Pectobacterium Species Research Articles

First Report of Pectobacterium aroidearum Causing Soft Rot in Olecranon Honey Peach (Prunus persica) in China.

Olecranon honey peach (Prunus persica L.) is a popular fruit tree cultivated in Guangdong Province of China. Due to its excellent economic values and popularity, it has recently been widely adopted and planted in several other southern Provinces and Autonomous Region in China, including Yunnan, Hunan, Jiangxi, Guizhou, and Guangxi. In Lianping County of Guangdong Province alone, the annual peach fruit production was about 78,800 tonnes (Xie et al. 2017). In July 2021, peach fruits showing soft rot symptoms were collected from an olecranon honey peach plantation in Lechang, Guangdong, China. Symptoms included tissue disintegration with bacterial oozes and rotting smells. To isolate the causal agent of soft rot in the peach fruits, the bacterial oozes from various rotted fruits were streaked on the modified YEB agar plate (Huang et al. 2021), and 21 bacterial colonies were selected for PCR amplification using the primers targeting the conserved region of 16S rDNA gene (Wei et al. 2020). A blastN analysis of the DNA sequences of the obtained PCR fragments in NCBI website indicated that 17 isolates named as ZL strains were potential bacterial species of Pectobacterium with about 99% similarity (Genbank accession number of ZL1: OK189602) to Pectobacterium aroidearum SCRI 109T (Genbank accession number: NR_159926). Three of them (ZL1, ZL2 and ZL3) were selected for assay of pathogenicity. The bacterial suspensions (10 μl, 1×106 CFU/ml) of strains ZL1, ZL2 and ZL3 were injected into olecranon honey peach fruits by using a syringe. A portion of peach fruits were similarly injected with sterile distilled water as the negative control. After 18 h incubation at 25 °C, the typical symptom of soft rot, i.e., tissue decay, became visible on the peach fruits inoculated with the bacterial suspensions. After inoculation for 42 h, bacterial oozes were exuded from rotting tissues. Peach fruits without injuries were also sprayed with the bacterial suspensions under the same conditions, but decay symptoms were not observed, suggesting that the bacterial infection needs the wounding or injuries. To fulfill the Koch's postulates, bacterial colonies were re-isolated from bacterial oozes, and their conserved region of 16S rDNA fragments were amplified and sequenced. Bioinformatics analysis of the DNA sequence data confirmed that all the isolated colonies were Pectobacterium strains. Using the Biolog Gen III system, the representative strain ZL1 was identified as Pectobacterium (SIM 0.56). Transmission electron microscopy analysis showed that the bacterial cells of strain ZL1 were rod-shaped with peripheral flagella. To further determine the species of ZL strains, eight housekeeping genes (acnA, gapA, icd, mdh, mtlD, pgi, proA and rpoS) were analyzed by the methods described previously (Nabhan et al. 2013). The amplified DNA sequences analyzed by the blastN program in NCBI showed that the sequences of eight housekeeping genes from strains ZL1, ZL2 and ZL3 were identical to each other (Genbank accession number: OK274248 to OK274255), and most of the gene sequences shared over 99% similarity to their counterparts in P. aroidearum L6 (Genbank accession number: NZ_CP065044) (Xu et al. 2021), except that the acnA and proA genes showed about 98% and 96% similarity respectively to the corresponding genes of P. aroidearum L6. In addition, the multi-locus sequence analysis (MLSA) using DNA sequences of above eight housekeeping genes showed that ZL strains were grouped with other P. aroidearum strains. Taken together, the results of molecular and biochemical assays confirmed that ZL strains isolated from olecranon honey peach fruits were P. aroidearum. To our knowledge, this is the first report of P. aroidearum causing soft rot disease in olecranon honey peach in China. P. aroidearum is a relatively newly described soft rot pathogen (Nabhan et al. 2013). More recently, the pathogen was found causing soft rot infections in lettuce, Chinese cabbage, pepper (Capsicum annuum) fruits, konjac, carrot and Syngonium podophyllum (Barroso et al. 2019; Moraes et al. 2020; Sun et al. 2019; Tang et al. 2020; Xu et al. 2021). The results of this study add a new plant species to the host range of P. aroidearum.

High-Quality Complete Genome Resource of Pectobacterium parvum Isolate FN20211 CausingAerial Stem Rot of Potato.

High-Quality Complete Genome Resource of Pectobacterium parvum Isolate FN20211 CausingAerial Stem Rot of Potato.

Selection and evaluation of suitable reference genes for quantitative gene expression analysis during infection of Cucumis sativus with Pectobacterium brasiliense.

Bacterial soft rot caused by Pectobacterium brasiliense (Pbr) has resulted in severe economic losses of cucumber production in northern China. Quantitative reverse transcription PCR (RT-qPCR) is widely used to determine the fold change in the expression of genes of interest, and an appropriate reference gene played a critical role in the evaluation of genes expression. However, the suitable reference genes for transcript normalization during the interaction between cucumber and Pbr have not yet been systematically validated. In this study, we aimed to identify the suitable reference genes for accurate and reliable normalization of cucumber and Pbr RT-qPCR data. We selected 14 candidate reference genes for cucumber and 10 candidate reference genes for Pbr were analysed by using four algorithms (the deltaCt method, BestKeeper, NormFinder and geNorm). Furthermore, five genes in cucumber involved in plant resistance and five genes in Pbr related to the virulence were selected to confirm the reliability of the reference genes by RT-qPCR. CsARF (ADP-ribosylation factor 1) and pgi (glucose-6-phosphate isomerase) were suggested as the most suitable reference genes for cucumber and Pbr respectively. Our results suggested that CsARF (ADP-ribosylation factor 1) and pgi (glucose-6-phosphate isomerase) could be as the reference genes to normalize expression data for cucumber and Pbr during the process of pathogen-host interaction respectively. To our knowledge, this is the first systematic study of the optimal reference genes specific to cucumber and Pbr, which could help advance the molecular interactions research in Cucurbitaceae vegetables and Pectobacterium species pathosystems.

Isolation and Genome Analysis of Pectobacterium colocasium sp. nov. and Pectobacterium aroidearum, Two New Pathogens of Taro.

Bacterial soft rot is one of the most destructive diseases of taro (Colocasia esculenta) worldwide. In recent years, frequent outbreaks of soft rot disease have seriously affected taro production and became a major constraint to the development of taro planting in China. However, little is known about the causal agents of this disease, and the only reported pathogens are two Dickeya species and P. carotovorum. In this study, we report taro soft rot caused by two novel Pectobacterium strains, LJ1 and LJ2, isolated from taro corms in Ruyuan County, Shaoguan City, Guangdong Province, China. We showed that LJ1 and LJ2 fulfill Koch’s postulates for taro soft rot. The two pathogens can infect taro both individually and simultaneously, and neither synergistic nor antagonistic interaction was observed between the two pathogens. Genome sequencing of the two strains indicated that LJ1 represents a novel species of the genus Pectobacterium, for which the name “Pectobacterium colocasium sp. nov.” is proposed, while LJ2 belongs to Pectobacterium aroidearum. Pan-genome analysis revealed multiple pathogenicity-related differences between LJ1, LJ2, and other Pectobacterium species, including unique virulence factors, variation in the copy number and organization of Type III, IV, and VI secretion systems, and differential production of plant cell wall degrading enzymes. This study identifies two new soft rot Pectobacteriaceae (SRP) pathogens causing taro soft rot in China, reports a new case of co-infection of plant pathogens, and provides valuable resources for further investigation of the pathogenic mechanisms of SRP.

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

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. 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 10fg genomic DNA and 1CFUml-1 bacterial culture. No change in the detection threshold (1CFUml-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. 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. The developed assay can be adopted for multiple purposes such as seed certification programmes, surveillance, biosecurity, microbial forensics, quarantine, border protection, inspections and epidemiology.

First Report of Pectobacterium versatile Causing Potato Soft Rot in Oregon and Washington.

Potato (Solanum tuberosum cv. Norkotah) tubers with symptoms of soft rot were submitted to Oregon State University, Hermiston Agricultural Research and Extension Center Plant Clinic in 2019. One submission in May, originated from a field with poor emergence and seed piece decay (~20% affected) in Umatilla County, Oregon. The second submission, in September, originated from a field in Washington. From each submission, ~100 mg tissue at the margin of infection was washed with distilled water, excised, macerated in 500 L sterile distilled water for 5 minutes. The resulting solution was streaked on crystal violet pectate (CVP) medium and incubated at 28°C for 24 hours. One colony, representative of the many white colonies that formed depressions on CVP plates, was isolated from each submission. Bacterial isolates from Oregon and Washington were named JB56A and JB133A, respectively, and preserved in Luria-Bertani (LB) broth with 15% glycerol at -80°C for long-term storage. Genomic DNA was extracted from JB56A and JB133A cultures grown in LB broth overnight at 30°C using the Wizard SV Genomic DNA kit. The partial dnaX gene (537 bp) was amplified from genomic DNA of each isolate using dnaXf/dnaXr primers (Slawiak et al. 2009) and sequenced. These sequences were deposited to the NCBI GenBank Database, accession numbers MW930747 (JB56A) and MW930748 (JB133A). BLAST analyses (Altschul et al. 1990) using default parameters indicated that the dnaX sequences of JB56A and JB133A were 99.2% (533/537) and 98.7% (530/537) identical to that of P. versatile SCC1 (CP021894). A condensed maximum likelihood tree was built using the partial dnaX sequence of the two query strains, twelve Pectobacterium reference strains to include all known species of Pectobacterium, and four Dickeya species as an outgroup (Fig. S1). JB56A and JB133A formed a monophyletic clade with P. versatile SCC1. Potato (cv. Upstate Abundance) tuber and stem bioassays (Ma et al. 2018) were conducted twice to assess the pathogenicity of these isolates. Tubers were wounded with a sterile 2 mm wide wooden applicator stick and 5 μl culture grown in LB broth overnight (~109 CFU) was pipetted into the wound. Tubers were incubated at 29°C for 24 hours and cut through puncture sites to observe symptoms. Stems of four- or five-week-old plants were wounded with a sterile toothpick about 10 cm above the soil line and a smear of JB56A or JB133A grown on LB agar was inserted into the wound using a toothpick and incubated in a greenhouse for 72 hours. Positive controls (D. dianthicola ME23) and negative controls (no bacteria) were included in both assays. Tubers and stems exhibited disease symptoms after 24 and 72 hours, respectively, following inoculation with JB56A, JB133A, and D. dianthicola ME23. No symptoms were observed for negative controls. The identity of bacteria re-isolated from the margin of stem lesions was confirmed by partial dnaX sequence analyses. P. versatile was recently described as a distinct species based on whole genome sequence comparisons (Portier et al. 2019). In 2018, we isolated P. versatile from potato stems with blackleg disease in New York, and a recent study found that it was isolated in the US from an iris in 1946 (Ma et al. 2021; Portier et al. 2019). However, the geographic distribution and importance of this pathogen in the US remains largely unknown. To our knowledge, this is the first report of potato soft rot caused by P. versatile in Oregon and Washington, two important potato producing states.

Comparative Genomic Analysis of Pathogenic Factors of Pectobacterium Species Isolated in South Korea Using Whole-Genome Sequencing.

In this study, we conducted whole-genome sequencing with six species of Pectobacterium composed of seven strains, JR1.1, BP201601.1, JK2.1, HNP201719, MYP201603, PZ1, and HC, for the analysis of pathogenic factors associated with the genome of Pectobacterium. The genome sizes ranged from 4,724,337 bp to 5,208,618 bp, with the GC content ranging from 50.4% to 52.3%. The average nucleotide identity was 98% among the two Pectobacterium species and ranged from 88% to 96% among the remaining six species. A similar distribution was observed in the carbohydrate-active enzymes (CAZymes) class and extracellular plant cell wall degrading enzymes (PCWDEs). HC showed the highest number of enzymes in CAZymes and the lowest number in the extracellular PCWDEs. Six strains showed four subsets, and HC demonstrated three subsets, except hasDEF, in type I secretion system, while the type II secretion system of the seven strains was conserved. Components of human pathogens, such as Salmonella pathogenicity island 1 type type III secretion system (T3SS) and effectors, were identified in PZ1; T3SSa was not identified in HC. Two putative effectors, including hrpK, were identified in seven strains along with dspEF. We also identified 13 structural genes, six regulator genes, and five accessory genes in the type VI secretion system (T6SS) gene cluster of six Pectobacterium species, along with the loss of T6SS in PZ1. HC had two subsets, and JK2.1 had three subsets of T6SS. With the GxSxG motif, the phospholipase A gene did locate among tssID and duf4123 genes in the T6SSa cluster of all strains. Important domains were identified in the vgrG/paar islands, including duf4123, duf2235, vrr-nuc, and duf3396.

New PCR-Based Assay for the Identification of Pectobacterium carotovorum Causing Potato Soft Rot

Soft rot on potato tuber is a destructive disease caused by pathogenic bacterial species of the genera Pectobacterium and Dickeya. Accurate identification of the causal agent is necessary to ensure adequate disease management because different species may have distinct levels of aggressiveness and host range. One of the most important potato pathogens is Pectobacterium carotovorum, a highly heterogeneous species capable of infecting multiple hosts. The complexity of this species, until recently divided into several subspecies, has made it difficult to develop precise diagnostic tests. This study proposes a PCR assay based on the new pair of primers Pcar1F/R to facilitate the identification of potato isolates of P. carotovorum according to the most recent taxonomic description of this species. The new primers were designed on a variable segment of the 16S rRNA gene and the intergenic spacer region of available DNA sequences from classical and recently established species in the genus Pectobacterium. The results of the PCR analysis of genomic DNA from 32 Pectobacterium and Dickeya strains confirmed that the Pcar1F/R primers have sufficient nucleotide differences to discriminate between P. carotovorum and other Pectobacterium species associated with damage to potato crops, with the exception of Pectobacterium versatile, which improves the specificity of the currently available primers. The proposed assay was originally developed as a conventional PCR but was later adapted to the real-time PCR format for application in combination with the existing real-time PCR test for the potato-specific pathogen Pectobacterium parmentieri. This should be useful for the routine diagnosis of potato soft rot.

First Report of Pectobacterium versatile Causing Aerial Stem Rot of Potato in China.

Pectobacterium species cause blackleg, soft rot and stem rot in potato and many other vegetable crops (Charkowski 2015). In July 2020, potato plants showing characteristic symptoms of aerial stem rot were observed in a field (cv. Xisen 6) in Fengning Manchu Autonomous County, Chengde, Hebei Province (North China). The disease incidence in that field (5 ha in size) was more than 50%. Putative pectolytic bacteria were obtained from symptomatic stem tissues (light brown and water-soaked stem sections) by culturing on the crystal violet pectate (CVP) medium. Bacterial colonies producing pits, were restreaked and purified on Luria-Bertani (LB) agar. The isolates causing stem rot were gram negative and rod shaped, negative for oxidase, urease, indole production, gelatin liquefaction and acid production from maltose and D-sorbitol. All isolates were catalase positive, produced acid from lactose, rhamnose, saccharose, raffinose and D-arabinose, and were tolerant to 5% NaCl, and able to utilize citrate. The bacterial gDNA was extracted using the EasyPure Bacteria Genomic DNA Kit (TransGen Biotech). The 16S rDNA region was amplified by PCR using the universal primer pair 27F/1492R and sequenced. Result of the Blastn analysis of the 16S rDNA amplicons (MZ379788, MZ379789) suggested that the isolates FN20111 and FN20121 belonged to the genus Pectobacterium. To determine the species of the stem rot Pectobacterium isolates, multi-locus sequence analysis (MLSA) was performed with six housekeeping genes acnA, gapA, icdA, mdh, proA and rpoS (MZ403781-MZ403792), and phylogenetic tree was reconstructed using RAxML v8.2.12 (https://github.com/stamatak/standard-RAxML). The result of phylogenetic analysis showed that the stem rot Pectobacterium isolates FN20111 and FN20121 clustered with P. versatile (syn. 'Candidatus Pectobacterium maceratum') strains CFBP6051T (Portier et al. 2019), SCC1 (Niemi et al. 2017) and F131 (Shirshikov et al. 2018). And the isolates FN20111 and FN20121 were more closely related to the type strain CFBP6051T than to strains SCC1 and F131. Potato seedlings (cv. Xisen 6 and Favorita) were inoculated with the isolates FN20111 and FN20121 by injecting 100 µl of bacterial suspensions (108 CFU·mL-1) into the upper parts of the stems of potato plants, or injected with 100 µl of 0.9% saline solution as control. The seedlings were grown at 28°C and 50% relative humidity. Three days post-inoculation, only the bacteria-inoculated seedlings showed diseased symptoms resembling to those observed in the field. Bacterial colonies were obtained from the infected stems and were identified using the same PCR primers of housekeeping genes as described above, fulfill Koch's postulates. P. versatile causing soft rot and blackleg on potato plants has been reported in Finland (Niemi et al. 2017), Russia (Shirshikov et al. 2018), Netherlands (Portier et al. 2019), Poland (Waleron et al. 2019) and in New York State (Ma et al. 2021). To our knowledge, this is the first report of P. versatile causing aerial stem rot of potato in China.

Pathogenicity and Relative Abundance of Dickeya and Pectobacterium Species in Switzerland: An Epidemiological Dichotomy.

Pectobacterium and Dickeya species are the causal agents of blackleg and soft rot diseases in potatoes. The main pathogenic species identified so far on potatoes are Dickeya dianthicola, Dickeya solani, Pectobacterium atrosepticum, Pectobacterium brasiliense, Pectobacterium carotovorum, and Pectobacterium parmentieri. Ten years ago, the most prevalent Soft Rot Pectobacteriaceae in Europe were the Dickeya species, P. atrosepticum and P. carotovorum, with some variations among countries. Since then, a drastic increase in the abundance of P. brasiliense has been observed in most European countries. This shift is difficult to explain without comparing the pathogenicity of all Dickeya and Pectobacterium species. The pathogenicity of all the above-mentioned bacterial species was assessed in field trials and in vitro tuber slice trials in Switzerland. Two isolates of each species were inoculated by soaking tubers of cv. Desiree in a suspension of 105 CFU/mL, before planting in the field. For all trials, the Dickeya species were the most virulent ones, but long-term strain surveys performed in Switzerland indicate that P. brasiliense is currently the most frequent species detected. Our results show that the pathogenicity of the species is not the main factor explaining the high prevalence of P. brasiliense and P. parmentieri in the Swiss potato fields.

Quantitative Real-Time PCR Assay for the Detection of Pectobacterium parmentieri, a Causal Agent of Potato Soft Rot.

Pectobacterium parmentieri is a plant-pathogenic bacterium, recently attributed as a separate species, which infects potatoes, causing soft rot in tubers. The distribution of P. parmentieri seems to be global, although the bacterium tends to be accommodated to moderate climates. Fast and accurate detection systems for this pathogen are needed to study its biology and to identify latent infection in potatoes and other plant hosts. The current paper reports on the development of a specific and sensitive detection protocol based on a real-time PCR with a TaqMan probe for P. parmentieri, and its evaluation. In sensitivity assays, the detection threshold of this protocol was 102 cfu/mL on pure bacterial cultures and 102–103 cfu/mL on plant material. The specificity of the protocol was evaluated against P. parmentieri and more than 100 strains of potato-associated species of Pectobacterium and Dickeya. No cross-reaction with the non-target bacterial species, or loss of sensitivity, was observed. This specific and sensitive diagnostic tool may reveal a wider distribution and host range for P. parmentieri and will expand knowledge of the life cycle and environmental preferences of this pathogen.

Characterization of Pectobacterium brasiliense strains from potato and vegetables in Israel

AbstractPectobacterium brasiliense (Pbr) infects a wide range of crops worldwide, causing potato blackleg and soft rot and vegetable soft rots. This study aimed to characterize the genetic diversity and virulence variability among 68 Pbr strains isolated from either symptomless potato progeny tubers, diseased potato plants, ware potatoes wash water, or vegetables grown in Israel, as well as strains isolated from symptomless seed tubers grown in Europe, or diseased potato plants grown in France. The collection was typed using PCR and TaqMan real‐time PCR analyses, dnaX sequence analysis, pulsed‐field gel electrophoresis (PFGE), and pectolytic activity. dnaX phylogeny grouped almost all strains in a common genetic clade related to Pbr, which was distinct from the other Pectobacterium species. PFGE analysis identified two main clusters, including one major group of 47 strains with 95%–100% similarity. Maceration assays on two potato cultivars showed significant differences between strains but with no correlations with the source of the strains nor the status of the host (with/without symptoms). Molecular (dnaX sequences and PFGE profiles) and phenotypic analyses (tuber maceration tests) showed that the tested Pbr strains are not a homogeneous group. Analysis of the tested Pbr strains isolated from potato and vegetables grown in fields with a history of potato cultivation suggests that seed tubers imported from Europe may be the main source for Pbr in Israel. To the best of our knowledge, this is the first study that describes biodiversity and population structure of P. brasiliense isolated from potato and vegetables under hot climate conditions.

Species of Dickeya and Pectobacterium Isolated during an Outbreak of Blackleg and Soft Rot of Potato in Northeastern and North Central United States.

An outbreak of bacterial soft rot and blackleg of potato has occurred since 2014 with the epicenter being in the northeastern region of the United States. Multiple species of Pectobacterium and Dickeya are causal agents, resulting in losses to commercial and seed potato production over the past decade in the Northeastern and North Central United States. To clarify the pathogen present at the outset of the epidemic in 2015 and 2016, a phylogenetic study was made of 121 pectolytic soft rot bacteria isolated from symptomatic potato; also included were 27 type strains of Dickeya and Pectobacterium species, and 47 historic reference strains. Phylogenetic trees constructed based on multilocus sequence alignments of concatenated dnaJ, dnaX and gyrB fragments revealed the epidemic isolates to cluster with type strains of D. chrysanthemi, D. dianthicola, D. dadantii, P. atrosepticum, P. brasiliense, P. carotovorum, P. parmentieri, P. polaris, P. punjabense, and P. versatile. Genetic diversity within D. dianthicola strains was low, with one sequence type (ST1) identified in 17 of 19 strains. Pectobacterium parmentieri was more diverse, with ten sequence types detected among 37 of the 2015–2016 strains. This study can aid in monitoring future shifts in potato soft rot pathogens within the U.S. and inform strategies for disease management.

Antibacterial Properties of Citric Acid/β-Alanine Carbon Dots against Gram-Negative Bacteria.

While multi-drug resistance in bacteria is an emerging concern in public health, using carbon dots (CDs) as a new source of antimicrobial activity is gaining popularity due to their antimicrobial and non-toxic properties. Here we prepared carbon dots from citric acid and β-alanine and demonstrated their ability to inhibit the growth of diverse groups of Gram-negative bacteria, including E. coli, Salmonella, Pseudomonas, Agrobacterium, and Pectobacterium species. Carbon dots were prepared using a one-pot, three-minute synthesis process in a commercial microwave oven (700 W). The antibacterial activity of these CDs was studied using the well-diffusion method, and their minimal inhibitory concentration was determined by exposing bacterial cells for 20 h to different concentrations of CDs ranging from 0.5 to 10 mg/mL. Our finding indicates that these CDs can be an effective alternative to commercially available antibiotics. We also demonstrated the minimum incubation time required for complete inhibition of bacterial growth, which varied depending on bacterial species. With 15-min incubation time, A. tumefaciens and P. aeruginosa were the most sensitive strains, whereas E. coli and S. enterica were the most resistant bacterial strains requiring over 20 h incubation with CDs.

First report of soft rot of cabbage caused by Pectobacterium wasabiae in Japan.

Cabbage (Brassica oleracea var. capitata) is one of the important vegetables in Japan. In the summer of 2019, some cabbages with soft rot were found in commercial fields in Hokkaido, the northern island in Japan. All diseased plants showed grey to brown discoloration and expanding water-soaked lesions on leaves. We obtained two independent strains (NACAB191 and NACAB192) from diseased leaves. DNA from these strains yielded an expected single size amplicon with the primer set of PhF/PhR for P. wasabiae (De Boer et al. 2012) by PCR, but did not yield the expected amplicon with the primer set of BR1f/L1r for P. carotovorum subsp. brasiliense (Duarte et al. 2004) and Eca1f/Eca2r for P. atrosepticum (De Boer et al., 1995) by PCR. These two strains grew at 37°C, and their ability to utilize raffinose and lactose. These bacterial strains were gram-negative and rod-shaped. The bacterium was positive for O-nitrophenyl-beta-D-galactopyranoside, N-acetylglucosaminyl transferase, gelatin liquefaction, and acid production from D-galactose, lactose, melibiose, raffinose, citrate, and trehalose. The bacterium was negative for indole production and acid production from maltose, α-methyl-D-glucoside, sorbitol, D-arabitol, inositol, inulin, and melezitose. All strains exhibited pectolytic activity on potato slices. The sequence analysis of 16S rDNA (LC597897 and LC597898) showed more than 98% identities to P. wasabiae strain (e.g. HAFL01 in Switzerland) by BLAST analysis. In addition, Multi-locus sequence analysis (Ma et al. 2007) was performed by MEGA10 (Kumer et al. 2018) using concatenated DNA sequences of seven housekeeping genes (aconitate hydratase(acnA, LC597923 and LC597924), glyceraldehyde-3-phosphate dehydrogenase A(gapA, LC597970 and LC597971), isocitrate dehydrogenase (icdA, LC597996 and LC597997), malate dehydrogenase(mdh, LC598022 and LC598023), mannitol-1-phosphate dehydrogenase (mtlD, LC598048 and LC598049), glucose-6-phosphate isomerase (pgi, LC598074 and LC598075) and gamma-glutamyl phospate reductase (proA, LC598079 and LC598080)), and all clustered NACAB191 and NACAB192 into a clade containing other confirmed strains of P. wasabiae. As a result, these two strains shared high identity with each other (>98%, E-Values showed 0). The clade containing these two strains was consistently placed in a larger clade with the other P. wasabiae and 100% bootstrap support for its separation from other Pectobacterium species available in GenBank when the consensus tree constructed using Maximum Likelihood method. Pathogenicity of these strains against cabbage (cv. 'Rakuen') was confirmed by the field experiments with five weeks growth plants sprayed with bacterial suspension (1×107cfu/ml). Thirty cabbages per strain were used in this study, 12 plants treated the suspension of NACAB191 and 16 plants treated the suspension of NACAB192 which died with the same soft rot symptoms about four weeks after inoculation. Whereas water-inoculated plants remained symptomless. Strains re-isolated from the artificially diseased stems were confirmed as P. wasabiae using the methods as biochemical characterization and multiple genetic analyses. Based on the disease symptoms, the cultural, molecular, and pathological features of the strains, we conclude that the soft rot symptoms of cabbage in Hokkaido in 2019 were caused by P. wasabiae. To our knowledge, this is the first report of P. wasabiae as the soft rot disease agent of cabbage in Japan.

First report of soft rot of onion caused by Pectobacterium wasabiae in Japan.

Onion (Allium cepa L.) is one of the important vegetables in Japan. In the summer of 2019, onions with soft rot were found in commercial fields in Hokkaido, the northern island in Japan. Diseased onion showed chlorosis, maceration of leaves, and rotted bulbs. We sampled some diseased onions and isolated three independent isolations (NAONI191, NAONI192 and NAONI193) from infected bulbs on LB medium. These strains were identified as Pectobacterium wasabiae based on their inability to grow at 37°C, and their ability to utilize raffinose and lactose. These bacterial strains were gram-negative, rod-shaped, N-acetylglucosaminyl transferase, gelatin liquefaction. The bacterium was positive for O-nitrophenyl-beta-D-galactopyranoside, N-acetylglucosaminyl transferase, gelatin liquefaction, and acid production from D-galactose, lactose, melibiose, raffinose, citrate, and trehalose. The bacterium was negative for indole production and acid production from maltose, α-methyl-D-glucoside, sorbitol, D-arabitol, inositol, inulin, and melezitose. All the strains exhibited pectolytic activity on potato slices. DNA from these strains yielded a single size amplicon with the primer set of PhF/PhR for P. wasabiae (De Boer et al. 2012) by PCR. However, DNA from these strains did not yield the expected amplicon with the primer set of BR1f/L1r for P. carotovorum subsp. brasiliense (Duarte et al. 2004) and Eca1f/Eca2r for P. atrosepticum (De Boer et al., 1995) by PCR. The sequence analysis of 16S rDNA (LC597917- LC597919) showed more than 98% identities to P. wasabiae strains (e.g. HAFL01 in Switzerland) by BLAST analysis. In addition, Multi-locus sequence analysis (Ma et al. 2007) was performed by MEGA6.06 using concatenated DNA sequences of seven housekeeping genes (aconitate hydratase(acnA, LC597925- LC597927), glyceraldehyde-3-phosphate dehydrogenase A(gapA, LC597972-LC597974), isocitrate dehydrogenase (icdA, LC597998- LC597998LC598000), malate dehydrogenase(mdh, LC598024- LC598026), mannitol-1-phosphate dehydrogenase (mtlD, LC598050- LC598052), glucose-6-phosphate isomerase (pgi, LC598076- LC598078) and gamma-glutamyl phospate reductase (proA, LC598099- LC598101)), and all clustered into a clade containing other confirmed strains of P. wasabiae. As a result, these three strains shared high identity with each other (>98%, E-Values showed 0). The clade containing these three strains was consistently placed in a larger clade with the other P. wasabiae and 100% bootstrap support for its separation from other Pectobacterium species available in GenBank when the consensus tree constructed using Maximum Likelihood method. Pathogenicity of these strains against onion (cv. 'Hayate') was confirmed by the field experiments with 5 weeks growth plants sprayed with bacterial suspension (1×107cfu/ml) resulting in soft rot on the plants about four weeks after inoculation, whereas water-inoculated plants remained symptomless. Strains re-isolated from the artificially diseased stems were confirmed as P. wasabiae using the methods as biochemical characterization and multiple genetic analyses. Based on the disease symptoms, the cultural, molecular, and pathological features of the strains, we conclude that the soft rot symptoms of onion in Hokkaido in 2019 were caused by P. wasabiae. To our knowledge, this is the first report of P. wasabiae as the soft rot disease agent of onion in Japan.

Diversity and Pathogenicity of Pectobacterium Species Responsible for Causing Soft Rot and Blackleg of Potato in the Columbia Basin

Diversity and Pathogenicity of Pectobacterium Species Responsible for Causing Soft Rot and Blackleg of Potato in the Columbia Basin

Analysis of the Taxonomy and Pathogenic Factors of Pectobacterium aroidearum L6 Using Whole-Genome Sequencing and Comparative Genomics.

Soft rot pectobacteria are devastating plant pathogens with a global distribution and a broad host range. Pectobacterium aroidearum L6, previously isolated from leaves of Syngonium podophyllum, is a pectolytic bacterial pathogen that causes typical soft rot on S. podophyllum. There is a shortage for genome data of P. aroidearum, which seriously hinders research on classification and pathogenesis of Pectobacterium. We present here the complete genome sequence of P. aroidearum L6. The L6 strain carries a single 4,995,896-bp chromosome with 53.10% G + C content and harbors 4,306 predicted protein-coding genes. We estimated in silico DNA–DNA hybridization and average nucleotide identity values in combination with the whole-genome-based phylogeny from 19 Pectobacterium strains including P. aroidearum L6. The results showed that L6 and PC1 formed a population distinct from other populations of the Pectobacterium genus. Phylogenetic analysis based on 16S rRNA and genome sequences showed a close evolutionary relationship among Pectobacterium species. Overall, evolutionary analysis showed that L6 was in the same branch with PC1. In comparison with 18 Pectobacterium spp. reference pathogens, strain L6 had 2,712 gene families, among which 1,632 gene families were identified as orthologous to those strains, as well as 1 putative unique gene family. We discovered 478 genes, 10.4% of the total of predicted genes, that were potentially related to pathogenesis using the Virulence Factors of Pathogenic Bacteria database. A total of 25 genes were related to toxins, 35 encoded plant cell-wall degrading enzymes, and 122 were involved in secretion systems. This study provides a foundation for a better understanding of the genomic structure of P. aroidearum and particularly offers information for the discovery of potential pathogenic factors and the development of more effective strategies against this pathogen.

The First Polish Isolate of a Novel Species Pectobacterium aquaticum Originates from a Pomeranian Lake.

Pectinolytic bacteria from the genus Pectobacterium cause high economic losses in various crops, vegetables, and ornamentals including potato. Thus far, these strains have been isolated from distinct environments such as rotten or asymptomatic plants, soil, and waterways. The prevalence of soft rot Pectobacteriaceae in different depths of Pomeranian lakes was performed by a qualified scuba diver over 2 years of monitoring. It allowed for the isolation and broad characterization of a strain from the newly established species Pectobacterium aquaticum. Phylogenetic analysis on the sequences of dnaX and recA genes revealed the highest similarity of this strain to P. aquaticum CFBP 8637T. In addition to the determination of analytical profile index (API 20E), we discovered that this strain possesses a smooth form of a lipopolysaccharide with O-polysaccharide consisting of mannose, glucose, and abequose. Moreover, the characterized strain, described as P. aquaticum IFB5637, produced plant-cell–wall-degrading enzymes, such as pectinases, cellulases, proteases, and was capable of macerating potato and chicory tissues under laboratory conditions. In view of more frequent irrigation of seed potato fields resulting from the ongoing climate warming, it is important to monitor the occurrence of potential disease-causing agents in natural waterways.

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

BackgroundBacterial 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.ResultsHere 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.ConclusionThe 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.