Impact of Homologous Recombination on Core Genome Evolution and Host Adaptation of Pectobacterium parmentieri.

Homologous recombination is a major force mechanism driving bacterial evolution, host adaptability, and acquisition of novel virulence traits. Pectobacterium parmentieri is a plant bacterial pathogen distributed worldwide, primarily affecting potatoes, by causing soft rot and blackleg diseases. The goal of this investigation was to understand the impact of homologous recombination on the genomic evolution of P. parmentieri. Analysis of P. parmentieri genomes using Roary revealed a dynamic pan-genome with 3,742 core genes and over 55% accessory genome variability. Bayesian population structure analysis identified 7 lineages, indicating species heterogeneity. ClonalFrameML analysis displayed 5,125 recombination events, with the lineage 4 exhibiting the highest events. fastGEAR analysis identified 486 ancestral and 941 recent recombination events ranging from 43 bp to 119 kb and 36 bp to 13.96 kb, respectively, suggesting ongoing adaptation. Notably, 11% (412 genes) of the core genome underwent recent recombination, with lineage 1 as the main donor. The prevalence of recent recombination (double compared to ancient) events implies continuous adaptation, possibly driven by global potato trade. Recombination events were found in genes involved in vital cellular processes (DNA replication, DNA repair, RNA processing, homeostasis, and metabolism), pathogenicity determinants (type secretion systems, cell-wall degrading enzymes, iron scavengers, lipopolysaccharides (LPS), flagellum, etc.), antimicrobial compounds (phenazine and colicin) and even CRISPR-Cas genes. Overall, these results emphasize the potential role of homologous recombination in P. parmentieri's evolutionary dynamics, influencing host colonization, pathogenicity, adaptive immunity, and ecological fitness.

Loop-mediated isothermal amplification (LAMP) assay for specific and rapid detection of Dickeya fangzhongdai targeting a unique genomic region.

Dickeya fangzhongdai, a bacterial pathogen of taro (Colocasia esculenta), onion (Allium sp.), and several species in the orchid family (Orchidaceae) causes soft rot and bleeding canker diseases. No field-deployable diagnostic tool is available for specific detection of this pathogen in different plant tissues. Therefore, we developed a field-deployable loop-mediated isothermal amplification (LAMP) assay using a unique genomic region, present exclusively in D. fangzhongdai. Multiple genomes of D. fangzhongdai, and other species of Dickeya, Pectobacterium and unrelated genera were used for comparative genomic analyses to identify an exclusive and conserved target sequence from the major facilitator superfamily (MFS) transporter gene region. This gene region had broad detection capability for D. fangzhongdai and thus was used to design primers for endpoint PCR and LAMP assays. In-silico validation showed high specificity with D. fangzhongdai genome sequences available in the NCBI GenBank genome database as well as the in-house sequenced genome. The specificity of the LAMP assay was determined with 96 strains that included all Dickeya species and Pectobacterium species as well as other closely related genera and 5 hosts; no false positives or false negatives were detected. The detection limit of the assay was determined by performing four sensitivity assays with tenfold serially diluted purified genomic DNA of D. fangzhongdai with and without the presence of crude host extract (taro, orchid, and onion). The detection limit for all sensitivity assays was 100 fg (18-20 genome copies) with no negative interference by host crude extracts. The assays were performed by five independent operators (blind test) and on three instruments (Rotor-Gene, thermocycler and dry bath); the assay results were concordant. The assay consistently detected the target pathogen from artificially inoculated and naturally infected host samples. The developed assay is highly specific for D. fangzhongdai and has applications in routine diagnostics, phytosanitary and seed certification programs, and epidemiological studies.

Elevation of Clavibacter michiganensis subsp. californiensis to species level as Clavibacter californiensis sp. nov., merging and re-classification of Clavibacter michiganensis subsp. chilensis and Clavibacter michiganensis subsp. phaseoli as Clavibacter phaseoli sp. nov. based on complete genome in silico analyses.

The Gram-positive genus Clavibacter is currently divided into seven species (Clavibacter michiganensis, Clavibacter nebraskensis, Clavibacter capsici, Clavibacter sepedonicus, Clavibacter tessellarius, Clavibacter insidiosus and Clavibacter zhangzhiyongii) and three subspecies (C. michiganensis subsp. californiensis, C. michiganensis subsp. chilensis and C. michiganensis subsp. phaseoli). Recent studies have indicated that the taxonomic rank of the subspecies must be re-evaluated. In this research, we assessed the taxonomic position of the three C. michiganensis subspecies and clarified the taxonomic nomenclature of other 75 Clavibacter strains. The complete genomes of the type strains of the three Clavibacter subspecies, the type strain of C. tessellarius and C. nebraskensis A6096 were sequenced using PacBio RSII technology. Application of whole-genome-based computational approaches such as average nucleotide identity (ANI), digital DNA-DNA hybridization, multi-locus sequence analysis of seven housekeeping genes (acnA, atpD, bipA, icdA, mtlD, recA and rpoB), a phylogenomic tree reconstructed from 1 028 core genes, and ANI-based phylogeny provided sufficient justification for raising C. michiganensis subsp. californiensis to the species level. These results led us to propose the establishment of Clavibacter californiensis sp. nov. as a species with its type strain C55T (=CFBP 8216T=ATCC BAA-2691T). Moreover, the orthologous and in silico dot plot analyses, along with the above described bioinformatic strategies, revealed a high degree of similarity between C. michiganensis subsp. chilensis and C. michiganensis subsp. phaseoli. Based on these analyses, we propose that both subspecies be combined into a single taxon and elevated to the species level as Clavibacter phaseoli sp. nov., with LPPA 982T (= CECT 8144T= LMG 27667T) as the type strain.

Development of a multiplex TaqMan qPCR targeting unique genomic regions for the specific and sensitive detection of Pectobacterium species and P. parmentieri.

AIM: The newly defined species Pectobacterium parmentieri has emerged as an aggressive pathogen that causes soft rot and blackleg diseases on potato and has been widely disseminated across the globe, jeopardizing the productivity and potato food safety. The implementation of a fast and accurate detection tool is imperative to control, monitor and prevent further spread of these pathogens. The objective of this work was to develop a specific and sensitive multiplex TaqMan qPCR to detect P. parmentieri and distinguish it from all known Pectobacterium species. A universal internal control was included to enhance the reliability of the assay. METHODS AND RESULTS: A comparative genomics approach was used to identify O-acetyltransferase and the XRE family transcriptional regulator as specific targets for primers/probe design for the detection of the Pectobacterium genus and P. parmentieri, respectively. Specificity was assessed with 35 and 25 strains included in the inclusivity and exclusivity panels, respectively, isolated from different geographical locations and sources. The assay specifically detected all 35 strains of Pectobacterium sp. and all 15 P. parmentieri strains. No cross-reactivity was detected during assay validation. Our assay detected up to 10 fg genomic DNA and 1 CFU ml-1 bacterial culture. No change in the detection threshold (1 CFU ml-1 ) was observed in spiked assays after adding host tissue to the reactions. The assay was validated with naturally and artificially infected host tissues and soil rhizosphere samples. All infected plant samples containing the target pathogens were accurately amplified. CONCLUSION: The presented multiplex TaqMan qPCR diagnostic assay is highly specific, sensitive, reliable for the detection of Pectobacterium species and P. parmentieri with no false positives or false negatives. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed assay can be adopted for multiple purposes such as seed certification programmes, surveillance, biosecurity, microbial forensics, quarantine, border protection, inspections and epidemiology.

Genome-informed loop-mediated isothermal amplification assay for specific detection of Pectobacterium parmentieri in infected potato tissues and soil.

Pectobacterium parmentieri (formerly Pectobacterium wasabiae), which causes soft rot disease in potatoes, is a newly established species of pectinolytic bacteria within the family Pectobacteriaceae. Despite serious damage caused to the potato industry worldwide, no field-deployable diagnostic tests are available to detect the pathogen in plant samples. In this study, we aimed to develop a reliable, rapid, field-deployable loop-mediated isothermal amplification (LAMP) assay for the specific detection of P. parmentieri. Specific LAMP primers targeting the petF1 gene region, found in P. parmentieri but no other Pectobacterium spp., were designed and validated in silico and in vitro using extensive inclusivity (15 strains of P. parmentieri) and exclusivity (94 strains including all other species in the genus Pectobacterium and host DNA) panels. No false positives or negatives were detected when the assay was tested directly with bacterial colonies, and with infected plant and soil samples. Sensitivity (analytical) assays using serially diluted bacterial cell lysate and purified genomic DNA established the detection limit at 10 CFU/mL and 100 fg (18-20 genome copies), respectively, even in the presence of host crude DNA. Consistent results obtained by multiple users/operators and field tests suggest the assay's applicability to routine diagnostics, seed certification programs, biosecurity, and epidemiological studies.

Genomic and Phenotypic Biology of Novel Strains of Dickeya zeae Isolated From Pineapple and Taro in Hawaii: Insights Into Genome Plasticity, Pathogenicity, and Virulence Determinants.

Dickeya zeae, a bacterial plant pathogen of the family Pectobacteriaceae, is responsible for a wide range of diseases on potato, maize, rice, banana, pineapple, taro, and ornamentals and significantly reduces crop production. D. zeae causes the soft rot of taro (Colocasia esculenta) and the heart rot of pineapple (Ananas comosus). In this study, we used Pacific Biosciences single-molecule real-time (SMRT) sequencing to sequence two high-quality complete genomes of novel strains of D. zeae: PL65 (size: 4.74997 MB; depth: 701x; GC: 53.6%) and A5410 (size: 4.7792 MB; depth: 558x; GC: 53.5%) isolated from economically important Hawaiian crops, taro, and pineapple, respectively. Additional complete genomes of D. zeae representing three additional hosts (philodendron, rice, and banana) and other species used for a taxonomic comparison were retrieved from the NCBI GenBank genome database. Genomic analyses indicated the truncated type III and IV secretion systems (T3SS and T4SS) in the taro strain, which only harbored one and two genes of T3SS and T4SS, respectively, and showed high heterogeneity in the type VI secretion system (T6SS). Unlike strain EC1, which was isolated from rice and recently reclassified as D. oryzae, neither the genome PL65 nor A5410 harbors the zeamine biosynthesis gene cluster, which plays a key role in virulence of other Dickeya species. The percentages of average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) between the two genomes were 94.47 and 57.00, respectively. In this study, we compared the major virulence factors [plant cell wall-degrading extracellular enzymes and protease (Prt)] produced by D. zeae strains and evaluated the virulence on taro corms and pineapple leaves. Both strains produced Prts, pectate lyases (Pels), and cellulases but no significant quantitative differences were observed (p > 0.05) between the strains. All the strains produced symptoms on taro corms and pineapple leaves, but the strain PL65 produced symptoms more rapidly than others. Our study highlights the genetic constituents of pathogenicity determinants and genomic heterogeneity that will help to understand the virulence mechanisms and aggressiveness of this plant pathogen.

Genome-Wide Analyses Revealed Remarkable Heterogeneity in Pathogenicity Determinants, Antimicrobial Compounds, and CRISPR-Cas Systems of Complex Phytopathogenic Genus Pectobacterium.

The Pectobacterium genus comprises pectolytic enterobacteria defined as the causal agents of soft rot, blackleg, and aerial stem rot diseases of potato and economically important crops. In this study, we undertook extensive genome-wide comparative analyses of twelve species that conform the Pectobacterium genus. Bioinformatics approaches outlined a low nucleotide identity of P. parmentieri and P. wasabiae with other species, while P. carotovorum subsp. odoriferum was shown to harbor numerous pseudogenes, which suggests low coding capacity and genomic degradation. The genome atlases allowed for distinguishing distinct DNA structures and highlighted suspicious high transcription zones. The analyses unveiled a noteworthy heterogeneity in the pathogenicity determinants. Specifically, phytotoxins, polysaccharides, iron uptake systems, and the type secretion systems III-V were observed in just some species. Likewise, a comparison of gene clusters encoding antimicrobial compounds put in evidence for high conservation of carotovoricin, whereas a few species possessed the phenazine, carbapenem, and carocins. Moreover, three clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) systems: I-E, I-F, and III-A were identified. Surrounding some CRISPR-Cas regions, different toxin and antitoxin systems were found, which suggests bacterial suicide in the case of an immune system failure. Multiple whole-genome alignments shed light on to the presence of a novel cellobiose phosphotransferase system (PTS) exclusive to P. parmenteri, and an unreported T5SS conserved in almost all species. Several regions that were associated with virulence, microbe antagonism, and adaptive immune systems were predicted within genomic islands, which underscored the essential role that horizontal gene transfer has imparted in the dynamic evolution and speciation of Pectobacterium species. Overall, the results decipher the different strategies that each species has developed to infect their hosts, outcompete for food resources, and defend against bacteriophages. Our investigation provides novel genetic insights that will assist in understanding the pathogenic lifestyle of Pectobacterium, a genus that jeopardizes the agriculture sustainability of important crops worldwide.

Development of a robust, field-deployable loop-mediated isothermal amplification (LAMP) assay for specific detection of potato pathogen Dickeya dianthicola targeting a unique genomic region.

Destructive maceration, a wide host range, and longevity in non-plant substrates has established Dickeya dianthicola (blackleg of potato) as a significant threat to potato industries worldwide. To protect these businesses, a specific and sensitive point-of-care D. dianthicola detection tool is necessary. We have developed a loop-mediated isothermal amplification (LAMP) assay for specific, sensitive, and rapid detection of D. dianthicola, which can be streamlined for point-of-care use. The developed LAMP assay targets a unique gene, alcohol dehydrogenase, of D. dianthicola. Assay specificity was assessed using strains present in inclusivity (16 D. dianthicola strains) and exclusivity panels (56 closely related, potato pathogenic, and other bacterial strains). Amplification with strains of inclusivity panel occurred, and cross-reactivity with non-target DNA was not observed. The limit of detection (LOD) was 10 CFU/ml when dilutions were made before isolating the genomic DNA; however, LOD was determined as 1 pg using 10-fold serially diluted D. dianthicola genomic DNA. Similar LOD of 1 pg was observed when serially diluted target genomic DNA was mixed with host genomic DNA. LOD (1 pg) was also calculated with 10-fold serially diluted synthetic DNA fragments containing primer target sites. Naturally and artificially inoculated plant samples were used for field adaptability tests with the field-deployable Optigene Plant Material Lysis Kit and a heat block (65°C); the results were obtained within 20 minutes. Despite the lack of method precision, no false positives or false negatives were observed. Therefore, with prepared reactions and a steady heat source, this assay can be used for rapid point-of-care detection, which is imperative for quarantine, eradication, disease management, and border protection.