GENOMIC BIOLOGY AND INTERACTIONS OF PECTINOLYTIC BACTERIA

Abstract

The soft rot Pectobacteriaceae, the Dickeya and Pectobacterium species, cause economically important diseases on vegetables, horticultural and other crops worldwide, resulting in high economic losses. Dickeya oryzae is the causal agent of bacterial heart rot, fruit collapse of pineapple, and bacterial foot rot of rice, and significantly reduces crop production. This study used the Multi-Locus Sequence Typing (MLST) method to define the phylogenetic relationships among the strains isolated from different sources, including kale, pineapples, corn, taro, and irrigation water. The housekeeping genes, dnaA, gapA, gyrB, atpD, and purA were selected after in silico screening for more SNPs and resolution capacity. A total of 119 strains of Dickeya and Pectobacterium species were evaluted. The phylogenetic and ClonalFrame analyses of the concatenated genes (total length ~4,625 bp) demonstrated that the pineapple strains from Hawaii were grouped with D. oryzea. The strains found associated with kale and taro presented close relationships with the D. zeae strains. ClonalFrame outcomes indicated 1.4- and 1.6-times higher recombination impact than point mutation (r/m 1. 364473 and 1. 576925) for D. zeae and D. oryzae data sets, respectively, and both species evolved from the same ancestor. Additionally, 24 selected strains of Dickeya and Pectobacterium species were compared to determine the main virulence factors, carbon utilization, and the ability to cause disease to different hosts. Tested Dickeya and Pectobacterium strains exhibited higher activities of cell wall degrading enzymes and motility at 28 °C. The kale strain (PL47) exhibited a distinct pattern of carbon utilization among Dickeya species. The Hawaiian taro strain showed the highest host specificity with taro from where it was originally isolated. We aimed to understand the genomic constituents of diverse D. zeae strains through comparative genomic analyses. We used Pacific Biosciences SMRT sequencing to sequence two high-quality complete genomes of novel strains of D. zeae: PL65 (size—4.74997 MB; depth—701x; GC—53.3%) and A5410 (size—4.7792 MB; depth—558x; GC—53.6%) isolated from economically important Hawaiian crops, taro, and pineapple, respectively. The genomic analyses indicated truncated type III and IV secretion systems in the taro strain and showed high heterogeneity in the type VI secretion system. Our study highlights genetic constituents of pathogenicity determinants and genomic heterogeneity that will help understand the virulence mechanisms and aggressiveness of this plant pathogen. We recently isolated the soft rot bacteria associated with kale and post-harvest taro, and both are economically important crops for Hawaii and the United States. The isolated strain (PL47) from kale showing soft rot symptom was identified as Dickeya zeae, and three strains (PL64T, PL63, and PL48) were identified as Pectobacterium, but the species designation of these strains were unclear. The pectinolytic bacterial strain PL65T isolated from an infected taro corm was identified as Dickeya zeae, but the species designation of this strain was unclear. The pectinolytic bacterial strains PL65T, isolated from an infected taro corm, PL64T, PL63, and PL48, isolated from infected kale, were subjected to polyphasic analysis to determine genomic phenotypic characteristics. Two representative strains, PL64T and PL65T were used for high-quality complete genome studies. The whole genome was sequenced using the Oxford Nanopore MinION and the Illumina Next Genome Sequencing (NGS) system and Pacific Biosciences SMRT. Multi-locus sequence analyses (MLSA) revealed strains PL64T and PL65T were in a novel clade separated from the other Dickeya zeae and Pectobacteium brasiliense strains, respectively. Phylogenetic analysis based on core gene sequences clearly showed two potentially new species within the Pectorobactericeae family. The in-silico DNA– DNA hybridization value of strain PL65T with other type strains of Dickeya species was <68%. Average nucleotide identity (ANI) analysis revealed that PL65T was at the margin of the species delineation cut-off values with a 96% ANI value. in-silico DNA–DNA hybridization value of strain PL64T with other type strains of Pectobacterium species was <60.2%. Average nucleotide identity (ANI) analysis revealed that PL64T was at the margin of the species delineation cut-off values with a ~94% ANI value. Based on the results of polyphasic approaches, including genome-to-genome comparisons presented in this report, we propose the establishment of two new species, Pectobacterium hawaiiense sp. nov. with PL64T as the type strain and Dickeya colocasia sp. nov. with PL65T as the type strain. We aimed to develop a rapid, field-deployable recombinase polymerase amplification (RPA) for specific and rapid detection of Dickeya spp. using lateral flow strips. A unique genomic region (mglA/mglC genes) conserved among Dickeya spp. was used to design highly specific, robust primers and probes for an RPA assay. Assay specificity was validated with 34 strains from Dickeya spp. and 24 strains from other genera and species; no false positives or negatives were detected. An RPA assay targeting the internal transcribed spacer region of the host genome was included to enhance the reliability and accuracy of the Dickeya assay. The detection limit of 1 fg was determined by both sensitivity and spiked sensitivity assays; no inhibitory effects were observed. The developed RPA assay is rapid, highly accurate, sensitive, and fully field deployable. It has numerous applications in routine diagnostics, surveillance, biosecurity, and disease management.