272 isolated during pineapple heart rot outbreak in Hawaii. Phytofrontiers, https://doi.org/10.1094/PHYTOFR-01-25-0001-A
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2024
Citation:
Invited Speaker: Arif M. Emerging strains and their multi-trophic interactions threatening food safety and biosecurity. XX International Plant Protection Congress (IPPC), Athens, Greece (July 29, 2024)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2024
Citation:
Invited Speaker: Arif M. Emerging strains and multi-trophic interactions: a serious threat to food safety and biosecurity, at Plant Biosecurity in Theory and Practice workshop, Biosecurity Research Institute, Kansas State University, Manhattan, KS, USA (May 23, 2024)
- Type:
Other Journal Articles
Status:
Published
Year Published:
2025
Citation:
Arizala D, Dobhal S, Alvarez AM, Arif M (2025). Description of two novel non-pathogenic tomato-associated Clavibacter species: Clavibacter seminis sp. nov. and Clavibacter quasicaliforniensis sp. nov. bioRxiv, doi: 10.1101/2025.03.25.645327.
- Type:
Peer Reviewed Journal Articles
Status:
Accepted
Year Published:
2025
Citation:
Dobhal S, Hugouvieux-Cotte-Pattat, N, Arizala D, Sari, GB, Chaung SC, Alvarez AM, Arif M (2024). Dickeya ananatis sp. nov., pectinolytic bacterium isolated from pineapple (Ananas comosus). bioRxiv, doi.org/10.1101/2024.10.29.620964 (Accepted for publication in International Journal of Systematic and Evolutionary Microbiology)
- Type:
Book Chapters
Status:
Published
Year Published:
2025
Citation:
Charkowski A, Arif M (2024). Pectobacterium. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 4th ed., edited by Jones JB, Sundin GW, Miller SA, Newberry EA, Sharma A. American Phytopathological Society Press, 2025.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2024
Citation:
Bhandari S, Marabella M, Howard J, Do S, Montoya-Pimolwatana M, Dou Y, Arizala D, Montesinos S, Dobhal S, Arif M (2024). Loop-mediated Isothermal Amplification (LAMP) assay for reliable detection of Xanthomonas axonopodis pv. vasculorum. APS Plant Health 2024, July 27-30, Memphis, Tennessee.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2024
Citation:
Bhandari S, Arif M, Ma LM, Dobhal S. (2024). Multi-trophic interactions between soft rot-causing bacteria, foodborne pathogen, and their host plant. APS Plant Health 2024, July 27-30, Memphis, Tennessee.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2025
Citation:
Invited Plenary Session Speaker: Arif M. Emerging pathogens and their multi-trophic interactions: Pioneering technologies to strengthen food safety and biosecurity. IPS 2025, Nagpur, India (Jan 20, 2025).
- Type:
Other
Status:
Other
Year Published:
2025
Citation:
Invited lecture on Soft rot bacteria Pectobacterium and Dickeya (PLPTH 840: Plant Pathogenic Bacteria) at Department of Plant Pathology, Kansas State University.
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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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