Pleiotropic effects of N-acylhomoserine lactone synthase ExpI on virulence, competition, and transmission in Pectobacterium carotovorum subsp. carotovorum Pcc21.

BACKGROUND: Pectobacterium species are necrotrophic phytopathogenic bacteria that cause soft rot disease in economically important crops. The successful infection of host plants relies on interactions among virulence factors, competition, and transmission within hosts. Pectobacteria primarily produce and secrete plant cell-wall degrading enzymes (PCWDEs) for virulence. The regulation of PCWDEs is controlled by quorum sensing (QS). Thus, the QS system is crucial for disease development in pectobacteria through PCWDEs. RESULTS: In this study, we identified a Tn-insertion mutant, M2, in the expI gene from a transposon mutant library of P. carotovorum subsp. carotovorum Pcc21 (hereafter Pcc21). The mutant exhibited reduced production and secretion of PCWDEs, impaired flagellar motility, and increased sensitivity to hydrogen peroxide, resulting in attenuated soft rot symptoms in cabbage and potato tubers. Transcriptomic analysis revealed the down-regulation of genes involved in the production and secretion in the mutant, consistent with the observed phenotype. Furthermore, the Pcc21 wild-type transiently colonized in the gut of Drosophila melanogaster within 12 h after feeding, while the mutant compromised colonization phenotype. Interestingly, Pcc21 produces a bacteriocin, carocin D, to compete with other bacteria. The mutant exhibited up-regulation of carocin D-encoding genes (caroDK) and inhibited the growth of a closely related bacterium, P. wasabiae. CONCLUSION: Our results demonstrated the significance of ExpI in the overall pathogenic lifestyle of Pcc21, including virulence, competition, and colonization in plant and insect hosts. These findings suggest that disease outcome is a result of complex interactions mediated by ExpI across multiple steps. © 2023 Society of Chemical Industry.

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.

A Switch from Latent to Typical Infection during Pectobacterium atrosepticum-Tobacco Interactions: Predicted and True Molecular Players.

Phytopathogenic microorganisms, being able to cause plant diseases, usually interact with hosts asymptomatically, resulting in the development of latent infections. Knowledge of the mechanisms that trigger a switch from latent to typical, symptomatic infection is of great importance from the perspectives of both fundamental science and disease management. No studies to date have compared, at the systemic molecular level, the physiological portraits of plants when different infection types (typical and latent) are developed. The only phytopathogenic bacterium for which latent infections were not only widely described but also at least fluently characterized at the molecular level is Pectobacterium atrosepticum (Pba). The present study aimed at the comparison of plant transcriptome responses during typical and latent infections caused by Pba in order to identify and then experimentally verify the key molecular players that act as switchers, turning peaceful plant-Pba coexistence into a typical infection. Based on RNA-Seq, we predicted plant cell wall-, secondary metabolism-, and phytohormone-related genes whose products contributed to the development of the disease or provided asymptomatic plant-Pba interactions. By treatment tests, we confirmed that a switch from latent to typical Pba-caused infection is determined by the plant susceptible responses mediated by the joint action of ethylene and jasmonates.

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.

Pectobacterium jejuense sp. nov. Isolated from Cucumber Stem Tissue.

A single Pectobacterium-like strain named 13-115T was isolated from a specimen of diseased cucumber stem tissue collected on Jeju Island, South Korea. The strain presented a rod-like shape and was negative for Gram staining. When grown on R2A medium at 25 °C, strain 13-115T formed round, convex and white colonies. This strain showed growth at temperatures ranging from 10 to 30 °C and tolerated a pH range of 6-9. The strain could also tolerate NaCl concentrations up to 5%. Analysis of the 16S rRNA gene sequence revealed that strain 13-115T exhibited similarity of over 99% with Pectobacterium brasiliense, P. carotovorum, P. polaris, and P. parvum. By conducting multilocus sequence analyses using dnaX, leuS, and recA genes, a separate phylogenetic lineage was discovered between strain 13-115T and other members of the genus Pectobacterium. Moreover, the strain showed relatively low in silico DNA-DNA hybridization (<60.6%) and average nucleotide identity (ANI) (<94.9%) values with recognized Pectobacterium species. The isolate has a genome size of 5,069,478 bp and a genomic G + C content of 52.04 mol%. Major fatty acids identified in the strain included C16:0 (28.99%), summed feature 3 (C16:1 ω7c and/or C16:1 ω6c; 28.85%), and C18:1 ω7c (19.01%). Pathogenicity assay confirmed that the novel strain induced soft rot symptoms in cucumber plants and Koch's postulates were fulfilled. Molecular analysis and phenotypic data indicated that strain 13-115T could be classified as a new species within the Pectobacterium genus, which has been named Pectobacterium jejuense. The type strain is 13-115T (= KCTC 92800T = JCM 35940T).

Two species with a peculiar evolution within the genus Pectobacterium suggest adaptation to a new environmental niche.

Historically, research on Soft Rot Pectobacteriacea (SRP) has focused on economically important crops and ornamentals and knowledge of these bacteria outside the plant context remains poorly investigated. Recently, two closely related species Pectobacterium aquaticum and Pectobacterium quasiaquaticum were isolated from water and have not been isolated from any plant yet. To identify the distinctive characteristics of these two species, we performed a comparative genomic analysis of 80 genomes representing 19 Pectobacterium species and performed an evolutionary reconstruction. Both water species underwent a reduction in genome size associated with a high pseudogene content. A high gene loss was predicted at the emergence of both species. Among the 199 gene families missing from both P. aquaticum and P. quasiaquaticum genomes but present in at least 80% of other Pectobacterium genomes, COG analysis identified many genes involved in nutrient transport systems. In addition, many type II secreted proteins were also missing in both species. Phenotypic analysis revealed that both species had reduced pectinolytic activity, a biofilm formation defect, were highly motile and had reduced virulence on several plants. These genomic and phenotypic data suggest that the ecological niche of P. aquaticum and P. quasiaquaticum may differ from that of other Pectobacterium species.

Investigation of the Role of a Zinc Uptake Regulator (Zur) in the Virulence of Pectobacterium odoriferum.

Pectobacterium spp. infect many horticultural crops worldwide and lead to serious crop losses. Zinc-uptake-regulator (Zur) proteins are present widely in prokaryotes and play an important role in pathogenicity. To uncover the role of Zur in P. odoriferum, we constructed mutant (ΔZur) and overexpression [Po (Zur)] strains of a Zur, and a virulence assay showed that the Po (Zur) was of significantly lower virulence, while the ΔZur displayed significantly increased virulence on Chinese cabbage compared to their respective control strains, wild-type P. odoriferum (Po WT) and P. odoriferum harboring an empty vector (Po (EV)) (p < 0.05). The growth curves of the ΔZur and Po (Zur) showed no obvious differences from those of the control strains. Comparative transcriptome analysis showed that Zur overexpression in P. odoriferum induced differentially expressed genes (DEGs) related to flagellum and cell motility, while mutating Zur resulted in DEGs mainly corresponding to divalent-metal-ion transport and membrane transport. Phenotypic experiments on the Po (Zur) showed that flagellum numbers and cell motility were reduced in comparison with the control, while those of the ΔZur did not change. Collectively, these results show that the Zur negatively regulates the virulence of P. odoriferum and might function via a dual mechanism dependent on dose.

Unleashing the Influence of cAMP Receptor Protein: The Master Switch of Bacteriocin Export in Pectobacterium carotovorum subsp. carotovorum.

Pectobacterium carotovorum subsp. carotovorum (Pcc) is a Gram-negative phytopathogenic bacterium that produces carocin, a low-molecular-weight bacteriocin that can kill related strains in response to factors in the environment such as UV exposure or nutritional deficiency. The function of the catabolite activator protein (CAP), also known as the cyclic AMP receptor protein (CRP), as a regulator of carocin synthesis was examined. The crp gene was knocked out as part of the investigation, and the outcomes were assessed both in vivo and in vitro. Analysis of the DNA sequence upstream of the translation initiation site of carocin S3 revealed two putative binding sites for CRP that were confirmed using a biotinylated probe pull-down experiment. This study revealed that the deletion of crp inhibited genes involved in extracellular bacteriocin export via the flagellar type III secretion system and impacted the production of many low-molecular-weight bacteriocins. The biotinylated probe pull-down test demonstrated that when UV induction was missing, CRP preferentially attached to one of the two CAP sites while binding to both when UV induction was present. In conclusion, our research aimed to simulate the signal transduction system that controls the expression of the carocin gene in response to UV induction.

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.

Occurrence, Characteristics, and qPCR-Based Identification of Pectobacterium versatile Causing Soft Rot of Chinese Cabbage in China.

Pectobacterium is one of the most important genera of phytopathogenic bacteria. It can cause soft-rot diseases on a wide range of plant species across the world. In this study, three Pectobacterium strains (KC01, KC02, and KC03) were isolated from soft-rotted Chinese cabbage in Beijing, China. These three strains were identified as Pectobacterium versatile based on phylogenetic analysis of Pectobacterium 16S ribosomal RNA, pmrA, and 504 Pectobacterium core genes, as well as a genomic average nucleotide identity analysis. Their biochemical characteristics were found to be similar to the P. versatile type strain ICMP9168T but differed in response to citric acid, stachyose, D-glucuronic acid, dextrin, and N-acetyl-ß-D-mannosamine. All of the tested P. versatile strains showed different carbohydrate utilization abilities compared with P. carotovorum and P. odoriferum, particularly in their ability to utilize D-arabitol, L-rhamnose, and L-serine. Under laboratory conditions, the maceration ability of P. versatile on Chinese cabbage was the highest at 28°C, compared with those at 13, 28, 23, and 33°C. Additionally, P. versatile could infect all of the 17 known Pectobacterium host plants, except for Welsh onion (Allium fistulosum). A SYBR Green quantitative PCR (qPCR) detection system was developed to distinguish P. versatile from other soft-rot bacteria based on the combined performance of melting curve (with a single melting peak at around 85°C) and fluorescence curve (with cycle threshold <30) when the bacterial genomic DNA concentration was in the range of 10 pg/µl to 10 ng/µl. This study is the first to report the presence of P. versatile on Chinese cabbage in China, as well as a specific and sensitive qPCR assay that can be used to quickly identify P. versatile. The work contributes to a better understanding of P. versatile and will facilitate the effective diagnosis of soft-rot disease, ultimately benefitting commercial crop production.

A Report of Pectobacterium carotovorum subsp. actinidiae Causing Kiwifruit Bacterial Canker in Guangxi, China.

A bacterial pathogen strain was isolated from susceptible tissue of Hongyang variety kiwifruit in Zhongfeng Town, Ziyuan County, Guilin City, Guangxi, China. Due to the relatively single variety of kiwifruit in Guangxi, the control technology of fruit farmers is backward, and the climate is humid, which is suitable for the growth of pathogenic bacteria, resulting in frequent occurrence of diseases. In this study, the pathogen strain was identified based on morphological, physiological, and biochemical tests; 16S rRNA gene; PCR detection with specific primers; and Biolog analysis. The results showed that a tobacco allergic reaction could be induced by inoculation with the pathogenic bacteria. Additionally, brown necrotic plaques appeared on kiwifruit leaves, necrotic phloem lesions appeared, and wounds on kiwifruit branches turned brown. The characteristics identified by morphological, physiological, biochemical, and Biolog identification were similar to those caused by Pectobacterium sp. Through 16S rRNA gene sequence analysis and PCR identification with specific primers, bands with a size corresponding to target bands indicated that the pathogen was Pectobacterium carotovorum subsp. actinidiae. This is the first report of kiwifruit canker disease caused by P. carotovorum subsp. actinidiae in Guangxi, China. In addition, through this study, a preliminary understanding of the pathogen has been obtained, which will lay the foundation for the prevention and control of the disease in the future.

Complete Genome Sequence Resource of Pectobacterium colocasium Strain F1-1 that Causes Soft Rot Disease of Taro.

Pectobacterium colocasium is a recently named, narrow-host-range phytopathogenic bacterium causing soft rot of taro (Colocasium esculenta). It is found on the Chinese mainland and the island of Taiwan. Taro is a domesticated crop with a long history of cultivation in Taiwan and the Pacific islands. However, not much was known about Pectobacterium spp. from taro, especially from the islands in the Pacific. Herein, we report a high-quality, completely annotated genome sequence of P. colosacium strain F1-1. The 4,816,345 bp genome, which was assembled with Illumina and Nanopore reads with 217× and 311× coverage, respectively, consists of one chromosome and no plasmid. This completely circularized genome will aid future studies in comparative genomics, evolution, and pathogenicity of P. colocasium. This genome resource will also be helpful for developing strategies to control P. colocasium in taro.[Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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.

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.

Pectobacterium versatile Bacteriophage Possum: A Complex Polysaccharide-Deacetylating Tail Fiber as a Tool for Host Recognition in Pectobacterial Schitoviridae.

Novel, closely related phages Possum and Horatius infect Pectobacterium versatile, a phytopathogen causing soft rot in potatoes and other essential plants. Their properties and genomic composition define them as N4-like bacteriophages of the genus Cbunavirus, a part of a recently formed family Schitoviridae. It is proposed that the adsorption apparatus of these phages consists of tail fibers connected to the virion through an adapter protein. Tail fibers possess an enzymatic domain. Phage Possum uses it to deacetylate O-polysaccharide on the surface of the host strain to provide viral attachment. Such an infection mechanism is supposed to be common for all Cbunavirus phages and this feature should be considered when designing cocktails for phage control of soft rot.

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.

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.

Genetic and Phenotypic Study of the Pectobacterium versatile Beta-Lactamase, the Enzyme Most Similar to the Plasmid-Encoded TEM-1.

Genus Pectobacterium bacteria include important agricultural pathogens. Pectobacterium versatile isolates contain a chromosome-borne beta-lactamase, PEC-1. This enzyme is the closest relative of TEM-1, a plasmid-borne beta-lactamase widespread in the Enterobacterales. We performed bioinformatics and phenotypic analyses to investigate the genetic and phenotypic features of PEC-1 and its frequency and ability to spread within genus Pectobacterium. We also compared the characteristics of PEC-1 and TEM-1 and evaluated the likelihood of transfer. We found that blaPEC-1 was present principally in a small number of genetic environments in P. versatile. Identical blaPEC-1 genetic environments were present in closely related species, consistent with the high frequency of genetic exchange within the genus Pectobacterium. Despite the similarities between PEC-1 and TEM-1, their genetic environments displayed no significant identity, suggesting an absence of recent transfer. Phenotypic analyses on clonal constructs revealed similar hydrolysis spectra. Our results suggest that P. versatile is the main reservoir of PEC-1, which seems to transfer to closely related species. The genetic distance between PEC-1 and TEM-1, and the lack of conserved elements in their genetic environments, suggest that any transfer that may have occurred must have taken place well before the antibiotic era. IMPORTANCE This study aimed to compare the chromosomal beta-lactamase from Pectobacterium versatile, PEC-1, with the well-known and globally distributed TEM-1 in terms of genetic and functional properties. Despite the similarities between the enzymes, we obtained no definitive proof of gene transfer for the emergence of blaPEC-1 from blaTEM-1. Indeed, given the limited degree of sequence identity and the absence of a common genetic environment, it seems unlikely that any transfer of this gene has occurred recently. However, although blaPEC-1 was found mostly in one specific clade of the P. versatile species, certain isolates from other closely related species, such as Pectobacterium brasiliense and Pectobacterium polaris, may also carry this gene inserted into common genetic environments. This observation suggests that genetic exchanges are frequent, accounting for the diffusion of blaPEC-1 between isolates from different Pectobacterium species and, potentially, to exogenous mobile genetic elements.

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.