Multilocus sequence and phenotypic analysis of Pectobacterium and Dickeya type strains for identification of soft rot Pectobacteriaceae from symptomatic potato stems and tubers in Pennsylvania.

Outbreaks of potato blackleg and soft rot caused by Pectobacterium species and more recently Dickeya species across the U.S. mid-Atlantic region have caused yield loss due to poor emergence as well as losses from stem and tuber rot. To develop management strategies for soft rot diseases, we must first identify which members of the soft rot Pectobacteriaceae are present in regional potato plantings. However, the rapidly expanding number of soft rot Pectobacteriaceae species and the lack of readily available comparative data for type strains of Pectobacterium and Dickeya hinder quick identification. This manuscript provides a comparative analysis of soft rot Pectobacteriaceae and a comprehensive comparison of type strains from this group using rep-PCR, MLSA and 16S sequence analysis, as well as phenotypic and physiological analyses using Biolog GEN III plates. These data were used to identify isolates cultured from symptomatic potato stems collected between 2016 and 2018. The isolates were characterized for phenotypic traits and by sequence analysis to identify the bacteria from potatoes with blackleg and soft rot symptoms in Pennsylvania potato fields. In this survey, P. actinidiae, P. brasiliense, P. polonicum, P. polaris, P. punjabense, P. parmentieri, and P. versatile were identified from Pennsylvania for the first time. Importantly, the presence of P. actinidiae in Pennsylvania represents the first report of this organism in the U.S. As expected, P. carotorvorum and D. dianthicola were also isolated. In addition to a resource for future work studying the Dickeya and Pectobacterium associated with potato blackleg and soft rot, we provide recommendations for future surveys to monitor for quarantine or emerging soft rot Pectobacteriace regionally.

Pseudomonas fluorescens MFE01 delivers a putative type VI secretion amidase that confers biocontrol against the soft-rot pathogen Pectobacterium atrosepticum.

The type VI secretion system (T6SS) is a contractile nanomachine widespread in Gram-negative bacteria. The T6SS injects effectors into target cells including eukaryotic hosts and competitor microbial cells and thus participates in pathogenesis and intermicrobial competition. Pseudomonas fluorescens MFE01 possesses a single T6SS gene cluster that confers biocontrol properties by protecting potato tubers against the phytopathogen Pectobacterium atrosepticum (Pca). Here, we demonstrate that a functional T6SS is essential to protect potato tuber by reducing the pectobacteria population. Fluorescence microscopy experiments showed that MFE01 displays an aggressive behaviour with an offensive T6SS characterized by continuous and intense T6SS firing activity. Interestingly, we observed that T6SS firing is correlated with rounding of Pectobacterium cells, suggesting delivery of a potent cell wall targeting effector. Mutagenesis coupled with functional assays then revealed that a putative T6SS secreted amidase, Tae3Pf , is mainly responsible for MFE01 toxicity towards Pca. Further studies finally demonstrated that Tae3Pf is toxic when produced in the periplasm, and that its toxicity is counteracted by the Tai3Pf inner membrane immunity protein.

Nano-metals forming bacteria in Egypt. II. Efficacy towards biomolecules, ultrastructure, growth parameters, and eco-friendly therapeutic of soft rot/blackleg genera.

The nanoparticles (NPs) formed by Enterococcus thailandicus, Pseudomonas putida, Marinobacter hydrocarbonoclasticus, and P. geniculate were tested against soft rot/blackleg genera. The effects of NPs recorded on bacterial DNA, proteins, and carbohydrates concentration of Pectobacterium carotovorum subsp. carotovorum, Enterobacter cloacae (soft rot), and Dickeya solani (soft rot/blackleg). Treated cells showed degradation in isolated DNA, decreased proteins and carbohydrates concentration compared with untreated cells. Using Scanning Electron Microscope (SEM), the treated cells showed collapsed and small pits in the cell wall. Using Transmission Electron Microscope (TEM), internal changes showed penetration of NPs inside the tested bacterial cells, the appearance of periplasmic space, formation of vacuoles, and condensation of cytoplasm. Disease severity ex vivo of potato tuber infected with tested genera demonstrated that NPs treatment didn't show any rotted tissue compared with untreated. The ability to uptake and accumulate FeNPs from the soil in potato (Solanum tuberosum) seedlings; Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES) was used. It recorded an increase in iron content of treated potato (Solanum tuberosum) seedlings with NPs, compared with untreated. FeNPs can be used to control soft rot/blackleg diseases, instead of copper pesticides. It could be a new, approach for disease management and increase the plant's nutritional value.

Genome Sequence Resource of Pectobacterium polaris QK413-1 that Causes Blackleg on Potato in Fujian Province, China.

The Pectobacterium pathogens cause soft rot and blackleg diseases on many plants and crops, including potatoes. Here, we first report a high-quality genome assembly and announcement of the P. polaris strain QK413-1, which causes blackleg disease in potatoes in China. The QK413-1 genome was sequenced and assembled using the PacBio Sequel II and Illumina sequencing platform. The assembled genome has a total size of 5,005,507 bp with a GC content of 51.81%, encoding 4,782 open reading frames, including 639 virulence genes, 273 drug resistance genes, and 416 secreted proteins. The QK413-1 genome sequence provides a valuable resource for the control of potato blackleg and research into its mechanism.

Microbe Profile: Pectobacterium atrosepticum: an enemy at the door.

Pectobacterium atrosepticum is part of a larger family of soft rot bacteria (Pectobacteriaceae) that cause disease on a wide range of crops worldwide. They are closely related to members of the Enterobacteriaceae and, as the plant pathogens and plant associated members of the group, form part of a continuum towards opportunistic and more devastating animal and human pathogens. Many of the horizontally acquired islands present in the genome of P. atrosepticum are directly responsible for life on plants. These include genes for a plethora of plant cell wall degrading enzymes, plant toxins, siderophores etc., which are exported by multiple secretion systems under a highly coordinated regulation system.

Oak bark (Quercus sp. cortex) protects plants through the inhibition of quorum sensing mediated virulence of Pectobacterium carotovorum.

Bacterial intercellular communication mediated by small diffusible molecules, known as quorum sensing (QS), is a common mechanism for regulating bacterial colonisation strategies and survival. Influence on QS by plant-derived molecules is proposed as a strategy for combating phytopathogens by modulating their virulence. This work builds upon other studies that have revealed plant-derived QS inhibitors extracted from oak bark (Quercus sp.). It was found that co-incubation of Pectobacterium carotovorum VKM-B-1247 with oak bark extract (OBE) reduced the production of acyl-HSL. This was accompanied by a dose-dependent decrease in the bacterial cellulolytic and protease activity. At the transcriptomic level, the OBE treatment suppressed the main QS-related genes expR/expI. Potato tubers pre-treated with OBE showed resistance to a manifestation of soft-rot symptoms. Analysis of the component composition of the OBE identified several biologically active molecules, such as n-hexadecanoic acid, 2,6-di-tert-butyl-4-methylphenol, butylated hydroxytoluene (BHT), gamma-sitosterol, lupeol, and others. Molecular docking of the binding energy between identified molecules and homology models of LuxR-LuxI type proteins allow to identify potential inhibitors. Collectively, obtained results figure out great potential of widely distributed oak-derived plant material for bacterial control during storage of potato.

Insights into complex infection by two Pectobacterium species causing potato blackleg and soft rot.

Pectobacterium spp. are causative agents of blackleg and soft rot of potato. However, little is known about the relationship between the pathogenicity of mixed infections of different Pectobacterium spp. at different temperatures. In this study, two pectinolytic strains of Pectobacterium spp. were isolated from the same potato plant with typical symptoms of blackleg and identified as P. brasiliense and P. carotovorum by multilocus sequence analysis (MLSA), whole-genome phylogenetic tree construction, average nucleotide identity (ANI) analysis and digital DNA-DNA hybridization (dDDH). Plant cell wall degrading enzyme, including pectinases, cellulases and proteases, as the most important virulence factors, as well as pathogenicity toward potato tuber, were compared between the strains P. brasiliense BL-2 and P. carotovorum BL-4 at 28 â„ƒ. The results showed that P. carotovorum had higher cell wall-degrading enzyme activities and brought more severe disease symptoms to potato tubers than P. brasiliense. Moreover, the pathogenicity of P. carotovorum and P. brasiliense increased with increasing temperature (20, 25, 28, 32 â„ƒ). The pathogenicity was more severe when P. carotovorum strain BL-4 was co-inoculated with P. brasiliense strain BL-2, especially when the former exhibited an advantage in bacterial number at the initial time. The results of this study provide new insight for understanding the pathogenicity caused by mixed infections with different species of Pectobacterium spp., and they may provide some guidance for controlling potato blackleg and soft rot.

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.

High-Quality Complete Genome Resource of Pathogenic Bacterium Pectobacterium atrosepticum Strain Green1 Isolated from Potato (Solanum tuberosum L.) in Greenland.

Pectobacterium atrosepticum is a narrow-host-range, pectinolytic, plant-pathogenic bacterium causing blackleg of potato (Solanum tuberosum L.) worldwide. Till present, several P. atrosepticum genomes have been sequenced and characterized in detail; however, all of these genomes have come from P. atrosepticum isolates from plants grown in temperate zones, not from hosts cultivated under different climatic conditions. Herewith, we present the first complete, high-quality genome of the P. atrosepticum strain Green1 isolated from potato plants grown under a subarctic climate in Greenland. The genome of P. atrosepticum strain Green1 consists of one chromosome of 4,959,719 bp, with a GC content of 51% and no plasmids. The genome contains 4,531 annotated features, including 4,179 protein-coding genes, 22 ribosomal RNA genes, 70 transfer RNA genes, 8 noncoding RNA genes, 2 CRISPRs, and 126 pseudogenes. We believe that the information in this first high-quality, complete, closed genome of P. atrosepticum strains isolated from host plants grown in a subarctic agricultural region will provide resources for comparative genomic studies and for analyses targeting climatic adaptation and ecological fitness mechanisms present in P. atrosepticum.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Pectobacterium parmentieri SCC 3193 Mutants with Altered Synthesis of Cell Surface Polysaccharides Are Resistant to N4-Like Lytic Bacteriophage ϕA38 (vB_Ppp_A38) but Express Decreased Virulence in Potato (Solanum tuberosum L.) Plants.

Pectobacterium parmentieri is a Gram-negative plant-pathogenic bacterium able to infect potato (Solanum tuberosum L.). Little is known about lytic bacteriophages infecting P. parmentieri and how phage-resistance influences the environmental fitness and virulence of this species. A lytic phage vB_Ppp_A38 (ϕA38) has been previously isolated and characterized as a potential biological control agent for the management of P. parmentieri. In this study, seven P. parmentieri SCC 3193 Tn5 mutants were identified that exhibited resistance to infection caused by vB_Ppp_A38 (ϕA38). The genes disrupted in these seven mutants encoded proteins involved in the assembly of O-antigen, sugar metabolism, and the production of bacterial capsule exopolysaccharides. The potential of A38-resistant P. parmentieri mutants for plant colonization and pathogenicity as well as other phenotypes expected to contribute to the ecological fitness of P. parmentieri, including growth rate, use of carbon and nitrogen sources, production of pectinolytic enzymes, proteases, cellulases, and siderophores, swimming and swarming motility, presence of capsule and flagella as well as the ability to form biofilm were assessed. Compared to the wild-type P. parmentieri strain, all phage-resistant mutants exhibited a reduced ability to colonize and to cause symptoms in growing potato (S. tuberosum L.) plants. The implications of bacteriophage resistance on the ecological fitness of P. parmentieri are discussed.

Genome-Wide Analyses of the Temperature-Responsive Genetic Loci of the Pectinolytic Plant Pathogenic Pectobacterium atrosepticum.

Temperature is one of the critical factors affecting gene expression in bacteria. Despite the general interest in the link between bacterial phenotypes and environmental temperature, little is known about temperature-dependent gene expression in plant pathogenic Pectobacterium atrosepticum, a causative agent of potato blackleg and tuber soft rot worldwide. In this study, twenty-nine P. atrosepticum SCRI1043 thermoregulated genes were identified using Tn5-based transposon mutagenesis coupled with an inducible promotorless gusA gene as a reporter. From the pool of 29 genes, 14 were up-regulated at 18 °C, whereas 15 other genes were up-regulated at 28 °C. Among the thermoregulated loci, genes involved in primary bacterial metabolism, membrane-related proteins, fitness-corresponding factors, and several hypothetical proteins were found. The Tn5 mutants were tested for their pathogenicity in planta and for features that are likely to remain important for the pathogen to succeed in the (plant) environment. Five Tn5 mutants expressed visible phenotypes differentiating these mutants from the phenotype of the SCRI1043 wild-type strain. The gene disruptions in the Tn5 transposon mutants caused alterations in bacterial generation time, ability to form a biofilm, production of lipopolysaccharides, and virulence on potato tuber slices. The consequences of environmental temperature on the ability of P. atrosepticum to cause disease symptoms in potato are discussed.

Complete genome sequences of novel Berlinvirus and novel Certrevirus lytic for Pectobacterium sp. causing soft rot and black leg disease of potato.

Two novel dsDNA bacteriophages named Pectobacterium virus CB251 (PcCB251) and Pectobacterium virus CB7V (PcCB7V) targeting plant pathogen Pectobacterium parmentieri have been isolated and sequenced. The PcCB251 genome consists of 40,557 bp with G+C content of 48.6% and contains 47 predicted genes on a single strand. The phage is classified in genus Berlinvirus, family Autographiviridae. The PcCB7V phage has a circular dsDNA genome of 146,054 bp with G+C content of 50.4% and contains 269 predicted protein genes on both strands and 13 tRNA genes. The PcCB7V phage can be classified in genus Certrevirus, subfamily Vequintavirinae. Both novel bacteriophages have narrow host ranges, but they extend the list of candidates for phage-based control of pectolytic bacteria causing soft rot disease of potato.

The Pectobacterium pangenome, with a focus on Pectobacterium brasiliense, shows a robust core and extensive exchange of genes from a shared gene pool.

BACKGROUND: Bacterial plant pathogens of the Pectobacterium genus are responsible for a wide spectrum of diseases in plants, including important crops such as potato, tomato, lettuce, and banana. Investigation of the genetic diversity underlying virulence and host specificity can be performed at genome level by using a comprehensive comparative approach called pangenomics. A pangenomic approach, using newly developed functionalities in PanTools, was applied to analyze the complex phylogeny of the Pectobacterium genus. We specifically used the pangenome to investigate genetic differences between virulent and avirulent strains of P. brasiliense, a potato blackleg causing species dominantly present in Western Europe. RESULTS: Here we generated a multilevel pangenome for Pectobacterium, comprising 197 strains across 19 species, including type strains, with a focus on P. brasiliense. The extensive phylogenetic analysis of the Pectobacterium genus showed robust distinct clades, with most detail provided by 452,388 parsimony-informative single-nucleotide polymorphisms identified in single-copy orthologs. The average Pectobacterium genome consists of 47% core genes, 1% unique genes, and 52% accessory genes. Using the pangenome, we zoomed in on differences between virulent and avirulent P. brasiliense strains and identified 86 genes associated to virulent strains. We found that the organization of genes is highly structured and linked with gene conservation, function, and transcriptional orientation. CONCLUSION: The pangenome analysis demonstrates that evolution in Pectobacteria is a highly dynamic process, including gene acquisitions partly in clusters, genome rearrangements, and loss of genes. Pectobacterium species are typically not characterized by a set of species-specific genes, but instead present themselves using new gene combinations from the shared gene pool. A multilevel pangenomic approach, fusing DNA, protein, biological function, taxonomic group, and phenotypes, facilitates studies in a flexible taxonomic context.

Identification of Pectobacterium versatile Causing Blackleg of Potato in New York State.

Soft rot bacteria classified in the Pectobacteriaceae (SRP), including Pectobacterium and Dickeya spp., are responsible for soft rot and blackleg diseases of potato. Since 2014, blackleg outbreaks caused by D. dianthicola have increased in the United States and Canada. Our previous study found that the most abundant causal organisms of blackleg disease in New York State were P. parmentieri and D. dianthicola, with the latter being the only Dickeya species reported. In the present study, we identified and characterized pathogenic SRP bacteria from 19 potato samples collected in New York State during the 2017 growing season. We used genome sequence comparison to determine the pathogens' species. We found eight P. versatile, one P. atrosepticum, two P. carotovorum, two P. parmentieri, and six D. dianthicola isolates in our 2017 SRP collection. This is the first time that P. versatile has been reported to cause potato blackleg disease in New York State. We determined the phylogenetic relationships between the SRP strains by using 151 single-copy orthologous gene sequences shared among the set of bacteria in our analysis, which provided better resolution than phylogenies constructed with the dnaX gene.

Complete Genome Sequence Resource for the Necrotrophic Plant-Pathogenic Bacterium Pectobacterium carotovorum WPP14.

Pectobacterium spp. are a major cause of loss in vegetable and ornamental plant production. One of these species, Pectobacterium carotovorum, can cause soft rot disease on many plants, particularly potato. These diseases lead to significant economic loss and pose food security threats by reducing crop yields in the field, in transit, and during storage. The Gram-negative enterobacterium P. carotovorum WPP14 is a particularly virulent strain for which there is no available closed genome, limiting the molecular research for this important pathogen. Here, we report a high-quality complete and annotated genome sequence of P. carotovorum WPP14. The 4,892,225-bp genome was assembled with Nanopore reads and polished with Illumina reads, yielding 394× and 164× coverage, respectively. This closed genome provides a resource for research on improved detection and biology of P. carotovorum, which could translate into improved disease management.

Antibacterial activity and mode of action of potassium tetraborate tetrahydrate against soft-rot bacterial plant pathogens.

Bacterial soft rot caused by the bacteria Dickeya and Pectobacterium is a destructive disease of vegetables, as well as ornamental plants. Several management options exist to help control these pathogens. Because of the limited success of these approaches, there is a need for the development of alternative methods to reduce losses. In this study, we evaluated the effect of potassium tetraborate tetrahydrate (PTB) on the growth of six Dickeya and Pectobacterium spp. Disc diffusion assays showed that Dickeya spp. and Pectobacterium spp. differ in their sensitivity to PTB. Spontaneous PTB-resistant mutants of Pectobacterium were identified and further investigation of the mechanism of PTB resistance was conducted by full genome sequencing. Point mutations in genes cpdB and supK were found in a single Pectobacterium atrosepticum PTB-resistant mutant. Additionally, point mutations in genes prfB (synonym supK) and prmC were found in two independent Pectobacterium brasiliense PTB-resistant mutants. prfB and prmC encode peptide chain release factor 2 and its methyltransferase, respectively. We propose the disruption of translation activity due to PTB leads to Pectobacterium growth inhibition. The P. atrosepticum PTB-resistant mutant showed altered swimming motility. Disease severity was reduced for P. atrosepticum-inoculated potato stems sprayed with PTB. We discuss the potential risk of selecting for bacterial resistance to this chemical.

Pectobacterium punjabense sp. nov., isolated from blackleg symptoms of potato plants in Pakistan.

Pectobacterium isolates SS95T, SS54 and SS56 were collected from a potato field in the Chiniot district in the plains of the Punjab province, Pakistan. Sequencing of the gapA barcode revealed that these strains belong to a novel phylogenetic group separated from P.ectobacterium wasabiae and Pectobacterium parmentieri species. Furthermore, multilocus sequence analyses of 13 housekeeping genes (fusA, rpoD, acnA, purA, gyrB, recA, mdh, mtlD, groEL, secY, glyA, gapA and rplB) clearly distinguished the type strain, SS95T, from its closest relatives, i.e. P. parmentieri RNS 08-42-1AT and P. wasabiae CFBP3304T, as well as from all the other known Pectobacteriumspecies. In silico DNA-DNA hybridization (<44.1 %) and average nucleotide identity (<90.75 %) values of strain SS95T compared with other Pectobacterium type strains supported the delineation of a new species. Genomic and phenotypic comparisons permitted the identification of additional traits that distinguished the Pakistani isolates from all other known Pectobacterium type strains. The name Pectobacterium punjabense sp. nov. is proposed for this taxon with the type strain SS95T (=CFBP 8604T=LMG 30622T).

Pectobacterium and Dickeya Responsible for Potato Blackleg Disease in New York State in 2016.

Beginning in 2014, outbreaks of blackleg disease compromised potato (Solanum tuberosum) production in the northeastern United States. Disease severity was atypical for plantings with certified seed. During 2016, 43 samples with blackleg symptoms were analyzed, originating from more than 20 farms operating in New York State. A combination of techniques was employed to identify the blackleg pathogens: isolation in vitro, diagnostic PCR assays for Pectobacterium and Dickeya sp., pathogenicity assays, and DNA sequencing. Twenty-three bacterial isolates were obtained, the majority of which were designated D. dianthicola or P. parmentieri; two of the isolates were designated P. atrosepticum. All isolates were pathogenic in stem lesion and tuber soft rot assays and exhibited pectin degrading activity (pitting) in crystal violet pectate agar medium. Phylogenetic analyses of dnaX gene sequences placed all but one of the isolates into clades corresponding to D. dianthicola, P. parmentieri, or P. atrosepticum. One atypical isolate clustered with P. carotovorum subspecies. Data are consistent with the hypothesis that D. dianthicola from New York and the northeast are part of a single clade, and at least three different soft rot bacteria were associated with blackleg during 2016 in New York.