Clonality and Diversity in the Soft Rot Dickeya solani Phytopathogen.

Bacterial diversity analyses often suffer from a bias due to sampling only from a limited number of hosts or narrow geographic locations. This was the case for the phytopathogenic species Dickeya solani, whose members were mainly isolated from a few hosts-potato and ornamentals-and from the same geographical area-Europe and Israel, which are connected by seed trade. Most D. solani members were clonal with the notable exception of the potato isolate RNS05.1.2A and two related strains that are clearly distinct from other D. solani genomes. To investigate if D. solani genomic diversity might be broadened by analysis of strains isolated from other environments, we analysed new strains isolated from ornamentals and from river water as well as strain CFBP 5647 isolated from tomato in the Caribbean island Guadeloupe. While water strains were clonal to RNS05.1.2A, the Caribbean tomato strain formed a third clade. The genomes of the three clades are highly syntenic; they shared almost 3900 protein families, and clade-specific genes were mainly included in genomic islands of extrachromosomal origin. Our study thus revealed both broader D. solani diversity with the characterisation of a third clade isolated in Latin America and a very high genomic conservation between clade members.

Insight into biodiversity of the recently rearranged genus Dickeya.

The genus Dickeya includes plant pathogenic bacteria attacking a wide range of crops and ornamentals as well as a few environmental isolates from water. Defined on the basis of six species in 2005, this genus now includes 12 recognized species. Despite the description of several new species in recent years, the diversity of the genus Dickeya is not yet fully explored. Many strains have been analyzed for species causing diseases on economically important crops, such as for the potato pathogens D. dianthicola and D. solani. In contrast, only a few strains have been characterized for species of environmental origin or isolated from plants in understudied countries. To gain insights in the Dickeya diversity, recent extensive analyzes were performed on environmental isolates and poorly characterized strains from old collections. Phylogenetic and phenotypic analyzes led to the reclassification of D. paradisiaca (containing strains from tropical or subtropical regions) in the new genus, Musicola, the identification of three water species D. aquatica, D. lacustris and D. undicola, the description of a new species D. poaceaphila including Australian strains isolated from grasses, and the characterization of the new species D. oryzae and D. parazeae, resulting from the subdivision of the species D. zeae. Traits distinguishing each new species were identified from genomic and phenotypic comparisons. The high heterogeneity observed in some species, notably for D. zeae, indicates that additional species still need to be defined. The objective of this study was to clarify the present taxonomy of the genus Dickeya and to reassign the correct species to several Dickeya strains isolated before the current classification.

The Broad Host Range Plant Pathogen Dickeya dianthicola Shows a High Genetic Diversity.

The wide host range phytopathogen D. dianthicola, first described in ornamentals in the 1950s, rapidly became a threat for potato production in Europe and, more recently, worldwide. Previous genomic analyses, mainly of strains isolated from potato, revealed little sequence diversity. To further analyse D. dianthicola genomic diversity, we used a larger genome panel of 41 isolates encompassing more strains isolated from potato over a wide time scale and more strains isolated from other hosts. The phylogenetic and pan-genomic trees revealed a large cluster of highly related genomes but also the divergence of two more distant strains, IPO 256 and 67.19, isolated from potato and impatiens, respectively, and the clustering of the three strains isolated from Kalanchoe with one more distinct potato strain. An SNP-based minimal spanning tree highlighted both diverse clusters of (nearly) clonal strains and several strains scattered in the MST, irrespective of country or date of isolation, that differ by several thousand SNPs. This study reveals a higher diversity in D. dianthicola than previously described. It indicates the clonal spread of this pathogen over long distances, as suspected from worldwide seed trading, and possible multiple introductions of D. dianthicola from alternative sources of contaminations.

Diversity within the Dickeya zeae complex, identification of Dickeya zeae and Dickeya oryzae members, proposal of the novel species Dickeya parazeae sp. nov.

The genus Dickeya comprises plant pathogens that cause diseases in a large range of economically important crops and ornamentals. Strains previously assigned to the species Dickeya zeae are major pathogens attacking vital crops such as maize and rice. They are also frequently isolated from surface water. The newly described species Dickeya oryzae is closely related to D. zeae members, so that the limit between the two species can be difficult to define. In order to clearly distinguish the two species, globally described by the term 'D. zeae complex', we sequenced the genome of four new water isolates and compared them to 14 genomes available in databases. Calculation of average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values confirmed the phylogenomic classification into the two species D. zeae and D. oryzae. It also allowed us to propose a new species, Dickeya parazeae sp. nov., to characterize a clade distinct from those containing the D. zeae type strain NCPPB2538T. Strain S31T (CFBP 8716T=LMG 32070T) isolated from water in France is proposed as the type strain of the new species. Phenotypic analysis of eight publically available strains revealed traits common to the five tested D. oryzae members but apparently not shared by the D. oryzae type strain. Genomic analyses indicated that a simple distinction between the species D. zeae, D. parazeae and D. oryzae can be obtained on the basis of the recA sequence. D. oryzae can be distinguished from the two other species by growth on l-tartaric acid. Based on the recA marker, several strains previously identified as D. zeae were re-assigned to the species D. parazeae or D. oryzae. This study also highlighted the broad host range diversity of these three species.

Early Emergence of Dickeya solani Revealed by Analysis of Dickeya Diversity of Potato Blackleg and Soft Rot Causing Pathogens in Switzerland.

Blackleg and soft rot in potato caused by Pectobacterium and Dickeya enterobacteral genera are among the most destructive bacterial diseases in this crop worldwide. In Europe, over the last century, Pectobacterium spp. were the predominant causal agents of these diseases. As for Dickeya, before the large outbreak caused by D. solani in the 2000s, only D. dianthicola was isolated in Europe. The population dynamics of potato blackleg causing soft rot Pectobacteriaceae was, however, different in Switzerland as compared to that in other European countries with a high incidence (60 up to 90%) of Dickeya species (at the time called Erwinia chrysanthemi) already in the 1980s. To pinpoint what may underlie this Swiss peculiarity, we analysed the diversity present in the E. chrysanthemi Agroscope collection gathering potato isolates from 1985 to 2000s. Like elsewhere in Europe during this period, the majority of Swiss isolates belonged to D. dianthicola. However, we also identified a few isolates, such as D. chrysanthemi and D. oryzeae, two species that have not yet been reported in potatoes in Europe. Interestingly, this study allowed the characterisation of two "early" D. solani isolated in the 1990s. Genomic comparison between these early D. solani strains and strains isolated later during the large outbreak in the 2000s in Europe revealed only a few SNP and gene content differences, none of them affecting genes known to be important for virulence.

Dickeya undicola sp. nov., a novel species for pectinolytic isolates from surface waters in Europe and Asia.

Strains 2B12T, FVG1-MFV-O17 and FVG10-MFV-A16 were isolated from fresh water samples collected in Asia and Europe. The nucleotide sequences of the gapA barcodes revealed that all three strains belonged to the same cluster within the genus Dickeya. Using 13 housekeeping genes (fusA, rpoD, rpoS, glyA, purA, groEL, gapA, rplB, leuS, recA, gyrB, infB and secY), multilocus sequence analysis confirmed the existence of a new clade. When the genome sequences of these three isolates and other Dickeya species were compared, the in silico DNA-DNA hybridization and average nucleotide identity values were found to be no more than 45.50 and 91.22 %, respectively. The closest relative species was Dickeya fangzhongdai. Genome comparisons also highlighted genetic traits differentiating the new strains from D. fangzhongdai strains DSM 101947T (=CFBP 8607T) and B16. Phenotypical tests were performed to distinguish the three strains from D. fangzhongdai and other Dickeya species. The name Dickeya undicola sp. nov. is proposed with strain 2B12T (=CFBP 8650T=LMG 30903T) as the type strain.

Genomic characterisation of the new Dickeya fangzhongdai species regrouping plant pathogens and environmental isolates.

BACKGROUND: The Dickeya genus is part of the Pectobacteriaceae family that is included in the newly described enterobacterales order. It comprises a group of aggressive soft rot pathogens with wide geographic distribution and host range. Among them, the new Dickeya fangzhongdai species groups causative agents of maceration-associated diseases that impact a wide variety of crops and ornamentals. It affects mainly monocot plants, but D. fangzhongdai strains have also been isolated from pear trees and water sources. Here, we analysed which genetic novelty exists in this new species, what are the D. fangzhongdai-specific traits and what is the intra-specific diversity. RESULTS: The genomes of eight D. fangzhongdai strains isolated from diverse environments were compared to 31 genomes of strains belonging to other Dickeya species. The D. fangzhongdai core genome regroups approximately 3500 common genes, including most genes that encode virulence factors and regulators characterised in the D. dadantii 3937 model strain. Only 38 genes are present in D. fangzhongdai and absent in all other Dickeyas. One of them encodes a pectate lyase of the PL10 family of polysaccharide lyases that is found only in a few bacteria from the plant environment, soil or human gut. Other D. fangzhongdai-specific genes with a known or predicted function are involved in regulation or metabolism. The intra-species diversity analysis revealed that seven of the studied D. fangzhongdai strains were grouped into two distinct clades. Each clade possesses a pool of 100-150 genes that are shared by the clade members, but absent from the other D. fangzhongdai strains and several of these genes are clustered into genomic regions. At the strain level, diversity resides mainly in the arsenal of T5SS- and T6SS-related toxin-antitoxin systems and in secondary metabolite biogenesis pathways. CONCLUSION: This study identified the genome-specific traits of the new D. fangzhongdai species and highlighted the intra-species diversity of this species. This diversity encompasses secondary metabolites biosynthetic pathways and toxins or the repertoire of genes of extrachromosomal origin. We however didn't find any relationship between gene content and phenotypic differences or sharing of environmental habitats.

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.

Transcriptome analysis of the Dickeya dadantii PecS regulon during the early stages of interaction with Arabidopsis thaliana.

PecS is one of the major global regulators controlling the virulence of Dickeya dadantii, a broad-host-range phytopathogenic bacterium causing soft rot on several plant families. To define the PecS regulon during plant colonization, we analysed the global transcriptome profiles in wild-type and pecS mutant strains during the early colonization of the leaf surfaces and in leaf tissue just before the onset of symptoms, and found that the PecS regulon consists of more than 600 genes. About one-half of these genes are down-regulated in the pecS mutant; therefore, PecS has both positive and negative regulatory roles that may be direct or indirect. Indeed, PecS also controls the regulation of a few dozen regulatory genes, demonstrating that this global regulator is at or near the top of a major regulatory cascade governing adaptation to growth in planta. Notably, PecS acts mainly at the very beginning of infection, not only to prevent virulence gene induction, but also playing an active role in the adaptation of the bacterium to the epiphytic habitat. Comparison of the patterns of gene expression inside leaf tissues and during early colonization of leaf surfaces in the wild-type bacterium revealed 637 genes modulated between these two environments. More than 40% of these modulated genes are part of the PecS regulon, emphasizing the prominent role of PecS during plant colonization.

Extension of the transposable bacterial virus family: two genomic organisations among phages and prophages with a Tn552-related transposase.

Mu-like transposable phages and prophages have been isolated from, or predicted, in a wide range of bacterial phyla. However, related B3-like transposable phages, which differ in their genome organisation and the DDE transposase and transposition activator they code for have thus far been restricted to a very limited set of hosts. Through sequence similarity searches, we have now expanded the number of predicted B3-like prophages and uncovered a third genomic organisation. These new genomes, although only prophages, further illustrate the previously reported mosaicism existing in the proposed "Saltoviridae" family of Caudovirales, and further support the proposal to move morphology criteria (contractile vs. flexible or short tail, i.e. Myo-vs. Sipho- or Podoviridae) from the family to the subfamily level in the taxonomic classification of the Caudovirales.

Use of RP4::Mini-Mu for Gene Transfer.

Gene cloning is an invaluable technique in genetic analysis and exploitation of genetic properties of a broad range of bacteria. Numerous in vitro molecular cloning protocols have been devised but the efficiency of these techniques relies on the frequency with which the recombinant DNA can be introduced in the recipient strain. Here, we describe an in vivo gene transfer and cloning technique based on transposable bacteriophage Mu property to rearrange its host genome. This technique uses the broad host range plasmid RP4 carrying a transposable mini-MuA+ derivative and was successfully used as well in enteric as in environmental nonenteric bacteria.

Manipulation of ABA Content in Arabidopsis thaliana Modifies Sensitivity and Oxidative Stress Response to Dickeya dadantii and Influences Peroxidase Activity.

The production of reactive oxygen species (ROS) is one of the first defense reactions induced in Arabidopsis in response to infection by the pectinolytic enterobacterium Dickeya dadantii. Previous results also suggest that abscisic acid (ABA) favors D. dadantii multiplication and spread into its hosts. Here, we confirm this hypothesis using ABA-deficient and ABA-overproducer Arabidopsis plants. We investigated the relationships between ABA status and ROS production in Arabidopsis after D. dadantii infection and showed that ABA status modulates the capacity of the plant to produce ROS in response to infection by decreasing the production of class III peroxidases. This mechanism takes place independently of the well-described oxidative stress related to the RBOHD NADPH oxidase. In addition to this weakening of plant defense, ABA content in the plant correlates positively with the production of some bacterial virulence factors during the first stages of infection. Both processes should enhance disease progression in presence of high ABA content. Given that infection increases transcript abundance for the ABA biosynthesis genes AAO3 and ABA3 and triggers ABA accumulation in leaves, we propose that D. dadantii manipulates ABA homeostasis as part of its virulence strategy.

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.

A straightforward and reliable method for bacterial in planta transcriptomics: application to the Dickeya dadantii/Arabidopsis thaliana pathosystem.

Transcriptome analysis of bacterial pathogens is a powerful approach to identify and study the expression patterns of genes during host infection. However, analysis of the early stages of bacterial virulence at the genome scale is lacking with respect to understanding of plant-pathogen interactions and diseases, especially during foliar infection. This is mainly due to both the low ratio of bacterial cells to plant material at the beginning of infection, and the high contamination by chloroplastic material. Here we describe a reliable and straightforward method for bacterial cell purification from infected leaf tissues, effective even if only a small amount of bacteria is present relative to plant material. The efficiency of this method for transcriptomic analysis was validated by analysing the expression profiles of the phytopathogenic enterobacterium Dickeya dadantii, a soft rot disease-causing agent, during the first hours of infection of the model host plant Arabidopsis thaliana. Transcriptome profiles of epiphytic bacteria and bacteria colonizing host tissues were compared, allowing identification of approximately 100 differentially expressed genes. Requiring no specific equipment, cost-friendly and easily transferable to other pathosystems, this method should be of great interest for many other plant-bacteria interaction studies.

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.

Vfm a new quorum sensing system controls the virulence of Dickeya dadantii.

Dickeya dadantii is a plant pathogen that secretes cell wall-degrading enzymes (CWDE) that are responsible for soft-rot symptoms. Virulence genes are expressed in a concerted manner and culminate when bacterial multiplication slows. We identify a 25 kb vfm cluster required for D. dadantii CWDE production and pathogenesis. The vfm cluster encodes proteins displaying similarities both with enzymes involved in amino acid activation and with enzymes involved in fatty acid biosynthesis. These similarities suggest that the vfm genes direct the production of a metabolite. Cell-free supernatant from the D. dadantii wild-type strain restores CWDE production in vfm mutants. Collectively, our results indicate that vfm genes direct the synthesis of an extracellular signal and constitute a new quorum sensing system. Perception of the signal is achieved by the two-component system VfmH-VfmI, which activates the expression of the vfmE gene encoding an AraC regulator. VfmE then activates both the transcription of the CWDE genes and the expression of the vfm operons. The vfm gene cluster does not seem to be widespread among bacterial species but is conserved in other Dickeya species and could have been laterally transferred to Rahnella. This work highlights that entirely new families of bacterial languages remain to be discovered.

The role of secretion systems and small molecules in soft-rot Enterobacteriaceae pathogenicity.

Soft-rot Enterobacteriaceae (SRE), which belong to the genera Pectobacterium and Dickeya, consist mainly of broad host-range pathogens that cause wilt, rot, and blackleg diseases on a wide range of plants. They are found in plants, insects, soil, and water in agricultural regions worldwide. SRE encode all six known protein secretion systems present in gram-negative bacteria, and these systems are involved in attacking host plants and competing bacteria. They also produce and detect multiple types of small molecules to coordinate pathogenesis, modify the plant environment, attack competing microbes, and perhaps to attract insect vectors. This review integrates new information about the role protein secretion and detection and production of ions and small molecules play in soft-rot pathogenicity.

Modelling the onset of virulence in pathogenic bacteria.

Bacterial virulence is a multifactorial process. In this chapter, we review some known mechanisms used by bacteria to trigger their production of virulence factors. We develop the idea that although the onset of virulence shows up an abrupt transition, the modelling of this dynamics can be classified in two qualitatively distinct infectious transitions which are respectively called "shift" or "switch." We review methods enabling one to determine the types of behaviour that can be exhibited by a given model and we consider applications in three cases of virulence factor regulation. We conclude that in most cases a "successful" infection would require that the onset of virulence follows an irreversible switch behaviour.

PecS is an important player in the regulatory network governing the coordinated expression of virulence genes during the interaction between Dickeya dadantii 3937 and plants.

Successful infection of a pathogen relies on the coordinated expression of numerous virulence factor-encoding genes. In plant-bacteria interactions, this control is very often achieved through the integration of several regulatory circuits controlling cell-cell communication or sensing environmental conditions. Dickeya dadantii (formerly Erwinia chrysanthemi), the causal agent of soft rot on many crops and ornamentals, provokes maceration of infected plants mainly by producing and secreting a battery of plant cell wall-degrading enzymes. However, several other virulence factors have also been characterized. During Arabidopsis infection, most D. dadantii virulence gene transcripts accumulated in a coordinated manner during infection. This activation requires a functional GacA-GacS two-component regulatory system but the Gac system is not involved in the growth phase dependence of virulence gene expression. Here we show that, contrary to Pectobacterium, the AHL-mediated ExpIR quorum-sensing system does not play a major role in the growth phase-dependent control of D. dadantii virulence genes. On the other hand, the global regulator PecS participates in this coordinated expression since, in a pecS mutant, an early activation of virulence genes is observed both in vitro and in planta. This correlated with the known hypervirulence phenotype of the pecS mutant. Analysis of the relationship between the regulatory circuits governed by the PecS and GacA global regulators indicates that these two regulators act independently. PecS prevents a premature expression of virulence genes in the first stages of colonization whereas GacA, presumably in conjunction with other regulators, is required for the activation of virulence genes at the onset of symptom occurrence.

lpxC and yafS are the most suitable internal controls to normalize real time RT-qPCR expression in the phytopathogenic bacteria Dickeya dadantii.

BACKGROUND: Quantitative RT-PCR is the method of choice for studying, with both sensitivity and accuracy, the expression of genes. A reliable normalization of the data, using several reference genes, is critical for an accurate quantification of gene expression. Here, we propose a set of reference genes, of the phytopathogenic bacteria Dickeya dadantii and Pectobacterium atrosepticum, which are stable in a wide range of growth conditions. RESULTS: We extracted, from a D. dadantii micro-array transcript profile dataset comprising thirty-two different growth conditions, an initial set of 49 expressed genes with very low variation in gene expression. Out of these, we retained 10 genes representing different functional categories, different levels of expression (low, medium, and high) and with no systematic variation in expression correlating with growth conditions. We measured the expression of these reference gene candidates using quantitative RT-PCR in 50 different experimental conditions, mimicking the environment encountered by the bacteria in their host and directly during the infection process in planta. The two most stable genes (ABF-0017965 (lpxC) and ABF-0020529 (yafS) were successfully used for normalization of RT-qPCR data. Finally, we demonstrated that the ortholog of lpxC and yafS in Pectobacterium atrosepticum also showed stable expression in diverse growth conditions. CONCLUSIONS: We have identified at least two genes, lpxC (ABF-0017965) and yafS (ABF-0020509), whose expressions are stable in a wide range of growth conditions and during infection. Thus, these genes are considered suitable for use as reference genes for the normalization of real-time RT-qPCR data of the two main pectinolytic phytopathogenic bacteria D. dadantii and P. atrosepticum and, probably, of other Enterobacteriaceae. Moreover, we defined general criteria to select good reference genes in bacteria.