Atroseptica Strain Research Articles

RpoS-Regulated Genes and Phenotypes in the Phytopathogenic Bacterium Pectobacterium atrosepticum.

The alternative sigma factor RpoS is considered to be one of the major regulators providing stress resistance and cross-protection in bacteria. In phytopathogenic bacteria, the effects of RpoS have not been analyzed with regard to cross-protection, and genes whose expression is directly or indirectly controlled by RpoS have not been determined at the whole-transcriptome level. Our study aimed to determine RpoS-regulated genes and phenotypes in the phytopathogenic bacterium Pectobacterium atrosepticum. Knockout of the rpoS gene in P. atrosepticum affected the long-term starvation response, cross-protection, and virulence toward plants with enhanced immune status. The whole-transcriptome profiles of the wild-type P. atrosepticum strain and its ΔrpoS mutant were compared under different experimental conditions, and functional gene groups whose expression was affected by RpoS were determined. The RpoS promoter motif was inferred within the promoter regions of the genes affected by rpoS deletion, and the P. atrosepticum RpoS regulon was predicted. Based on RpoS-controlled phenotypes, transcriptome profiles, and RpoS regulon composition, the regulatory role of RpoS in P. atrosepticum is discussed.

Genotypic and phenotypic uniformity among the population of Pectobacterium atrosepticum strains isolated during three growing seasons from potato fields in Poland

Pectobacterium atrosepticum (Pba)are phytopathogenic, pectinolytic, non-sporulating, gram-negative rods causing blackleg and soft rot diseases in potato. They are ubiquitously present under temperate climate conditions and contribute to significant economic losses. Currently, there are no efficient control methods against soft rot Pectobacteriaceae (SRP). As limitation of the spread of SRP is solely prevention-based, we undertook broad characterization on both genotypic and phenotypic levels of the P. atrosepticum isolates acquired during monitoring of potato fields in Poland in 2013, 2014 and 2016. Of REP, ERIC and BOX-based genomic fingerprinting, the BOX-based approach turned out to be the most informative and allowed for classification of 118 Pba strains into six groups. The present study indicated that IV and I BOX profiles dominated among the investigated population. Finally, 23 Pba strains representing all BOX groups and originating from various growing seasons were selected for genotypic, phylogenetic and phenotypic studies. rpoS-based phylogeny revealed intraspecies variation (16 SNP sites) among the studied Pba strains, in contrast to the analyses relying on gyrA (1 SNP) and recA (0 SNP) sequences. Pba strains showed higher potency to macerate potato tissue at 20 °C than 28 °C. These isolates exhibited rather uniform production of plant cell wall degrading enzymes (pectinases, cellulases and proteases), lipases, siderophores and biofilm, however in the majority of cases inferior in comparison to Dickeya solani and Pectobacterium carotovorum strains. In conclusion, this study revealed genotypic and phenotypic uniformity in addition to high virulence potential of Pba strains isolated from potato fields during several growing seasons in Poland.

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.

Development of qPCR Detection Assay for Potato Pathogen Pectobacterium atrosepticum Based on a Unique Target Sequence.

The recent taxonomic diversification of bacterial genera Pectobacterium and Dickeya, which cause soft rot in plants, focuses attention on the need for improvement of existing methods for the detection and differentiation of these phytopathogens. This research presents a whole genome-based approach to the selection of marker sequences unique to particular species of Pectobacterium. The quantitative real-time PCR assay developed is selective in the context of all tested Pectobacterium atrosepticum strains and is able to detect fewer than 102 copies of target DNA per reaction. The presence of plant DNA extract did not affect the sensitivity of the assay.

Pectobacterium Phage Jarilo Displays Broad Host Range and Represents a Novel Genus of Bacteriophages Within the Family Autographiviridae

Background: Soft rot Pectobacteriaceae includes the genera Pectobacterium and Dickeya, which are important plant pathogens being responsible for diseases in a wide range of plant species, with potatoes as the main group. Both genera cause pre- and postharvest losses of potatoes, resulting in huge economic losses linked with the soft rot diseases. Materials and Methods: Organic waste was used to isolate phages, with Pectobacterium carotovorum subsp. carotovorum DSM 30170 as host. Complete genome sequencing, comparative genomics, and electron microscopy were used to characterize the phage. An adsorption assay was used to estimate adsorption rate. Twenty-three strains from the genera Pectobacterium and Dickeya were used to examine the host range of the phage. Results: Pectobacterium phage Jarilo represents a novel genus of bacteriophages within the family Autographiviridae, order Caudovirales. Jarilo possesses a double-stranded DNA genome of 40557 bp with a G+C% content of 50.08% and 50 predicted open reading frames. Gene synteny and products seem to be partly conserved between Pectobacterium phage Jarilo and Enterobacteria phage T7, but limited nucleotide similarity is found between Jarilo and other phages within the family Autographiviridae. The adsorption rate of phage Jarilo increased continuously for 1 h upon infection. Phage Jarilo was not able to infect any strains of P. carotovorum and Dickeya tested with the exception of the P. carotovorum strain used for isolation. However, phage Jarilo infected 10 of 16 Pectobacterium atrosepticum strains tested. Conclusion: We propose Pectobacterium phage Jarilo as the first member of a new genus of bacteriophages within the family Autographiviridae, order Caudovirales, displaying a broad host range within the genera of Pectobacterium.

A novel six-phage cocktail reduces Pectobacterium atrosepticum soft rot infection in potato tubers under simulated storage conditions

Pectobacterium atrosepticum is a species of plant pathogenic bacteria responsible for significant losses in potato production worldwide. Pectobacterium atrosepticum can cause blackleg disease on potato stems as well as the tuber disease termed potato soft rot. Methods for the effective control of these diseases are limited and are primarily based on good agricultural practices. Bacteriophages, viruses of bacteria, could be used as an alternative, environmentally friendly, control measure. Here, we describe the isolation and characterization of 29 phages virulent to P. atrosepticum. The phages belong to 12 different species based on a 95% sequence identity cut-off. Furthermore, based on sequence diversity and propagation results, we selected six of these phages to form a phage cocktail. The phages in the cocktail was tested on a number of P. atrosepticum strains in order to determine their host range. The phages was found to lyse 93% of the tested strains. The cocktail was subsequently tested for its effectiveness in combatting potato soft rot under simulated storage conditions. Use of the phage cocktail reduced both disease incidence and disease severity by 61% and 64%, respectively, strongly indicating that phage biocontrol has the potential to reduce the economic impact of soft rot in potato production.

A novel six-phage cocktail reducesPectobacterium atrosepticumsoft rot infection in potato tubers under simulated storage conditions

Pectobacterium atrosepticum is a species of plant pathogenic bacteria responsible for significant losses in potato production worldwide. Pectobacterium atrosepticum can cause blackleg disease on potato stems as well as the tuber disease termed potato soft rot. Methods for the effective control of these diseases are limited and are primarily based on good agricultural practices. Bacteriophages, viruses of bacteria, could be used as an alternative, environmentally friendly, control measure. Here, we describe the isolation and characterization of 29 phages virulent to P. atrosepticum. The phages belong to 12 different species based on a 95% sequence identity cut-off. Furthermore, based on sequence diversity and propagation results, we selected six of these phages to form a phage cocktail. The phages in the cocktail was tested on a number of P. atrosepticum strains in order to determine their host range. The phages was found to lyse 93% of the tested strains. The cocktail was subsequently tested for its effectiveness in combatting potato soft rot under simulated storage conditions. Use of the phage cocktail reduced both disease incidence and disease severity by 61% and 64%, respectively, strongly indicating that phage biocontrol has the potential to reduce the economic impact of soft rot in potato production.

Stimulation of defense reactions in potato against Pectobacterium sp.

The pesticides used to tackle potato pathogens, including bacteria, are environmentally damaging, so more sustainable treatments are needed. Because plant defense-related hormones such as methyl jasmonate and salicylic acid and the elicitor chitosan can stimulate plant defense systems, the effect of these compounds was tested by adding them to a watering solution 2 days before inoculating two S. tuberosum cultivars with either Pectobacterium carotovorum strain 5890 or P. atrosepticum strain 889. Growth and the production of several defense phenolic compounds were determined in treated and untreated plants, inoculated or not. P. carotovorum 5890 was more virulent than P. atrosepticum 5889 as reflected by higher levels of phenolic compounds in the plants. The defense-related hormone salicylic acid offered the most protection against the bacteria without interfering with plant growth. Because plants produce and accumulate secondary metabolites for protection against infection, higher production was accompanied by less pathogen damage and higher resistance.

Draft Genome Sequence of Pectobacterium atrosepticum PB72 and Complete Genome Sequence of the Specific Bacteriophage PP90.

We present the draft genome sequence of Pectobacterium atrosepticum strain PB72 infecting potatoes in Russia. PB72 is similar to the previously reported strain 21A. Considering potential biocontrol of this pathogen, an infectious bacteriophage was isolated and characterized. Phage vB_PatP_PP90 is a lytic podovirus of narrow host range belonging to the KP34virus genus.

Delineation of the functional and structural properties of the glutathione transferase family from the plant pathogen Erwinia carotovora

Erwinia carotovora, a widespread plant pathogen that causes soft rot disease in many plants, is considered a major threat in agriculture. Bacterial glutathione transferases (GSTs) play important roles in a variety of metabolic pathways and processes, such as the biodegradation of xenobiotics, protection against abiotic stress, and resistance against antimicrobial drugs. The GST family of canonical soluble enzymes from Erwinia carotovora subsp. atroseptica strain SCRI1043 (EcaGSTs) was investigated. Genome analysis showed the presence of six putative canonical cytoplasmic EcaGSTs, which were revealed by phylogenetic analysis to belong to the well-characterized GST classes beta, nu, phi, and zeta. The analysis also revealed the presence of two isoenzymes that were phylogenetically close to the omega class of GSTs, but formed a distinct class. The EcaGSTs were cloned and expressed in Escherichia coli, and their catalytic activity toward different electrophilic substrates was elucidated. The EcaGSTs catalyzed different types of reactions, although all enzymes were particularly active in reactions involving electrophile substitution. Gene and protein expression profiling conducted under normal culture conditions as well as in the presence of the herbicide alachlor and the xenobiotic 1-chloro-2,4-dinitrobenzene (CDNB) showed that the isoenzyme EcaGST1, belonging to the omega-like class, was specifically induced at both the protein and mRNA levels. EcaGST1 presumably participates in counteracting the xenobiotic toxicity and/or abiotic stress conditions, and may therefore represent a novel molecular target in the development of new chemical treatments to control soft rot diseases.

Complete genome sequence and pathogenic genes analysis of Pectobacterium atroseptica JG10-08

Pectobacterium atroseptica is known as a rod-shaped gram-negative bacterial pathogen associated with the blackleg of potato. P. atroseptica has been widely identified as the predominant agent causing tuber rot in temperate regions, a disease that leads to severe economic losses to potato industry. In this study, we provide the complete genome sequence of P. atroseptica JG10-08, which revealed that P. atroseptica strain JG10-08 carries a single 5,004,926 bp chromosome with 51.15% G+C content and harbors 4252 predicted coding genes. Phylogenetic analysis based on the genome sequences showed a close evolutionary relationship between P. atroseptica and Pectobacterium wasabiae. We discovered total 168 genes were potentially related to pathogenesis including 9 strain-specific genes encoding toxins on the genome of JG10-08. Further comparison with other species in Pectobacterium revealed a better understanding of pathogenic factors, especially secretion systems in P. atroseptica JG10-08. Collectively, the results of this research provide a solid foundation for discovering the underlying pathogenic mechanisms of P. atroseptica and offer the information to develop more effective strategies against blackleg of potatoes.

Comprehensive overexpression analysis of cyclic-di-GMP signalling proteins in the phytopathogen Pectobacterium atrosepticum reveals diverse effects on motility and virulence phenotypes.

Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a ubiquitous bacterial signalling molecule produced by diguanylate cyclases of the GGDEF-domain family. Elevated c-di-GMP levels or increased GGDEF protein expression is frequently associated with the onset of sessility and biofilm formation in numerous bacterial species. Conversely, phosphodiesterase-dependent diminution of c-di-GMP levels by EAL- and HD-GYP-domain proteins is often accompanied by increased motility and virulence. In this study, we individually overexpressed 23 predicted GGDEF, EAL or HD-GYP-domain proteins encoded by the phytopathogen Pectobacterium atrosepticum strain SCRI1043. MS-based detection of c-di-GMP and 5'-phosphoguanylyl-(3'-5')-guanosine in these strains revealed that overexpression of most genes promoted modest 1-10-fold changes in cellular levels of c-di-GMP, with the exception of the GGDEF-domain proteins ECA0659 and ECA3374, which induced 1290- and 7660-fold increases, respectively. Overexpression of most EAL domain proteins increased motility, while overexpression of most GGDEF domain proteins reduced motility and increased poly-β-1,6-N-acetyl-glucosamine-dependent flocculation. In contrast to domain-based predictions, overexpression of the EAL protein ECA3549 or the HD-GYP protein ECA3548 increased c-di-GMP concentrations and reduced motility. Most overexpression constructs altered the levels of secreted cellulases, pectinases and proteases, confirming c-di-GMP regulation of virulence in Pe. atrosepticum. However, there was no apparent correlation between virulence-factor induction and the domain class expressed or cellular c-di-GMP levels, suggesting that regulation was in response to specific effectors within the network, rather than total c-di-GMP concentration. Finally, we demonstrated that the cellular localization patterns vary considerably for GGDEF/EAL/HD-GYP proteins, indicating it is a likely factor restricting specific interactions within the c-di-GMP network.

Virulence Function of Pectobacterium atrosepticum Secretion System Mutants on Evaluation of Some Solanum tuberosum Resistance Genes

Background: P. atrosepticum is a commercially important pathogen. It causes blackleg in the field and soft rot of tubers after the harvest. This effect is due to secretion of depolymerases and other virulence factors by several mechanisms including T3SS Objectives: The effect of bacterial T3SS on Solanum tuberosum (S. tuberosum) varieties and its relationship with S. tuberosum resistance gene expression were studied. Materials and Methods: A P. atrosepticum HrpW gene was cloned, sequenced and constructed a phylogenic tree with some phytopathogen. The virulence properties of P. atrosepticum strains were investigated and then S. tuberosum varieties were tested for their sensitivity against P. atrosepticum. PR-5 and HIN genes copy-number for infiltrated S. tuberosum tubers were assessed. Results: The results show that infiltrated tubers of P. atrosepticum T3SS mutants were significantly more macerated than the wild type ones. Conclusions: PR-5 and HIN expression amounts were depended on bacterial T3SS function.

Inactivation of PbTopo IIIβ causes hyper‐excision of the Pathogenicity Island HAI2 resulting in reduced virulence of Pectobacterium atrosepticum

Topoisomerase III enzymes are present only in a limited set of bacteria and their physiological role remains unclear. Here, we show that PbTopo IIIβ, a homologue of topoisomerase III encoded on the chromosome of Pectobacterium atrosepticum strain SCRI1043 (Pba SCRI1043), is involved in excision of HAI2, a discrete ~100 kb region, from the Pba SCRI1043 chromosome. HAI2 is a Pathogenicity Island (PAI) that encodes coronafacic acid (Cfa), a major virulence determinant required for infection of potato. PAIs are horizontally acquired genetic elements that in some instances are able to excise from the chromosome of their host cell to form a circular episome prior to transfer to a recipient bacterium. We demonstrate excision of HAI2 from the chromosome, a process that is independent of growth phase and that results in the production of a circular intermediate. Inactivation of PbTopo IIIβ causes a 10(3) - to 10(4) -fold increase in excision, leading to reduced fitness in vitro and a decrease in the virulence of Pba SCRI1043 on potato. These results suggest that PbTopo IIIβ is required for stable maintenance of HAI2 in the chromosome of Pba SCRI1043 and may control as yet unidentified genes involved in viability and virulence of Pba SCRI1043 on potato.

Isolation and identification of N-acylhomoserin lactone degrading bacteria from potato rhizosphere

In many Gram-negative bacteria, including a number of pathogens such asPectobacterium carotovorum, virulence factor production and many functions are linked to the quorum sensing (QS) systems that use diffusible N-acyl homoserine lactones (AHLs) as intercellular messenger molecules. A number of organisms also contain genes that hydrolyze AHLs into inactive products, thereby blocking the quorum-sensing systems. In this study, the diversity of bacteria that inactivate N-AHL signal in soil and potato rhizoshere was investigated. Among 139 isolated strains, eighteen N-AHL degrading isolates were finally identified as genera Bacillus, Arthrobacter, Mesorhizobium,Pseudomonas and Streptomyces using polyphasic approaches. All these isolates were capable to degraded both synthetic and natural N-AHL produced by Pectobacterium atrosepticum strain SM1. In quenching experiments selected isolates, especially Bacillussp. EM84, were markedly reduced the pathogenicity of PaSM1 in potato tubers and totally suppressed tissue maceration on potato tubers. These results reveal the diversity of the QS interfering bacteria in the rhizosphere and demonstrate the validity of targeting QS signal molecules to control pathogens with natural bacterial isolates. Key words: Quorum sensing, acyl-homoserin lactone, quorum quenching, biocontrol.

Soft Rot Disease Severity Is Affected by Potato Physiology and Pectobacterium taxa.

Pectobacterium species cause disease worldwide in many crop and ornamental plants, including potato. A new Pectobacterium subspecies, P. carotovorum subsp. brasiliensis was recently described in Brazil and later found in the United States, Israel, and South Africa. Its virulence traits and host range remain unknown. A comparison of three taxa commonly found on potato showed that both P. carotovorum subsp. carotovorum and subsp. brasiliensis are more aggressive in causing tuber and stem soft rot than P. atrosepticum. Also, despite bacterial growth inhibition in vitro of P. carotovorum subsp. carotovorum and P. atrosepticum strains by P. carotovorum subsp. brasiliensis, this new subspecies and P. carotovorum subsp. carotovorum are able to co-colonize in the same infected tissue. Both subspecies were motile in lesions. Pathogenesis assays showed that host ranges of all three overlap, but are not identical. The host ranges of individual strains of P. carotovorum subsp. carotovorum and subsp. brasiliensis are limited, whereas P. atrosepticum can macerate many plant species in addition to potato. There was high variability in virulence assays with potato tuber; thus physiological factors were investigated. Tuber size, maturity, and field location had significant effects on susceptibility to soft rot, with larger, more mature tubers being more susceptible.

Two mobilePectobacterium atrosepticumprophages modulate virulence

The Pectobacterium atrosepticum strain SCRI1043 genome contains two complete prophage sequences. One, ECA41, is Mu-like and is able to integrate into, and excise from, various genomic locations. The other, ECA29, is a P2 family prophage, and is also able to excise from the genome. Excision of both prophages is rare and we were unable to induce lysis of cultures. Deletion of the entire prophages, both separately and in combination, did not affect the growth rate or the secretion of plant cell wall-degrading enzymes, but swimming motility was decreased. The virulence of prophage deletion strains in the potato host was decreased.

Genes responsible for coronatine synthesis in Pseudomonas syringae present in the genome of soft rot bacteria

Primers for the PCR amplification of homologous genes encoding polyketide coronafacic acid and coronafacic ligase in the cells of Pectobacterium atrosepticum SCRI1043 (BX950851) were developed to study the presence of these genes in the genome of Pectobacterium sp. and Dickeya sp. Coronafacic ligase catalyses the formation of coronatine from polyketide coronafacic acid and coronamic acid. Coronatine is a toxin produced by Pseudomonas syringae and is one of the major virulence factors in this bacterium. This study using several strains of P. atrosepticum, P. carotovorum subsp. carotovorum and Dickeya sp. isolated in different countries, indicated that all strains of P. atrosepticum possess genes coding coronafacic acid (cfa gene cluster) and coronafacic ligase (cfl). However, these genes were present only in the genome of five out of 50 tested P. carotovorum subsp. carotovorum strains and two out of 34 strains of Dickeya sp. tested. The PCR products homologous to the sequence of cfa7 and cfl gene fragments were sequenced in order to check the level of homology between genes of P. atrosepticum, P. carotovorum subsp. carotovorum and Dickeya sp. The sequences of the gene fragments amplified from all P. atrosepticum strains were almost identical (100% and 99.97%, respectively). The homology of the sequences obtained for P. atrosepticum and sequences of five P. carotovorum subsp. carotovorum and two Dickeya sp. was lower, between 89.69% to 95.00% for the cfl gene fragment, and about 94% for the cfa7 gene fragment.

Biochemical and genetic diversity of pectolytic enterobacteria causing soft rot disease of potatoes in New Zealand

The diversity of 89 pectolytic enterobacteria collected from potatoes in New Zealand was investigated by comparing their physiological, biochemical and genetic characteristics with those of type strains of Pectobacterium and strains previously identified as isolates of the genus Dickeya. Biochemical and physiological analyses demonstrated that the predominant pectolytic enterobacterium present in New Zealand is Pectobacterium carotovorum subspecies carotovorum. Isolates of Pectobacterium atrosepticum and strains closely related to Dickeya were also detected. Species and subspeciesspecific PCR confirmed the identity of the majority of the P. carotovorum subsp. carotovorum and P. atrosepticum strains. However, several atypical isolates classified by Biolog as P. carotovorum subsp. carotovorum failed to grow at 37C or to amplify the pectate lyase-encoding gene (pel gene) characteristic of this subspecies. Restriction fragment length polymorphism (RFLP) analyses of the 16S–23S intergenic transcribed spacer (ITS) also identified atypical isolates with unique ITS-RFLP profiles. Further characterisation of the atypical strains, by phylogenetic analysis of their 16S rRNA sequences, clustered them into four groups distinct from pectolytic enterobacteria previously found on potato. Several of the atypical strains proved highly virulent on both tubers and stems of potato, suggesting that a variety of novel species or subspecies are responsible for soft rot of potatoes in New Zealand, including Pectobacterium wasabiae.

STUDIES ON PECTOLYTIC ERWINIA SPP. IN PORTUGAL REVEAL UNUSUAL STRAINS OF E. CAROTOVORA SUBSP. ATROSEPTICA

The bacterial pathogens Erwinia carotovora subsp. atroseptica, E. carotovora subsp. carotovora and E. chrysanthemi are responsible for soft rot diseases, affecting plants in the field and/or in storage. Thirty-one strains from Portugal and elsewhere were characterised by classical, fatty acid methyl ester (FAME) and molecular methods (MSP-PCR and BOX-PCR). Ten strains were identified as E. carotovora subsp. atroseptica, 10 as E. carotovora subsp. carotovora, and 11 as E. chrysanthemi by classical methods. Strains of E.c. subsp. carotovora and E. chrysanthemi were phenotypically, chemotaxonomically, and genetically highly variable. E. c. subsp. atroseptica strains isolated from Portuguese fields had low molecular variability, but FAME analysis revealed high phenotypic variability. Fatty acid profiles of the Portuguese strains were compared with 27 E. c. subsp. atroseptica profiles obtained from the Dutch Plant Protection Service (PD) collection. The Portuguese E. c. subsp. atroseptica strains clustered separately from PD E. c. subsp. atroseptica strains. However, the clusters are not distant enough for Portuguese strains to be included in separate subspecies. Therefore, these Portuguese E. carotovora subsp. atroseptica strains may constitute an ecotype occurring in Portugal.