Recipient Organization
UNIV OF HAWAII
3190 MAILE WAY
HONOLULU,HI 96822
Performing Department
(N/A)
Non Technical Summary
Pectobacterium and Dickeya, two key genera, have been cited among the top ten bacterial plant pathogens limiting crop yields and threatening global food security.Species in both genera are globally distributedand cause considerable damage globally.Detection of both common and emerging species in propagative materials is essential to plant biosecurity.This proposal addresses the critical need to enhance our national crop biosecurity capacity by developing methods that can accurately detect, differentiate, and track origins of distinct species/strains of PectobacteriumandDickeya that threaten the potato and vegetable industries. The method includes bacterial genome sequencing, developing genome databaseand analyses that can accurately detect and differentiate the PectobacteriumandDickeyaspecies. The database and NextStrain pipeline can be used for global transmission ofPectobacteriumandDickeyaspecies. The ultimate goal of this project is todevelop a robust and highly accurate genome database and pipeline for plant pathogens to support farmers, seed producers, stakeholders, and government agencies.This project willstrengthen national crop biosecurity.
Animal Health Component
0%
Research Effort Categories
Basic
30%
Applied
70%
Developmental
(N/A)
Goals / Objectives
The goal of this project is to construct a genome database, including all type and reference strains from different geographical locations, and establish a pipeline that can use this database to accurately detect and identify the SRP in a sample.The specific objectives of this proposal are: 1) develop a genome database for SRPs to enhance capabilities for rapid detection and strain tracking; and 2) validation of "BacPath" pipeline for robust detection and differentiation of SRPs.The long-term goal of this research project is to develop a robust and highly accurate genome database and pipeline for plant pathogens to support farmers, seed producers, stakeholders, and government agencies.
Project Methods
OBJECTIVE 1: To develop a genome database for SRPs to enhance capabilities for rapid detection and strain tracking in global pathways.Genome sequencing and data validation: A total of 600 genomes of Dickeya and Pectobacterium genera are available in the GenBank but only 142 (Pectobacterium = 87; Dickeya = 55) are complete genomes. All complete genomes and some draft genomes (after validating for quality and metadata accuracy) will be included in the new database. Additionally, about 150 complete genomes (2-5 genomes for each species) representing different species and genomes of all type strains that are currently unavailable in the NCBI database, will be sequenced using Nanopore MinION and Illumina NovaSeq and hybrid assembliesusing TryCycler, which derives a consensus assembly after running multiple assemblers, will be prepared. Nanopore Medaka will be used to polish consensus assemblies first with the long reads and then with the Illumina reads. High-quality DNA will be isolated using Qiagen Genomic-tip 100/G following the manufacturer's instructions (Qiagen). The genomes will be annotated NCBI Prokaryotic Genome Annotation Pipelines and Rapid Prokaryotic Genome Annotation Tool (Prokka).NextStrain is a recent advancement for exploring the emergence, spread, evolution and transmission of pathogens. In our lab, we have established the NextStrain pipeline for bacterial pathogens that enables evaluation of newly emerged and high consequence strains. The procedure includes the generation of input VCF (variant calling format) data of core genomes using Snippy (https://github.com/tseemann/snippy). It integrates two major points: a phylodynamic analysis performed by Augur followed by an interactive visualization using Auspice platform. In this objective, we will perform NextStrain analyses for three species.OBJECTIVE 2: Validation of the "BacPath" pipeline for robust detection and differentiation of SRPs.The pipeline will be used for both short- and long-reads. Validation will be performed with DNA from all soft rot bacterial species and HTS data will be used to confirm specific detection. In depth validation, including in planta infection (potato plant and slices). The pipeline and detection methods will be validated by confirming LoD (10-fold dilutions of target DNA will be added in host DNA), specificity (with members of inclusivity and exclusivity panels; detection from infected samples),multi-instrument (Illumina and Oxford Nanopore), multi-operator ANDreproducibility. Mock data will also be created using MetaSim and used for validation.Data generated from the previous objective will be used in this objective. This pipeline will be compared with other pipelines for specificity (inclusivity and exclusivity data will be analyzed), time (running time), ease (methodology and procedure), cost (per sample $$), labor (cost and requirement). Both types of input data, short- and long-reads will be used. Minimum data required for reliable detection, using multiple platforms, with a different concentration of a pathogenwill be calculated.