Comprehensive phenomic and genomic studies of the species, Pectobacterium cacticida and proposal for reclassification as Alcorniella cacticida comb. nov.

Introduction: Pectobacterium cacticida was identified as the causative agent of soft rot disease in cacti. Due to a high potential of spread in the face of global warming, the species poses a significant threat to horticultural and crop industry. The aim of this study was to revise the genomic, physiology and virulence characteristics of P. cacticida and update its phylogenetic position within the Pectobacterium genus. Methods: Whole genome sequences of five P. cacticida strains were obtained and subjected to comprehensive genomic and phylogenomic data analyses. We assessed the presence of virulence determinants and genes associated with host and environmental adaptation. Lipidomic analysis, as well as biochemical and phenotypic assays were performed to correlate genomic findings. Results: Phylogenomic analysis revealed that P. cacticida forms a distinct lineage within the Pectobacterium genus. Genomic evaluation uncovered 516 unique proteins, most of which were involved in cellular metabolism. They included genes of carbohydrate metabolism and transport and ABC transporters. The main differing characteristics from other Pectobacterium species were the lack of a myo-inositol degradation pathway and the presence of the malonate decarboxylase gene. All tested strains were pathogenic towards Opuntia spp., chicory, Chinese cabbage, and potato, but exhibited only mild pathogenicity towards carrot. Discussion: This study sheds light into the genomic characteristics of P. cacticida and highlights the pathogenic potential of the species. Unique genes found in P. cacticida genomes possibly enhance the species' survival and virulence. Based on phylogenomic analyses, we propose the reclassification of P. cacticida to a new genus, Alcorniella comb. nov.

Identification and characterization of opportunistic pathogen Pectobacterium polonicum causing potato blackleg in China.

Blackleg and aerial stem rot of potato (Solanum tuberosum L.), caused by soft rot enterobacteria of the genera Pectobacterium and Dickeya, has recently increased years in Hebei Province, China. Field surveys were performed during the 2021 potato growing season in Hebei to identify and characterize bacterial pathogens. Sixteen potato plants showing blackleg or aerial stem rot were collected from three potato-producing areas, and ten representative pectinolytic bacteria were isolated from symptomatic plants. 16S rDNA sequencing and multilocus sequence analysis were performed to determine the taxonomic position of the bacterial isolates. The isolates belonged to the genus Pectobacterium, including Pectobacterium atrosepticum, Pectobacterium carotovorum, Pectobacterium brasiliense, and Pectobacterium parmentieri. The exceptions were isolates BY21311 and BY21312, which belonged to a new species of Pectobacterium polonicum previously found in groundwater. The taxonomy of isolate BY21311 was confirmed using whole genome-based analysis. P. polonicum has only been identified in potato plants on one farm in Baoding region in China. Isolates BY21311 and BY21312 displayed similar physiological and biochemical traits to the type strain DPMP315T. Artificial inoculation assays revealed that isolate BY21311 fulfilled Koch's postulates for potato blackleg. These findings represent the first time P. polonicum, a water-associated Pectobacterium species may be the cause of blackleg in the field. Interestingly, P. polonicum BY21311 has reduced ability to macerate potato tubers when compared to P. atrosepticum, P. brasiliense, P. versatile, and P. parvum, which is more virulent in tubers than the type strain DPMP315T. The host range of isolate BY21311 was determined by injection method, which can impregnate five plants. Although the genome of isolate BY21311 harbors gene clusters encoding a type III secretion system, it did not elicit a hypersensitive response (HR) in Nicotiana benthamiana or N. tabacum leaves. T3SS effector AvrE and T4SS effector PilN were obtained by predicting isolate BY21311 genome. P. polonicum appears to show significant variations in gene content between two genomes, and gene content varies between isolates BY21311 and DPMP315T, with strain specific-genes involved in many aspects, including lipopolysaccharide biosynthesis, substrate translocation, T4SS and T6SS among others, suggesting that isolates BY21311 and DPMP315T might represent distinct clades within the species.

Gene expression and phytohormone levels in the asymptomatic and symptomatic phases of infection in potato tubers inoculated with Dickeya solani.

Dickeya solani is a soft rot bacterium with high virulence. In potato, D. solani, like the other potato-infecting soft rot bacteria, causes rotting and wilting of the stems and rotting of tubers in the field and in storage. Latent, asymptomatic infections of potato tubers are common in harvested tubers, and if the storage conditions are not optimal, the latent infection turns into active rotting. We characterized potato gene expression in artificially inoculated tubers in nonsymptomatic, early infections 1 and 24 hours post-inoculation (hpi) and compared the results to the response in symptomatic tuber tissue 1 week (168 hpi) later with RNA-Seq. In the beginning of the infection, potato tubers expressed genes involved in the detection of the bacterium through pathogen-associated molecular patterns (PAMPs), which induced genes involved in PAMPs-triggered immunity, resistance, production of pathogenesis-related proteins, ROS, secondary metabolites and salicylic acid (SA) and jasmonic acid (JA) biosynthesis and signaling genes. In the symptomatic tuber tissue one week later, the PAMPs-triggered gene expression was downregulated, whereas primary metabolism was affected, most likely leading to free sugars fueling plant defense but possibly also aiding the growth of the pathogen. In the symptomatic tubers, pectic enzymes and cell wall-based defenses were activated. Measurement of hormone production revealed increased SA concentration and almost no JA in the asymptomatic tubers at the beginning of the infection and high level of JA and reduced SA in the symptomatic tubers one week later. These findings suggest that potato tubers rely on different defense strategies in the different phases of D. solani infection even when the infection takes place in fully susceptible plants incubated in conditions leading to rotting. These results support the idea that D. solani is a biotroph rather than a true necrotroph.

Dual Transcriptional Profiling of Carrot and 'Candidatus Liberibacter solanacearum' at Different Stages of Infection Suggests Complex Host-Pathogen Interaction.

The interactions between the phloem-limited pathogen 'Candidatus Liberibacter solanacearum' haplotype C and carrot (Daucus carota subsp. sativus) were studied at 4, 5, and 9 weeks postinoculation (wpi), by combining dual RNA-Seq results with data on bacterial colonization and observations of the plant phenotype. In the infected plants, genes involved in jasmonate biosynthesis, salicylate signaling, pathogen-associated molecular pattern- and effector-triggered immunity, and production of pathogenesis-related proteins were up-regulated. At 4 wpi, terpenoid synthesis-related genes were up-regulated, presumably as a response to the psyllid feeding, whereas at 5 and 9 wpi, genes involved in both the terpenoid and flavonoid production were down-regulated and phenylpropanoid genes were up-regulated. Chloroplast-related gene expression was down-regulated, in concordance with the observed yellowing of the infected plant leaves. Both the RNA-Seq data and electron microscopy suggested callose accumulation in the infected phloem vessels, likely to impair the transport of photosynthates, while phloem regeneration was suggested by the formation of new sieve cells and the upregulation of cell wall-related gene expression. The 'Ca. L. solanacearum' genes involved in replication, transcription, and translation were expressed at high levels at 4 and 5 wpi, whereas, at 9 wpi, the Flp pilus genes were highly expressed, suggesting adherence and reduced mobility of the bacteria. The 'Ca. L. solanacearum' genes encoding ATP and C4-dicarboxylate uptake were differentially expressed between the early and late infection stages, suggesting a change in the dependence on different host-derived energy sources. HPE1 effector and salicylate hydroxylase were expressed, presumably to suppress host cell death and salicylic acid-dependent defenses during the infection.[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 parvum sp. nov., having a Salmonella SPI-1-like Type III secretion system and low virulence.

Pectobacterium strains isolated from potato stems in Finland, Poland and the Netherlands were subjected to polyphasic analyses to characterize their genomic and phenotypic features. Phylogenetic analysis based on 382 core proteins showed that the isolates clustered closest to Pectobacterium polaris but could be divided into two clades. Average nucleotide identity (ANI) analysis revealed that the isolates in one of the clades included the P. polaris type strain, whereas the second clade was at the border of the species P. polaris with a 96 % ANI value. In silico genome-to-genome comparisons between the isolates revealed values below 70%, patristic distances based on 1294 core proteins were at the level observed between closely related Pectobacterium species, and the two groups of bacteria differed in genome size, G+C content and results of amplified fragment length polymorphism and Biolog analyses. Comparisons between the genomes revealed that the isolates of the atypical group contained SPI-1-type Type III secretion island and genes coding for proteins known for toxic effects on nematodes or insects, and lacked many genes coding for previously characterized virulence determinants affecting rotting of plant tissue by soft rot bacteria. Furthermore, the atypical isolates could be differentiated from P. polaris by their low virulence, production of antibacterial metabolites and a citrate-negative phenotype. Based on the results of a polyphasic approach including genome-to-genome comparisons, biochemical and virulence assays, presented in this report, we propose delineation of the atypical isolates as a novel species Pectobacterium parvum, for which the isolate s0421T (CFBP 8630T=LMG 30828T) is suggested as a type strain.

Effect of wet storage conditions on potato tuber transcriptome, phytohormones and growth.

BACKGROUND: Stored potato (Solanum tuberosum L.) tubers are sensitive to wet conditions that can cause rotting in long-term storage. To study the effect of water on the tuber surface during storage, microarray analysis, RNA-Seq profiling, qRT-PCR and phytohormone measurements were performed to study gene expression and hormone content in wet tubers incubated at two temperatures: 4 °C and 15 °C. The growth of the plants was also observed in a greenhouse after the incubation of tubers in wet conditions. RESULTS: Wet conditions induced a low-oxygen response, suggesting reduced oxygen availability in wet tubers at both temperatures when compared to that in the corresponding dry samples. Wet conditions induced genes coding for heat shock proteins, as well as proteins involved in fermentative energy production and defense against reactive oxygen species (ROS), which are transcripts that have been previously associated with low-oxygen stress in hypoxic or anoxic conditions. Wet treatment also induced senescence-related gene expression and genes involved in cell wall loosening, but downregulated genes encoding protease inhibitors and proteins involved in chloroplast functions and in the biosynthesis of secondary metabolites. Many genes involved in the production of phytohormones and signaling were also affected by wet conditions, suggesting altered regulation of growth by wet conditions. Hormone measurements after incubation showed increased salicylic acid (SA), abscisic acid (ABA) and auxin (IAA) concentrations as well as reduced production of jasmonate 12-oxo-phytodienoic acid (OPDA) in wet tubers. After incubation in wet conditions, the tubers produced fewer stems and more roots compared to controls incubated in dry conditions. CONCLUSIONS: In wet conditions, tubers invest in ROS protection and defense against the abiotic stress caused by reduced oxygen due to excessive water. Changes in ABA, SA and IAA that are antagonistic to jasmonates affect growth and defenses, causing induction of root growth and rendering tubers susceptible to necrotrophic pathogens. Water on the tuber surface may function as a signal for growth, similar to germination of seeds.

Genome sequence of the model plant pathogen Pectobacterium carotovorum SCC1.

Bacteria of the genus Pectobacterium are economically important plant pathogens that cause soft rot disease on a wide variety of plant species. Here, we report the genome sequence of Pectobacterium carotovorum strain SCC1, a Finnish soft rot model strain isolated from a diseased potato tuber in the early 1980's. The genome of strain SCC1 consists of one circular chromosome of 4,974,798 bp and one circular plasmid of 5524 bp. In total 4451 genes were predicted, of which 4349 are protein coding and 102 are RNA genes.

Genomic sequence of 'Candidatus Liberibacter solanacearum' haplotype C and its comparison with haplotype A and B genomes.

Haplotypes A and B of 'Candidatus Liberibacter solanacearum' (CLso) are associated with diseases of solanaceous plants, especially Zebra chip disease of potato, and haplotypes C, D and E are associated with symptoms on apiaceous plants. To date, one complete genome of haplotype B and two high quality draft genomes of haplotype A have been obtained for these unculturable bacteria using metagenomics from the psyllid vector Bactericera cockerelli. Here, we present the first genomic sequences obtained for the carrot-associated CLso. These two genomic sequences of haplotype C, FIN114 (1.24 Mbp) and FIN111 (1.20 Mbp), were obtained from carrot psyllids (Trioza apicalis) harboring CLso. Genomic comparisons between the haplotypes A, B and C revealed that the genome organization differs between these haplotypes, due to large inversions and other recombinations. Comparison of protein-coding genes indicated that the core genome of CLso consists of 885 ortholog groups, with the pan-genome consisting of 1327 ortholog groups. Twenty-seven ortholog groups are unique to CLso haplotype C, whilst 11 ortholog groups shared by the haplotypes A and B, are not found in the haplotype C. Some of these ortholog groups that are not part of the core genome may encode functions related to interactions with the different host plant and psyllid species.

Short oligogalacturonides induce pathogen resistance-associated gene expression in Arabidopsis thaliana.

BACKGROUND: Oligogalacturonides (OGs) are important components of damage-associated molecular pattern (DAMP) signaling and influence growth regulation in plants. Recent studies have focused on the impact of long OGs (degree of polymerization (DP) from 10-15), demonstrating the induction of plant defense signaling resulting in enhanced defenses to necrotrophic pathogens. To clarify the role of trimers (trimeric OGs, DP3) in DAMP signaling and their impact on plant growth regulation, we performed a transcriptomic analysis through the RNA sequencing of Arabidopsis thaliana exposed to trimers. RESULTS: The transcriptomic data from trimer-treated Arabidopsis seedlings indicate a clear activation of genes involved in defense signaling, phytohormone signaling and a down-regulation of genes involved in processes related to growth regulation and development. This is further accompanied with improved defenses against necrotrophic pathogens triggered by the trimer treatment, indicating that short OGs have a clear impact on plant responses, similar to those described for long OGs. CONCLUSIONS: Our results demonstrate that trimers are indeed active elicitors of plant defenses. This is clearly indicated by the up-regulation of genes associated with plant defense signaling, accompanied with improved defenses against necrotrophic pathogens. Moreover, trimers simultaneously trigger a clear down-regulation of genes and gene sets associated with growth and development, leading to stunted seedling growth in Arabidopsis.

Dickeya aquatica sp. nov., isolated from waterways.

Pectinolytic Gram-negative bacteria were isolated from different waterways in the UK and Finland. Three strains (174/2(T), 181/2 and Dw054) had the same 16S rRNA gene sequences which shared 99% sequence similarity to species of the genus Dickeya, and a phylogeny of related genera confirmed attribution to this genus. Fatty acid profile analysis of all three strains found a high proportion of C16 : 1ω7c/C16 : 1ω7c and C16 : 0 fatty acids, and library profile searches found closest matches to Dickeya chrysanthemi. Production of a concatenated phylogeny using six loci, recA, gapA, atpD, gyrB, infB and rpoB, provided a high-resolution phylogeny which placed strains 174/2(T) and 181/2 as a distinct clade, separated from the other species of the genus Dickeya by a relatively long branch-length. DNA-DNA hybridization analysis with a limited number of reference species also supported the distinctiveness of strains 174/2(T) and 181/2 within the genus Dickeya. All three strains could be phenotypically distinguished from other species of the genus by fermentation of melibiose and raffinose but not D-arabinose or mannitol. The name Dickeya aquatica sp. nov. is proposed for the new taxon; the type strain is 174/2(T) ( = NCPPB 4580(T) = LMG 27354(T)).

Dickeya solani sp. nov., a pectinolytic plant-pathogenic bacterium isolated from potato (Solanum tuberosum).

Pectinolytic bacteria have been recently isolated from diseased potato plants exhibiting blackleg and slow wilt symptoms found in a number of European countries and Israel. These Gram-reaction-negative, motile, rods were identified as belonging to the genus Dickeya, previously the Pectobacterium chrysanthemi complex (Erwinia chrysanthemi), on the basis of production of a PCR product with the pelADE primers, 16S rRNA gene sequence analysis, fatty acid methyl esterase analysis, the production of phosphatases and the ability to produce indole and acids from α-methylglucoside. Differential physiological assays used previously to differentiate between strains of E. chrysanthemi, showed that these isolates belonged to biovar 3. Eight of the isolates, seven from potato and one from hyacinth, were analysed together with 21 reference strains representing all currently recognized taxa within the genus Dickeya. The novel isolates formed a distinct genetic clade in multilocus sequence analysis (MLSA) using concatenated sequences of the intergenic spacer (IGS), as well as dnaX, recA, dnaN, fusA, gapA, purA, rplB, rpoS and gyrA. Characterization by whole-cell MALDI-TOF mass spectrometry, pulsed field gel electrophoresis after digestion of whole-genome DNA with rare-cutting restriction enzymes, average nucleotide identity analysis and DNA-DNA hybridization studies, showed that although related to Dickeya dadantii, these isolates represent a novel species within the genus Dickeya, for which the name Dickeya solani sp. nov. (type strain IPO 2222(T) = LMG25993(T) = NCPPB4479(T)) is proposed.

Expression of nipP.w of Pectobacterium wasabiae is dependent on functional flgKL flagellar genes.

While flagellum-driven motility is hypothesized to play a role in the virulence of Pectobacterium species, there is no direct evidence that genes involved in flagellum assembly regulate the synthesis of virulence factors. The purpose of this study was to identify genes that affect the production or secretion of necrosis-inducing protein (Nip) in the strain SCC3193. Transposon mutagenesis of an RpoS strain overexpressing NipP.w was performed, and a mutant associated with decreased necrosis of tobacco leaves was detected. The mutant contained a transposon in the regulatory region upstream of the flagellar genes flgK and flgL. Additional mutants were generated related to the flagellar genes fliC and fliA. The mutation in flgKL, but not those in fliC and fliA, inhibited nipP.w transcription. Moreover, the regulatory effect of the flgKL mutation on nipP.w transcription was partially dependent on the Rcs phosphorelay. Secretion of NipP.w was also dependent on a type II secretion mechanism. Overall, the results of this study indicate that the flgKL mutation is responsible for reduced motility and lower levels of nipP.w expression.

Draft genome sequences of 17 isolates of the plant pathogenic bacterium dickeya.

Dickeya (formerly Erwinia chrysanthemi) species cause diseases on a wide range of crops and ornamental plants worldwide. Here we present the draft sequences of 17 Dickeya isolates spanning four Dickeya species, including five isolates that are currently unassigned to a species.

Role and regulation of the Flp/Tad pilus in the virulence of Pectobacterium atrosepticum SCRI1043 and Pectobacterium wasabiae SCC3193.

In this study, we characterized a putative Flp/Tad pilus-encoding gene cluster, and we examined its regulation at the transcriptional level and its role in the virulence of potato pathogenic enterobacteria of the genus Pectobacterium. The Flp/Tad pilus-encoding gene clusters in Pectobacterium atrosepticum, Pectobacterium wasabiae and Pectobacterium aroidearum were compared to previously characterized flp/tad gene clusters, including that of the well-studied Flp/Tad pilus model organism Aggregatibacter actinomycetemcomitans, in which this pilus is a major virulence determinant. Comparative analyses revealed substantial protein sequence similarity and open reading frame synteny between the previously characterized flp/tad gene clusters and the cluster in Pectobacterium, suggesting that the predicted flp/tad gene cluster in Pectobacterium encodes a Flp/Tad pilus-like structure. We detected genes for a novel two-component system adjacent to the flp/tad gene cluster in Pectobacterium, and mutant analysis demonstrated that this system has a positive effect on the transcription of selected Flp/Tad pilus biogenesis genes, suggesting that this response regulator regulate the flp/tad gene cluster. Mutagenesis of either the predicted regulator gene or selected Flp/Tad pilus biogenesis genes had a significant impact on the maceration ability of the bacterial strains in potato tubers, indicating that the Flp/Tad pilus-encoding gene cluster represents a novel virulence determinant in Pectobacterium. Soft-rot enterobacteria in the genera Pectobacterium and Dickeya are of great agricultural importance, and an investigation of the virulence of these pathogens could facilitate improvements in agricultural practices, thus benefiting farmers, the potato industry and consumers.

Lack of RsmA-mediated control results in constant hypervirulence, cell elongation, and hyperflagellation in Pectobacterium wasabiae.

The posttranscriptional regulator RsmA controls the production of plant cell wall degrading enzymes (PCWDE) and cell motility in the Pectobacterium genus of plant pathogens. In this study the physiological role of gene regulation by RsmA is under investigation. Disruption of rsmA gene of the Pectobacterium wasabiae strain, SCC3193 resulted in 3-fold decrease in growth rate and increased virulence. The comparison of mRNA levels of the rsmA(-) mutant and wild-type using a genome-wide microarray showed, that genes responsible for successful infection, i.e. virulence factors, motility, butanediol fermentation, various secretion systems etc. were up-regulated in the rsmA(-) strain. The rsmA(-) strain exhibited a higher propensity to swarm and produce PCWDE compared to the wild-type strain. Virulence experiments in potato tubers demonstrated that in spite of its more efficient tissue maceration, the rsmA(-) strain's ability to survive within the host is reduced and the infection site is taken over by resident bacteria. Taken together, in the absence of RsmA, cells revert to a constitutively infective phenotype characterized by expression of virulence factors and swarming. We hypothesize that lack of control over these costly energetic processes results in decreased growth rate and fitness. In addition, our findings suggest a relationship between swarming and virulence in plant pathogens.

Revised phylogeny and novel horizontally acquired virulence determinants of the model soft rot phytopathogen Pectobacterium wasabiae SCC3193.

Soft rot disease is economically one of the most devastating bacterial diseases affecting plants worldwide. In this study, we present novel insights into the phylogeny and virulence of the soft rot model Pectobacterium sp. SCC3193, which was isolated from a diseased potato stem in Finland in the early 1980s. Genomic approaches, including proteome and genome comparisons of all sequenced soft rot bacteria, revealed that SCC3193, previously included in the species Pectobacterium carotovorum, can now be more accurately classified as Pectobacterium wasabiae. Together with the recently revised phylogeny of a few P. carotovorum strains and an increasing number of studies on P. wasabiae, our work indicates that P. wasabiae has been unnoticed but present in potato fields worldwide. A combination of genomic approaches and in planta experiments identified features that separate SCC3193 and other P. wasabiae strains from the rest of soft rot bacteria, such as the absence of a type III secretion system that contributes to virulence of other soft rot species. Experimentally established virulence determinants include the putative transcriptional regulator SirB, two partially redundant type VI secretion systems and two horizontally acquired clusters (Vic1 and Vic2), which contain predicted virulence genes. Genome comparison also revealed other interesting traits that may be related to life in planta or other specific environmental conditions. These traits include a predicted benzoic acid/salicylic acid carboxyl methyltransferase of eukaryotic origin. The novelties found in this work indicate that soft rot bacteria have a reservoir of unknown traits that may be utilized in the poorly understood latent stage in planta. The genomic approaches and the comparison of the model strain SCC3193 to other sequenced Pectobacterium strains, including the type strain of P. wasabiae, provides a solid basis for further investigation of the virulence, distribution and phylogeny of soft rot bacteria and, potentially, other bacteria as well.

Genome sequence of Pectobacterium sp. strain SCC3193.

We report the complete and annotated genome sequence of the plant-pathogenic enterobacterium Pectobacterium sp. strain SCC3193, a model strain isolated from potato in Finland. The Pectobacterium sp. SCC3193 genome consists of a 5,164,411-bp [corrected] chromosome, with no plasmids.

Hcp2, a secreted protein of the phytopathogen Pseudomonas syringae pv. tomato DC3000, is required for fitness for competition against bacteria and yeasts.

When analyzing the secretome of the plant pathogen Pseudomonas syringae pv. tomato DC3000, we identified hemolysin-coregulated protein (Hcp) as one of the secreted proteins. Hcp is assumed to be an extracellular component of the type VI secretion system (T6SS). Two copies of hcp genes are present in the P. syringae pv. tomato DC3000 genome, hcp1 (PSPTO_2539) and hcp2 (PSPTO_5435). We studied the expression patterns of the hcp genes and tested the fitness of hcp knockout mutants in host plant colonization and in intermicrobial competition. We found that the hcp2 gene is expressed most actively at the stationary growth phase and that the Hcp2 protein is secreted via the T6SS and appears in the culture medium as covalently linked dimers. Expression of hcp2 is not induced in planta and does not contribute to virulence in or colonization of tomato or Arabidopsis plants. Instead, hcp2 is required for survival in competition with enterobacteria and yeasts, and its function is associated with the suppression of the growth of these competitors. This is the first report on bacterial T6SS-associated genes functioning in competition with yeast. Our results suggest that the T6SS of P. syringae may play an important role in bacterial fitness, allowing this plant pathogen to survive under conditions where it has to compete with other microorganisms for resources.