Pattern and causes of the establishment of the invasive bacterial potato pathogen Dickeya solani and of the maintenance of the resident pathogen D. dianthicola.

Invasive pathogens can be a threat when they affect human health, food production or ecosystem services, by displacing resident species, and we need to understand the cause of their establishment. We studied the patterns and causes of the establishment of the pathogen Dickeya solani that recently invaded potato agrosystems in Europe by assessing its invasion dynamics and its competitive ability against the closely related resident D. dianthicola species. Epidemiological records over one decade in France revealed the establishment of D. solani and the maintenance of the resident D. dianthicola in potato fields exhibiting blackleg symptoms. Using experimentations, we showed that D. dianthicola caused a higher symptom incidence on aerial parts of potato plants than D. solani, while D. solani was more aggressive on tubers (i.e. with more severe symptoms). In co-infection assays, D. dianthicola outcompeted D. solani in aerial parts, while the two species co-existed in tubers. A comparison of 76 D. solani genomes (56 of which have been sequenced here) revealed balanced frequencies of two previously uncharacterized alleles, VfmBPro and VfmBSer , at the vfmB virulence gene. Experimental inoculations showed that the VfmBSer population was more aggressive on tubers, while the VfmBPro population outcompeted the VfmBSer population in stem lesions, suggesting an important role of the vfmB virulence gene in the ecology of the pathogens. This study thus brings novel insights allowing a better understanding of the pattern and causes of the D.solani invasion into potato production agrosystems, and the reasons why the endemic D. dianthicola nevertheless persisted.

Species Diversity of Dickeya and Pectobacterium Causing Potato Blackleg Disease in Pakistan.

Potato blackleg is caused by a diverse species of pectinolytic bacteria. In Pakistan, approximately 90% of the pathogens involved belong to Pectobacterium atrosepticum. Survey (2014 to 2017), sampling, and isolation from different potato growing areas of Punjab, Pakistan depicted an overall disease incidence of approximately 15%. Thirty-six pectinolytic strains confirmed through biochemical and pathogenicity testing were characterized via gapA gene to identify them at the species level. To further validate the identification, one strain from each species SS26 (P. atrosepticum), SS28 (Pectobacterium polaris), SS70 (Dickeya dianthicola), SS90 (Pectobacterium parmentieri), SS95 (Pectobacterium punjabense), and SS96 (Pectobacterium versatile) were selected for draft genome sequencing and multilocus sequence analysis of 13 housekeeping genes (fusA, rpoD, acnA, purA, gyrB, recA, mdh, mtlD, groEL, secY, glyA, gapA, and rplB). Phylogenetic analysis revealed considerable genetic diversity in the genus Pectobacterium. In silico DNA-DNA hybridization and average nucleotide identity values of the strains selected for genome sequencing were determined with other reference Pectobacterium and Dickeya strains. Moreover, all six representative strains were also phenotypically characterized on the basis of metabolism of different carbon sources. Overall, on the basis of genotypic and phenotypic characteristics, these 36 isolates were grouped into six species: P. atrosepticum, P. versatile, P. parmentieri, P. polaris, P. punjabense, and D. dianthicola.

Common and distinctive adaptive traits expressed in Dickeya dianthicola and Dickeya solani pathogens when exploiting potato plant host.

Blackleg and soft rot are devastating diseases on potato stem and tuber caused by Pectobacterium and Dickeya pectinolytic enterobacteria. In European potato cultures, D. dianthicola and D. solani species successively emerged in the past decades. Ecological traits associated to their settlement remain elusive, especially in the case of the recent invader D. solani. In this work, we combined genomic, metabolic and transcriptomic comparisons to unravel common and distinctive genetic and functional characteristics between two D. solani and D. dianthicola isolates. The two strains differ by more than a thousand genes that are often clustered in genomic regions (GRs). Several GRs code for transport and metabolism functions that correlate with some of the differences in metabolic abilities identified between the two Dickeya strains. About 800 D. dianthicola and 1100 D. solani genes where differentially expressed in macerated potato tubers as compared to when growing in rich medium. These include several genes located in GRs, pointing to a potential role in host interaction. In addition, some genes common to both species, including virulence genes, differed in their expression. This work highlighted distinctive traits when D. dianthicola and D. solani exploit the host as a resource.

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

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

Transfer of the potato plant isolates of Pectobacterium wasabiae to Pectobacterium parmentieri sp. nov.

Pectobacterium wasabiae was originally isolated from Japanese horseradish (Eutrema wasabi), but recently some Pectobacterium isolates collected from potato plants and tubers displaying blackleg and soft rot symptoms were also assigned to P. wasabiae. Here, combining genomic and phenotypical data, we re-evaluated their taxonomic position. PacBio and Illumina technologies were used to complete the genome sequences of P. wasabiae CFBP 3304T and RNS 08-42-1A. Multi-locus sequence analysis showed that the P. wasabiae strains RNS 08-42-1A, SCC3193, CFIA1002 and WPP163, which were collected from potato plant environment, constituted a separate clade from the original Japanese horseradish P. wasabiae. The taxonomic position of these strains was also supported by calculation of the in-silico DNA-DNA hybridization, genome average nucleotide indentity, alignment fraction and average nucleotide indentity values. In addition, they were phenotypically distinguished from P. wasabiae strains by producing acids from (+)-raffinose, α-d(+)-α-lactose, d(+)-galactose and (+)-melibiose but not from methyl α-d-glycopyranoside, (+)-maltose or malonic acid. The name Pectobacterium parmentieri sp. nov. is proposed for this taxon; the type strain is RNS 08-42-1AT (=CFBP 8475T=LMG 29774T).

Biocontrol of the Potato Blackleg and Soft Rot Diseases Caused by Dickeya dianthicola.

Development of protection tools targeting Dickeya species is an important issue in the potato production. Here, we present the identification and the characterization of novel biocontrol agents. Successive screenings of 10,000 bacterial isolates led us to retain 58 strains that exhibited growth inhibition properties against several Dickeya sp. and/or Pectobacterium sp. pathogens. Most of them belonged to the Pseudomonas and Bacillus genera. In vitro assays revealed a fitness decrease of the tested Dickeya sp. and Pectobacterium sp. pathogens in the presence of the biocontrol agents. In addition, four independent greenhouse assays performed to evaluate the biocontrol bacteria effect on potato plants artificially contaminated with Dickeya dianthicola revealed that a mix of three biocontrol agents, namely, Pseudomonas putida PA14H7 and Pseudomonas fluorescens PA3G8 and PA4C2, repeatedly decreased the severity of blackleg symptoms as well as the transmission of D. dianthicola to the tuber progeny. This work highlights the use of a combination of biocontrol strains as a potential strategy to limit the soft rot and blackleg diseases caused by D. dianthicola on potato plants and tubers.

Population genomics reveals additive and replacing horizontal gene transfers in the emerging pathogen Dickeya solani.

BACKGROUND: Dickeya solani is an emerging pathogen that causes soft rot and blackleg diseases in several crops including Solanum tuberosum, but little is known about its genomic diversity and evolution. RESULTS: We combined Illumina and PacBio technologies to complete the genome sequence of D. solani strain 3337 that was used as a reference to compare with 19 other genomes (including that of the type strain IPO2222(T)) which were generated by Illumina technology. This population genomic analysis highlighted an unexpected variability among D. solani isolates since it led to the characterization of two distinct sub-groups within the D. solani species. This approach also revealed different types of variations such as scattered SNP/InDel variations as well as replacing and additive horizontal gene transfers (HGT). Infra-species (between the two D. solani sub-groups) and inter-species (between D. solani and D. dianthicola) replacing HGTs were observed. Finally, this work pointed that genetic and functional variation in the motility trait could contribute to aggressiveness variability in D. solani. CONCLUSIONS: This work revealed that D. solani genomic variability may be caused by SNPs/InDels as well as replacing and additive HGT events, including plasmid acquisition; hence the D. solani genomes are more dynamic than that were previously proposed. This work alerts on precautions in molecular diagnosis of this emerging pathogen.

Genomic overview of the phytopathogen Pectobacterium wasabiae strain RNS 08.42.1A suggests horizontal acquisition of quorum-sensing genes.

The blackleg and soft-rot diseases caused by pectinolytic enterobacteria such as Pectobacterium and Dickeya are major causes of losses affecting potato crop in the field and upon storage. In this work, we report the isolation, characterization and genome analysis of the Pectobacterium wasabiae (formerly identified as Pectobacterium carotovorum subsp. carotovorum) strain RNS 08.42.1A, that has been isolated from a Solanum tuberosum host plant in France. Comparative genomics with 3 other P. wasabiae strains isolated from potato plants in different areas in North America and Europe, highlighted both a strong similarity at the whole genome level (ANI > 99 %) and a conserved synteny of the virulence genes. In addition, our analyses evidenced a robust separation between these four P. wasabiae strains and the type strain P. wasabiae CFBP 3304(T), isolated from horseradish in Japan. In P. wasabiae RNS 08.42.1A, the expI and expR nucleotidic sequences are more related to those of some Pectobacterium atrosepticum and P. carotovorum strains (90 % of identity) than to those of the other potato P. wasabiae strains (70 to 74 % of identity). This could suggest a recruitment of these genes in the P. wasabiae strain RNS 08.42.1A by an horizontal transfer between pathogens infecting the same potato host plant.

An increasing opine carbon bias in artificial exudation systems and genetically modified plant rhizospheres leads to an increasing reshaping of bacterial populations.

To investigate how exudation shapes root-associated bacterial populations, transgenic Arabidopsis thaliana plants that exuded the xenotopic compound octopine at low and high rates were grown in a nonsterile soil. Enumerations of both cultivable and octopine-degrading bacteria demonstrated that the ratios of octopine degraders increased along with octopine concentration. An artificial exudation system was also set up in which octopine was brought at four ratios. The density of octopine-degrading bacteria directly correlated with the input of octopine. Bacterial diversity was analysed by rrs amplicon pyrosequencing. Ensifer and Pseudomonas were significantly more frequently detected in soil amended with artificial exudates. However, the density of Pseudomonas increased as a response to carbon supplementation while that of Ensifer only correlated with octopine concentrations possibly in relation to two opposed colonization strategies of rhizosphere bacteria, that is, copiotrophy and oligotrophy.

Genomic and metabolic comparison with Dickeya dadantii 3937 reveals the emerging Dickeya solani potato pathogen to display distinctive metabolic activities and T5SS/T6SS-related toxin repertoire.

BACKGROUND: The pectinolytic enterobacteria of the Pectobacterium and Dickeya genera are causative agents of maceration-associated diseases affecting a wide variety of crops and ornamentals. For the past decade, the emergence of a novel species D. solani was observed in potato fields in Europe and the Mediterranean basin. The purpose of this study is to search by comparative genomics the genetic traits that could be distinctive to other Dickeya species and be involved in D. solani adaptation to the potato plant host. RESULTS: D. solani 3337 exhibits a 4.9 Mb circular genome that is characterized by a low content in mobile elements with the identification of only two full length insertion sequences. A genomic comparison with the deeply-annotated model D. dadantii 3937 strain was performed. While a large majority of Dickeya virulence genes are shared by both strains, a few hundreds genes of D. solani 3337, mostly regrouped in 25 genomic regions, are distinctive to D. dadantii 3937. These genomic regions are present in the other available draft genomes of D. solani strains and interestingly some of them were not found in the sequenced genomes of the other Dickeya species. These genomic regions regroup metabolic genes and are often accompanied by genes involved in transport systems. A metabolic analysis correlated some metabolic genes with distinctive functional traits of both D. solani 3337 and D. dadantii 3937. Three identified D. solani genomic regions also regroup NRPS/PKS encoding genes. In addition, D. solani encodes a distinctive arsenal of T5SS and T6SS-related toxin-antitoxin systems. These genes may contribute to bacteria-bacteria interactions and to the fitness of D. solani to the plant environment. CONCLUSIONS: This study highlights the genomic specific traits of the emerging pathogen D. solani and will provide the basis for studying those that are involved in the successful adaptation of this emerging pathogen to the potato plant host.

At a supra-physiological concentration, human sexual hormones act as quorum-sensing inhibitors.

N-acylhomoserine lactone (AHL)-mediated quorum-sensing (QS) regulates virulence functions in plant and animal pathogens such as Agrobacterium tumefaciens and Pseudomonas aeruginosa. A chemolibrary of more than 3500 compounds was screened using two bacterial AHL-biosensors to identify QS-inhibitors (QSIs). The purity and structure of 15 QSIs selected through this screening were verified using HPLC MS/MS tools and their activity tested on the A. tumefaciens and P. aeruginosa bacterial models. The IC50 value of the identified QSIs ranged from 2.5 to 90 µg/ml, values that are in the same range as those reported for the previously identified QSI 4-nitropyridine-N-oxide (IC50 24 µg/ml). Under the tested culture conditions, most of the identified QSIs did not exhibit bacteriostatic or bactericidal activities. One third of the tested QSIs, including the plant compound hordenine and the human sexual hormone estrone, decreased the frequency of the QS-regulated horizontal transfer of the tumor-inducing (Ti) plasmid in A. tumefaciens. Hordenine, estrone as well as its structural relatives estriol and estradiol, also decreased AHL accumulation and the expression of six QS-regulated genes (lasI, lasR, lasB, rhlI, rhlR, and rhlA) in cultures of the opportunist pathogen P. aeruginosa. Moreover, the ectopic expression of the AHL-receptors RhlR and LasR of P. aeruginosa in E. coli showed that their gene-regulatory activity was affected by the QSIs. Finally, modeling of the structural interactions between the human hormones and AHL-receptors LasR of P. aeruginosa and TraR of A. tumefaciens confirmed the competitive binding capability of the human sexual hormones. This work indicates potential interferences between bacterial and eukaryotic hormonal communications.

N,N'-alkylated Imidazolium-derivatives act as quorum-sensing inhibitors targeting the Pectobacterium atrosepticum-induced symptoms on potato tubers.

Bacteria belonging to the Pectobacterium genus are the causative agents of the blackleg and soft-rot diseases that affect potato plants and tubers worldwide. In Pectobacterium, the expression of the virulence genes is controlled by quorum-sensing (QS) and N-acylhomoserine lactones (AHLs). In this work, we screened a chemical library of QS-inhibitors (QSIs) and AHL-analogs to find novel QSIs targeting the virulence of Pectobacterium. Four N,N'-bisalkylated imidazolium salts were identified as QSIs; they were active at the µM range. In potato tuber assays, two of them were able to decrease the severity of the symptoms provoked by P. atrosepticum. This work extends the range of the QSIs acting on the Pectobacterium-induced soft-rot disease.

Quorum sensing signaling molecules produced by reference and emerging soft-rot bacteria (Dickeya and Pectobacterium spp.).

BACKGROUND: Several small diffusible molecules are involved in bacterial quorum sensing and virulence. The production of autoinducers-1 and -2, quinolone, indole and γ-amino butyrate signaling molecules was investigated in a set of soft-rot bacteria belonging to six Dickeya or Pectobacterium species including recent or emerging potato isolates. METHODOLOGY/PRINCIPAL FINDINGS: Using bacterial biosensors, immunoassay, and chromatographic analysis, we showed that soft-rot bacteria have the common ability to produce transiently during their exponential phase of growth the N-3-oxo-hexanoyl- or the N-3-oxo-octanoyl-l-homoserine lactones and a molecule of the autoinducer-2 family. Dickeya spp. produced in addition the indole-3-acetic acid in tryptophan-rich conditions. All these signaling molecules have been identified for the first time in the novel Dickeya solani species. In contrast, quinolone and γ-amino butyrate signals were not identified and the corresponding synthases are not present in the available genomes of soft-rot bacteria. To determine if the variations of signal production according to growth phase could result from expression modifications of the corresponding synthase gene, the respective mRNA levels were estimated by reverse transcriptase-PCR. While the N-acyl-homoserine lactone production is systematically correlated to the synthase expression, that of the autoinducer-2 follows the expression of an enzyme upstream in the activated methyl cycle and providing its precursor, rather than the expression of its own synthase. CONCLUSIONS/SIGNIFICANCE: Despite sharing the S-adenosylmethionine precursor, no strong link was detected between the production kinetics or metabolic pathways of autoinducers-1 and -2. In contrast, the signaling pathway of autoinducer-2 seems to be switched off by the indole-3-acetic acid pathway under tryptophan control. It therefore appears that the two genera of soft-rot bacteria have similarities but also differences in the mechanisms of communication via the diffusible molecules. Our results designate autoinducer-1 lactones as the main targets for a global biocontrol of soft-rot bacteria communications, including those of emerging isolates.

Efficient biostimulation of native and introduced quorum-quenching Rhodococcus erythropolis populations is revealed by a combination of analytical chemistry, microbiology, and pyrosequencing.

Degradation of the quorum-sensing (QS) signals known as N-acylhomoserine lactones (AHL) by soil bacteria may be useful as a beneficial trait for protecting crops, such as potato plants, against the worldwide pathogen Pectobacterium. In this work, analytical chemistry and microbial and molecular approaches were combined to explore and compare biostimulation of native and introduced AHL-degrading Rhodococcus erythropolis populations in the rhizosphere of potato plants cultivated in farm greenhouses under hydroponic conditions. We first identified gamma-heptalactone (GHL) as a novel biostimulating agent that efficiently promotes plant root colonization by AHL-degrading R. erythropolis population. We also characterized an AHL-degrading biocontrol R. erythropolis isolate, R138, which was introduced in the potato rhizosphere. Moreover, root colonization by AHL-degrading bacteria receiving different combinations of GHL and R138 treatments was compared by using a cultivation-based approach (percentage of AHL-degrading bacteria), pyrosequencing of PCR-amplified rrs loci (total bacterial community), and quantitative PCR (qPCR) of the qsdA gene, which encodes an AHL lactonase in R. erythropolis. Higher densities of the AHL-degrading R. erythropolis population in the rhizosphere were observed when GHL treatment was associated with biocontrol strain R138. Under this condition, the introduced R. erythropolis population displaced the native R. erythropolis population. Finally, chemical analyses revealed that GHL, gamma-caprolactone (GCL), and their by-products, gamma-hydroxyheptanoic acid and gamma-hydroxycaproic acid, rapidly disappeared from the rhizosphere and did not accumulate in plant tissues. This integrative study highlights biostimulation as a potential innovative approach for improving root colonization by beneficial bacteria.

Gamma-caprolactone stimulates growth of quorum-quenching Rhodococcus populations in a large-scale hydroponic system for culturing Solanum tuberosum.

Bacteria degrading quorum sensing (QS) signals have been proposed as biocontrol agents able to quench QS-dependent expression of virulence symptoms caused by Pectobacterium on potato plants. We report here that gamma-caprolactone (GCL) treatment stimulated growth of the native QS-degrading bacterial community in an industrial plant hydroponic system for culturing Solanum tuberosum. Post-GCL treatment, QS-degrading bacteria were mainly identified as Rhodococcus isolates, while Agrobacterium isolates dominated under similar untreated conditions. Most of the assayed Rhodococcus isolates exhibited efficient biocontrol activity for protecting potato tubers. Analytical chemistry approach revealed the rapid degradation of GCL introduced in the plant cultures.

Growth promotion of quorum-quenching bacteria in the rhizosphere of Solanum tuberosum.

Among 17 molecules structurally related to N-acylhomoserine lactone (NAHL), gamma-caprolactone (GCL), 6-caprolactone (6CL) and 4-heptanolide (HTN) were found to stimulate the degradation of NAHL by bacterial communities recovered from bulk and rhizospheric soils. In the 6CL-, GCL- and HTN-treated bacterial consortia, the NAHL-degrading bacteria were more abundant than in control (mannitol-treated) consortia. Moreover, the GCL- and HTN-consortia showed a biocontrol activity against Pectobacterium atrosepticum in soft rot assays with tubers of Solanum tuberosum. When GCL was applied to hydroponic cultures of S. tuberosum, a significant increase of the ratio of NAHL-degrading bacteria among total cultivable bacteria was observed in several independent experiments. Most of these bacteria, the growth of which was stimulated by GCL amendment, were also able to use GCL as a sole carbon source. They belong to the Rhodococcus and Delftia genera. DGGE analysis revealed that GCL treatments affected the structure of bacterial communities. This work highlights the possibility to manage the NAHL-degrading bacteria in a complex environment such as rhizosphere.

Involvement of N-acylhomoserine lactones throughout plant infection by Erwinia carotovora subsp. atroseptica (Pectobacterium atrosepticum).

Erwinia carotovora subsp. atroseptica is responsible for potato blackleg disease in the field and tuber soft rot during crop storage. The process leading to the disease occurs in two phases: a primary invasion step followed by a maceration step. Bacteria-to-bacteria communication is associated with a quorum-sensing (QS) process based on the production of N-acylhomoserine lactones (HSL). The role of HSL throughout plant infection was analyzed. To this purpose, HSL produced by a specific E. carotovora subsp. atroseptica wild-type strain, which was particularly virulent on potato, were identified. A derivative of this strain that expressed an HSL lactonase gene and produced low amounts of HSL was generated. The comparison of these strains allowed the evaluation of the role of HSL and QS in disease establishment and development. Bacterial growth and motility; activity of proteins secreted by type I, II, and III systems; and hypersensitive and maceration reactions were evaluated. Results indicated that HSL production and QS regulate only those traits involved in the second stage of the host plant infection (i.e., tissue maceration) and hypersensitive response in nonhost tobacco plants. Therefore, the use of QS quenching strategies for biological control in E. carotovora subsp. atroseptica cannot prevent initial infection and multiplication of this pathogen.