Dickeya dianthicola emerged from a potato culture area exhibiting a wide diversity of pectinolytic pathogens in northern Morocco

doi:10.21203/rs.2.22648/v1

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Dickeya dianthicola emerged from a potato culture area exhibiting a wide diversity of pectinolytic pathogens in northern Morocco

https://doi.org/10.21203/rs.2.22648/v1

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Background: Dickeya and Pectobacterium are pectinolytic pathogens that cause damage to many plants including major crops. Emergence of D. solani and re-emergence D. dianthicola were recently observed in potato fields in several continents. The purpose of this work was to describe the species diversity of Dickeya and Pectobacterium collected from potato fields in Northern Morocco, where Dickeya potato pathogens have not been isolated until recently.

Results: Along three years, 119 pathogens belonging to Pectobacterium and Dickeya genera were isolated from three potato culture areas and characterized using selective PCR and gapA gene sequencing. Out of them, 19% belonged to P. versatile, 3% to P. carotovorum, 5% to P. polaris, 56% to P. brasiliense, while 17% to D. dianthicola. The taxonomic assignations were confirmed by draft genome analyses of representative isolates belonging to the collected species. D. dianthicola were isolated from a unique area where a wide species diversity of pectinolytic pathogens was also observed. In potato maceration assay, D. dianthicola isolates were more aggressive than Pectobacterium isolates, a feature that should alert stakeholders of a potential threat linked to the emergence of D. dianthicola in this country. By combining Oxford Nanopore Technologies (ONT) and Illumina technologies, the sequence of the complete genome of D. dianthicola LAR.16.03.LID was obtained. A unique circular chromosome of 4,976,211 bp codes for 4,223 predicted proteins. Comparison of the three complete genomes of D. dianthicola strains RNS049, ME23 and LAR.16.03.LID revealed a highly conserved synteny and the occurrence of strain-specific regions associated with the presence of mobile elements.

Conclusion: By combining population sampling and genomics, this study highlighted the ecological context from which D. dianthicola emerged in Morocco. Furthermore, the first complete genome of a D. dianthicola strain isolated in Northern Morocco will constitute a reference genome for investigating the dynamics of the emerging D. dianthicola pathogens in this country.

Epigenetics & Genomics

Oxford Nanopore Technologies

Illumina

genomics

diversity

Pectobacterium

Dickeya

virulence

potato

Pectinolytic Pectobacterium and Dickeya spp. are causative agents of severe diseases on a wide range of plants with a high economic value [1, 2]. On potato tubers and stems, the diseases caused by pectinolytic pathogens are soft rot and blackleg, respectively. These pathogens produce a large set of extracellular enzymes that break down the plant cell wall, resulting in plant tissue decay and maceration [3]. These pathogens cause important losses to the production of potato tubers for food market, and all along the certification steps of tuber seeds. The pathogens may be acquired by plant hosts from soil and/or transmitted over generation because of contaminated tuber-seeds [4]. On plants, they remain at a low level in asymptomatic plant tissues and may be particularly destructive when environmental conditions favor their proliferation and the expression of virulence factors.

P. atrosepticum was considered as the primary pathogen responsible for rotting of stored potato tubers and wilting of growing potato plants under temperate climates [4]. Other Pectobacterium species frequently associated to damage on potato crops are P. carotovorum, P. brasiliense, P. parmentieri and P. polaris [5–9]. Some Pectobacterium species have also been characterized in some particular areas such as: P. peruviense strains from tubers in Peru and P. punjabense species from symptomatic potato plants in Pakistan [10, 11]. P. odoriferum exhibits a very wide host range including potato plants [12], while some other species were characterized by a more restricted host range in the culture fields: P. wasabiae was isolated from symptomatic Japanese horseradish [13]; P. betavasculorum is reported almost exclusively on sugar beet [14]; P. aroidearum exhibits with preference for some monocotyledonous plants [15]; P. zantedeschiae strains were isolated from Zantedeschia spp. (Calla lily) [16]; and, P. actinidiae from symptomatic Actinidia chinensis (kiwi fruit) [17]. Recently, some other species have also been described with bacterial strains isolated from surface waters : P. fontis, P. aquaticum and P. versatile [7, 18, 19]. Altogether, 16 Pectobacterium species are described so far [7].

A limited number of Dickeya species, i.e. D. dianthicola and D. solani, have been associated to symptoms on potato. D. dianthicola was first reported on potato in the Netherlands in the 1970s and then have been detected since in many other European countries [20]. D. solani emerged in potato cultivars in the 2000s in several European countries and Israel [21]. During the past decade, taxonomy of Dickeya species, as that of Pectobacterium, was revisited by genomics in combination with the exploitation of international culture collections and exploration of diverse ecosystems around the world [22, 23]. By now, the genus Dickeya encompasses ten species: D. aquatica, D. chrysanthemi, D. dadantii, D. dianthicola, D. fangzhongdai, D. lacustris, D. paradisiaca, D. solani, D. undicola, and D. zeae [21, 24–29]. Bacteria belonging to this genus cause plant disease symptoms in temperate, tropical and subtropical climates [30].

The unambiguous identification of Dickeya and Pectobacterium species is crucial for epidemiology purposes, for developing appropriate prophylactic approaches as well as for quality controls in national and international trade exchanges. Multi-Locus Sequence Analysis (MLSA) provides relevant information for a better understanding of speciation, hence for proposing pertinent taxa [15, 31]. MLSA may exploit gene sequences obtained by PCR-sequencing of each locus and whole genome sequencing. Among the loci commonly included in MLSA, the rrs sequence is poorly informative at a species level while the gapA gene appeared as an appropriate marker to discriminate the different Dickeya and Pectobacterium species [10, 18, 19, 26, 27, 32]. Taxonomy of Dickeya and Pectobacterium gained in precision and robustness with additional genome analyses that are ANI and isDDH [33]. Comparative genomics is also used to identify species-specific DNA regions. Analysis of the functions encoded by these DNA regions allows the prediction of species-specific metabolic traits: this knowledge contributes to the understanding of both the taxonomy and ecology of the Dickeya and Pectobacterium pathogens [21, 25–27, 34].

P. atrosepticum, P. carotovorum and P. brasiliense were described in Morocco as the main causative agents of blackleg and soft rot disease in potato crop [35–38]. In 2016, D. dianthicola was described for the first time in the North of Morocco [39]. In relation, the main objectives of this study were: (i) to investigate the species composition of the Moroccan Dickeya and Pectobacterium populations collected between 2015 and 2017 from diseased potato tubers and stems, (ii) to compare aggressiveness of some identified pathogens belonging to different species, and (iii) to propose a complete genome of the emerging pathogen D. dianthicola in Morocco that could be used for further studies as a reference genome.

Diversity of the pectinolytic Dickeya and Pectobacterium in the Northern Morocco

In 2015, 2016 and 2017, our field inspections revealed the occurrence of blackleg symptoms in several potato fields located in many townships distributed in three regions (Meknes, S1; Guigo, S2; Larache, S4) in Northern Morocco (Fig. 1). No symptoms were found in fields in the Boumia region (S3). Out of 200 strains isolated from plant symptoms, 140 provoked cavities on pectate-containing medium. These were tested by PCR to evaluate whether they belong to Pectobacterium and Dickeya genera: 119 isolates generated amplification signals for either Y1/Y2 or ADE1/ADE2 primer couples. Most of the isolates (83%) generated a signal with Pectobacterium primers Y1/Y2, while the others (17%) did so with Dickeya ADE1/ADE2 primers.

All these PCR-positive Pectobacterium/Dickeya isolates were further characterized at species level using the gapA gene (FigS1). The regional diversity of the Dickeya and Pectobacterium isolates is summarized in Fig. 1 (a detailed list in given in TableS2). Larache region (S4) showed the highest diversity of taxons with the presence of D. dianthicola (20 isolates), P. polaris (6 isolates), P. brasiliense (5 isolates) and P.carotovorum (23 isolates). On the other hand, our investigations revealed two species P. brasiliense (53 isolates) and P. carotovorum (3 isolates) in the Meknes region (S1) and a single one P. brasiliense (9 isolates) in Guigo region (S2).

From the farmers, we collected different information on the potato variety, origin of the tubers seeds (local production or importation), irrigation with surface water (from a dam) or underground water and geography (for the 3 sampled regions). We tested whether a correlation between these different parameters and the diversity of pathogens (the combination of Dickeya and Pectobacterium species) existed. We used the software SAS (Statistical Analysis System, version 9.00, SAS Institute, 2002, Cary, USA) in this analysis. Statistical analysis (Qui2 test with p < 0.05) revealed that the higher diversity of pathogens was associated to three confounding factors: geography (a unique Larache region), water surface irrigation and imported seed tubers.

Draft genomes of 10 pectinolytic bacteria from northern Morocco

A draft genome (Illumina technology) was used to consolidate the taxonomic position of ten isolates belonging to the collected taxons. Between 705,755 and 17,016,482 trimmed reads were used for the contig assembly of each of the ten genomes. Characteristics of the draft genomes are presented in the Table 1. Genome data were exploited to retrieve 14 housekeeping genes from each genome using Blast. The concatenated genes were used in MLSA. The MLSA tree (Fig. 2) showed a similar topology as that generated by the gapA analysis (FigS1).

Table 1

Draft genome sequences of Pectobacterium and Dickeya strains isolated from Morocco.
Organisme	Accession N°	Genome size	N50 (pb)	Nb of contigs	Coverage	Nb of CDS	Nb of tRNAs
Pectobacterium polaris S4.16.03.2B	QZDF00000000	4,862,009	155,865	65	41	4,355	54
Pectobacterium brasiliense S1.16.01.3 k	QZDG00000000	4,946,598	146,844	74	410	4,337	36
Pectobacterium brasiliense S1.15.11.2D	QZDH00000000	4,818,836	99,392	91	420	4,206	35
Pectobacterium brasiliense S4.16.03.1C	QZDI00000000	4,944,722	139,665	74	467	4,336	37
Pectobacterium carotovorum S1-A16	QZDJ00000000	4,835,633	255,206	37	55	4,261	63
Pectobacterium versatile S4.16.03.3I	QZDK00000000	4,854,084	82,62	108	246	4,247	34
Pectobacterium versatile S4.16.03.3K	QZDL00000000	4,870,940	90,195	106	237	4,262	34
Pectobacterium versatile S4.16.03.3F	QZDM00000000	4,852,595	89,731	114	143	4,244	40
Dickeya dianthicola S4.16.03.P2.4	QZDN00000000	4,865,147	92,028	101	415	4,238	37
Dickeya dianthicola S4.16.03.LID	QZDO00000000	4,863,939	71,707	108	344	4,238	37

Genomic data were also used to calculate ANI and DDH values. Most of the Moroccan strains exhibited an ANI value higher than 95% and a DDH value higher than 70% with the closest type strain, confirming their taxonomical assignation. Strains belonging to the P. versatile clade showed an in-silico DDH lower than 70% but an ANI value higher than 95% with the strain SCC1. Polyphasic analysis confirmed the classification of P. versatile S4.16.03.3I (CFBP8660) and P. versatile S4.16.03.3F (CBBP8659) as well as P. versatile SCC1 into P. versatile species [7].

Aggressiveness of the pectinolytic bacteria from northern Morocco

Of the ten Moroccan strains of which the genome was sequenced, nine were tested for aggressiveness on potato tubers. For each strain, ten tubers were inoculated and the resulting maceration symptoms classified into five categories (Fig. 3). The aggressiveness between all the strains were compared using Kruskal-wallis test. No significant difference was observed between strains belonging to a same species. In contrast, D. dianthicola strains showed a higher aggressiveness when compared with Pectobacterium strains (P value < 0.05).

Complete genome of D. dianthicola LAR.16.03.LID

The genome of D. dianthicola LAR.16.03.LID was the first complete genome of a D. dianthicola isolate collected in Morocco and the third D. dianthicola genome in the NCBI database that already hosted that of strains ME23 and RNS04.9 (Fig. 4). Phylogenetic relationships between all D. dianthicola genomes available in NCBI were determined using MLSA. In the phylogenetical tree, the two Moroccan strains LAR.16.03.LID and S4.16.03.P2.4 appeared to be tightly related. This could be explained by the close isolation locations that were two potato fields separated by a simple road. The two Moroccan D. dianthicola clustered with D. dianthicola ME23 which has been recently collected in a potato field in USA (Fig. 4). The genomic relationship between D. dianthicola ME23 and D. dianthicola LAR.16.03.LID was confirmed using SNP/InDels calling. The variants (SNP/InDels) number in LAR.16.03.LID genome reached 12259 and 16335 using ME23 or D. dianthicola RNS04.9 genome as a reference, respectively.

The LAR.16.03.LID genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline. A graphical genome map is provided in Fig. 5. The D. dianthicola LAR.16.03.LID genome exhibited a high synteny with that of D. dianthicola strains ME23 and RNS049, with the exception of some large insertion/deletions scattered in the genomes (Fig. 6). These regions contained strain-specific genes with no counterpart in the other D. dianthicola genomes. In strains LAR.16.03LID and ME23, the analyses evidenced one strain-specific region that contained some mobile elements such as genes from transposons and prophages (Fig. 6 and TableS3). Strain RNS049 exhibited four strain-specific regions that also contain mobile elements (Fig. 6 and TableS3). Phaster analysis suggested the presence of intact prophages (Fig. 6 and TableS5). The CRISPR elements are very important features of bacterial genomes as they provide acquired immunity against viruses and plasmids [49]. The three D. dianthicola genomes hosted three or four CRISPR loci (Fig. 6 and TableS6).

The D. dianthicola LAR.16.03.LID genome exhibited a similar arsenal of virulence genes as that described for D. dianthicola RNS049 [34]. All the pectinase-encoding genes described in the model strain D. dadantii 3937 [48] were conserved in the tree D. dianthicola genomes, with the noticeable exceptions of the pehK gene (that encodes a predicted polygalacturonase) and a truncated form of the pelA gene (that encodes

Pectate lyase A). Aside macerating enzymes, genes implicated in different stage of the host infection were conserved in the three D. dianthicola strains, including those involved in the resistance to oxidative stress, acidic pH (cfa, asr) and antimicrobial peptides (arnB-T, sapABCDF), synthesis of cell envelope components (such as bscABCD and wza-wzb-wzc) and siderophore synthesis and uptake (acsF-A and cbrABCDE for achromobactin and fct-cbsCEBA for chrysobactin) [48].

We strengthened this analysis using the resistance gene identifier (RGI) and AntiSMASH softwares. We searched for genes involved in the resistance to different families of antimicrobial compounds but no differences were observed between the three D. dianthicola genomes (TableS7). The AntiSMASH analysis identified many secondary metabolite biogenesis clusters in D. dianthicola genomes that were already described in several Dickeya species like those responsible for the synthesis of siderophores, cyanobactin with cytotoxic activity, bacteriocin, NRPS, arylpolyene … (Fig. 6 and Table S4).

The main objective of this study was to characterize the pectinolytic populations isolated from symptomatic potato plants in Morocco between 2015 and 2017. A set of 119 pectinolytic bacteria belonging to the genus Pectobacterium or Dickeya were isolated and characterized using the gapA gene marker in combination with MLSA, ANI and. Most of the isolates (83%) belonged to the Pectobacterium genus: P. brasiliense species dominated in the Meknes and Guigo regions, while D. dianthicola was identified in the Larache region only.

P. brasiliense caused major economic losses to several crops (Potato, Cucumber, paprika…) in many countries including Canada, USA, South Africa, China, Korea and New Zealand [50–55]. The wide host range of this pathogen could facilitate its surviving even in harsh environments by parasitizing many alternative host plants. In several studies, P. brasiliense isolates have been shown to be more aggressive than other Pectobacterium spp., except in the case of tree Canadian strains exhibiting a low aggressiveness [56]. In our study, the P. brasiliense isolates were as virulent as the other Pectobacterium strains. P. brasiliense was described in Morocco in 2012 and by now was the dominant species in two regions (S1 and S2). In the S2 region, the farmers use tuber seeds produced in the S1 region, confirming the effective adaptation of this pathogen to the northern parts of Morocco.

The northern region (S4) exhibited the highest diversity of the pathogens: P. versatile, P. polaris described for the first time in Morocco, P. brasiliense and D. dianthicola. In this region the majority of tuber seeds were imported and the irrigation water derived from a dam: either one or both agronomic practices would contribute to a diversity of pathogens wider in the Larache region (S4) than in the other investigated regions. While previous studies identified P. carotovorum species as the most prevalent soft rot pathogens in Morocco [36, 37], our study extended the diversity of the other Pectobacterium and Dickyea, including the recently described species P. versatile [7]. This species encompasses isolates, including P. versatile strain SCC1 isolated in 1980 in Poland [57], which had been collected from a wide diversity of environments (host plants, surface waters) and geographic areas [7]. The presence of members of this clade in the potato field could be linked to irrigation, as P. versatile is also able to survive in this environment. This hypothesis remains to be investigated by sampling water from the dam. In addition, our study extended to Morocco the worldwide distribution of this species.

The international distribution of the genus Pectobacterium increases fears about the economic losses caused by this bacterium to the potato growers. In this study all tested strains were able to macerate potato tubers, in agreement with their capability to form cavities on CVP plates containing pectin. A study in Algeria carried out between 2014 and 2015 has revealed the presence of pectinolytic bacteria that causes soft rot in potato that belonged to P. brasiliense and P. carotovorum as judged by MLSA [58]. Ozturk et al. reported the presence of P. atrosepticum, P. brasiliense, P. carotovorum and P. parmentieri species in Turkey [59]. In Europe, prevalence of different species belonging to the genus Pectobacterium and Dickeya detected in diseased potato plants differs from year to year and countries, five bacterial species being the main causative agents of blackleg, namely P. atrosepticum, P. parmentieri, P. brasilense, D. solani and D. dianthicola [60].

D. dianthicola species have been detected in Morocco in 2017 [39] and been described as the main species causing losses in potato in North America [61]. More studies are needed for monitoring the spread of this highly aggressive pathogen. To reach this objective, we used Illumina and ONT sequencing technologies to assemble a complete genome of one D. dianthicola isolate that could be used, in the future, as a reference for studying clonal variability of D. dianthicola populations in Morocco and elsewhere. Comparative genomic of the tree complete genomes available indicated the presence of several clusters encoding the biosynthesis of several secondary metabolites implicated in defense against stresses or possibly important during plant-bacteria interactions. For instance, bacteriocins are small molecules with bactericidal activity usually restricted to closely related species, increasing the competition during infection, while the production of arylpolyene implicated in the protection of the bacterium from reactive oxygen species [62] has been recently described as an outcome of the study of D. fangzhongdai genomes [63].

This work constitutes the first wide scale analysis of Pectobacterium and Dickeya potato pathogen in Morocco. It provided the first genomic descritpion of Pectobacterium and Dickeya species in this country. A complete genome of the emerging D. dianthicola pathogen in Morocco was achieved by combining ONT and Illumina technologies. These data could be used as a reference for monitoring the spread of this pathogen in northern Africa and elsewhere.

Sampling and isolation of pectinolytic bacteria

In 2015, 2016 and 2017, blackleg symptoms were searched in farmer’s potato fields in 4 regions (Meknes, Guigo, Boumia and Larache) in the northern part of Morocco. Pectinolytic bacteria were isolated from symptomatic plant tissues using crystal violet pectate (CVP) medium as described previously [40]. The CVP plates were incubated at 28 °C for 3 days and colonies that had formed pits were re-streaked onto Tryptone (5 g/L) Yeast extract (3 g/L) medium (TY). The purified isolates were spotted again on CVP to check pectinolytic activity. The obtained cultures from single colonies were stored in 25% glycerol at − 80 °C.

Molecular characterization of Pectobacterium and Dickeya isolates

The primer couples Y1/Y2 and ADE1/ADE2 (Table S1) were used for the identification of isolates belonging to Pectobacterium and Dickeya genera [41, 42]. The reaction was carried out in a final volume of 25 µl, containing 1 µl of bacterial DNA (50 ng/µl), 2.5 µl of PCR buffer (10×), 2 µl of MgCl2 (25 mM), 2.5 µl of dNTPs (1 mM), 1U Taq polymerase and 1 µl of each primer (1 µM) and water. The temperature set for PCR is the same as described by [41, 42]. The analysis of PCR products was done by electrophoresis in 2% agarose gel, using PCR products of P. atrosepticum CFBP1526^T and D. solani IPO2222^T as control along with the 1 Kb DNA ladder.

Positive strains for either Y1/Y2 or ADE1/ADE2 PCR were further characterized using the gapA barcode procedure [32]. All the gapA PCR products obtained with gapA-F/gapA-R primers (Table S1) were sequenced using Sanger technology (GATC Biotech, Germany). The sequences were trimmed using CLC genomic workbench (V10.1.1) and aligned with ClustalW. The phylogenetic analysis of gapA gene was performed: the evolutionary distances were computed using the maximum composite Likelihood method (Mega7 software) with 1000 bootstrap. The obtained sequences were deposited in GenBank (TableS2).

Genome Sequencing And Assembly

Extraction of DNA

DNA of ten isolates (list in Table 1) was extracted from overnight cultures in TY medium using Epicenter kits (Madison, WI, USA) followed by an ethanol precipitation. The quantity and quality control of the DNA was completed using a NanoDrop device and agarose gel electrophoresis at 1.0%.

ILLUMINA Sequencing

Paired-end libraries (500 bp in insert size) were constructed for each strain, and DNA sequencing was performed by Illumina NextSeq technology. Sequencing of the library was carried out using 2 × 75 bp paired-end read module. Illumina sequencing was performed at the I2BC sequencing platform (Gif-sur-Yvette, France).

Oxford Nanopore Technologies Sequencing

Library preparation of D. dianthicola LAR.16.03.LID and sequencing were performed at the GeT-PlaGe core facility, INRA Toulouse, according to the manufacturer’s instructions “1D Native barcoding genomic DNA” (EXP-NBD103 and SQK-LSK108)”. At each step, DNA was quantified using the Qubit dsDNA HS Assay Kit (Life Technologies). DNA purity was tested using the nanodrop (Thermofisher) and size distribution and degradation assessed using the Fragment analyzer (AATI) High Sensitivity DNA Fragment Analysis Kit. Purification steps were performed using AMPure XP beads (Beckman Coulter). 5 µg of each DNA (5 samples) were purified then sheared at 20 kb using the megaruptor1 system (diagenode). A DNA damage repair step was performed on 3 µg of sample. Then a END-repair and dA tail of double stranded DNA fragments was performed on 1 µg of each sample. Then specific index were ligated to each sample. The library was generated by an equimolar pooling of these barcoded samples. Then adapters were ligated to the library. Library was loaded on a R9.4.1 flowcell and sequenced on MinION instrument at 0.15 pmol within 48H.

Draft genome assembly of 10 Pectobacterium and Dickeya strains isolated from Morocco

Assembly of the Illumina sequences was performed using the CLC Genomics Workbench v10.1.1 software (CLCInc, Aarhus, Denmark). After quality (quality score threshold 0.05) and length (above 40 nucleotides) trimming of the sequences, contigs were generated by de novo assembly (CLC parameters: automatic determination of the word and bubble sizes with no scaffolding). The draft genome sequences of each strain were deposited at NCBI and annotated using NCBI Prokaryotic Genome Annotation Pipeline. Statistics of the all ten draft genomes are presented in Table 1.

Whole genome assembly of D. dianthicola LAR.16.03.LID strain

Fast5s from Nanopore sequencing were obtained with MinKNOW version 1.10.23 and were basecalled with ONT Albacore Sequencing Pipeline Software version 2.1.10 and reads passing the internal test were used for subsequent analysis. Porechop 0.2.1 (https://github.com/rrwick/Porechop) was used for adaptor trimming. Illumina PE reads were processed with trim_galore 0.4.0 (https://github.com/FelixKrueger/TrimGalore) to trim adaptor sequences.

Nanopore reads were assembled using Canu 1.7 [43] with the “genomeSize = 5 m” and “minReadLength = 3000” options. For Nanopore-only assembly, one output contig was obtained, then polished three times using Pilon 1.22 (https://github.com/broadinstitute/pilon) with the “--mindepth 25” option. The contig was finally circularized using Circlator 1.5.1 (https://github.com/sanger-pathogens/circlator).

Virulence Assay In Potato Tubers

Nine bacterial strains from Morocco were cultivated in TY broth for 24 h with 125 rpm at 28 °C, the strains P. brasiliense S4.16.03.1C has been isolated from the same field as P. brasiliense S1.16.01.3K and show 100 of ANI value was deleted from the aggressiveness test. Bacterial cultures were washed twice and suspended in NaCl 0.85%, and the optical density was adjusted to OD₆₀₀ = 1.0 using a spectrophotometer. Potato tubers (cv. Bintje) were surface-disinfected by submerging them into a 5% sodium hypochlorite solution for 5 min, and then rinsed in distilled water twice and air dried at room temperature one day before inoculation. Ten potato tubers were infected with 10 µl of cell suspension of each strain, along with 10 tubers with NaCl 0.8% alone as a negative control. After 5 days of incubation at 24 °C, the tubers were cut vertically through the inoculation points and classified into five aggressiveness categories. Virulence assays were statistically analyzed to infer the aggressiveness variability within strains on potato tubers. Significance of the observed differences was assessed using a Kruskal-Wallis test with p < 0.05.

Genome Analysis

Phylogenetic and molecular evolutionary analyses were conducted using MEGA, version 7. An MLSA was performed using 13 concatenated housekeeping genes (fusA, rpoD, acnA, purA, gyrB, recA, mdh, mtlD, groEL, secY, glyA, gapA, rplB) retrieved from all the Pectobacterium spp. and Dickeya spp. strains in order to confirm their phylogenetic position within the reference strains P. atrosepticum ICMP1526^T, P. betavasculorum NCPPB2795^T, P. parmentieri RNS 08-42.1A^T, P. wasabiae CFBP 3304^T, P. actinidiae KKH3, P. brasiliense LMG21371^T, P. odoriferum BCS7, P. aroidearum PC1, D. dianthicola NCPPB 453^T, D. dadantii NCPPB 898^T, D. solani IPO2222^T. The average nucleotide identity (ANI) value was calculated as previously proposed using the ANI calculator (http://enveomics.ce.gatech.edu/ani/). The in-silico DNA-DNA hybridization (isDDH) was evaluated using genome sequence-based species delineation (http://ggdc.dsmz.de/) (Table 2).

Table 2

Pairwise ANI and isDDH values of Pectobacterium and Dickeya strains isolated from Morocco.
	ANI values
Strains	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16
1-Pp NIBIO 1006^T		96.8	92.9	92.9	93.5	93.7	93.5	93.7	93.4	93.4	93.5	93.5	92.2	79.3	79	79.1
2-Pp S4.16.03.2B	73.30		93	92.9	93.5	93.7	93.5	93.7	93.4	93.5	93.5	93.4	92.2	78.9	79	79
3-Pc ICMP5702^T	52.3	52.10		97.2	92.6	92.9	92.8	92.8	95.1	95.2	95.2	95.2	94.8	78.9	78.9	78.9
4-Pc S1-A16	52.2	51.90	76.30		92.6	92.9	92.7	92.9	95.3	95.3	95.3	95.3	94.8	78.8	79.6	79.6
5-Pb LMG 21371^T	54.4	54.5	50.9	50.9		96.1	95.9	96.1	92.2	92.2	92.2	92.3	93.00	78.6	79.5	79.5
6-Pb S4.16.03.1C	56	55.7	52	51.7	68.5		96.3	100	92.3	91.2	91.1	91.2	91.9	78.7	79.6	79.6
7-Pb S1.15.11.2D	54.7	54.2	51.3	51	67.1	69.4		96.3	92.3	92.2	92.3	92.3	91.8	79.1	79.1	79.1
8-Pb S1.16.01.3K	56	55.7	76	51.7	68.5	100	71.4		92.4	92.4	92.4	92.3	91.9	79.6	79.6	79.7
9-Pv SCC1	54.3	54.3	63.5	63.9	48.9	49.8	49.5	49.3		99.5	99.5	99.5	94.7	79.2	79.1	79.3
10-Pv S4.16.03.3F	54.4	54.3	63.9	64.3	49.1	49.9	49.5	49.9	96.6		100	100	94.7	79.2	79.2	79.2
11-Pv S4.16.03.3 k	54.4	54.3	63.8	64.2	49.2	49.9	49.5	49.9	96.6	99.3		100	94.7	79.3	79.3	79.4
12-Pv S4.16.03.3I	54.4	54.3	63.8	64.2	49.1	49.9	49.5	49.8	96.5	100	99.3		94.7	79.3	79.3	79.3
13-Po BCS7	49.2	49	61.3	60.6	47.4	47.8	47.6	47.8	60.4	60.7	60.6	60.6		79.1	79.1	79.1
14-Ddi NCPPB453^T	21.1	20.7	20.5	20.7	20.9	21	20.6	21	21.2	21	21	21	21.1		99.5	99.5
15-Ddi S4.16.03.P2.4	20.8	20.6	20.4	20.9	21.1	20.9	20.7	20.9	21	21	21	21	20.8	95.6		100
16-Ddi S4.16.03.LID	20.8	20.6	20.4	21	21.1	20.9	20.6	20.9	21	21	21	21	20.8	95.6	100
	is-DDH
Strains: 1, P. polaris NIBIO1006^T ; 2, P. polaris S4.16.03.2B 3, P. carotovorum ICMP5702^T; 4, P. carotovorum S1-A16; 5, P. brasiliense LMG21371^T; 6, P. brasiliense S4.16.03.1C; 7, P. brasiliense S1.15.11.2D 8, P. brasiliense S1.16.01 3K; 9, P. versatile SCC1; 10, P. versatile S4.16.03.3F; 11, P. versatile S4.16.03.3 k 12, P. versatile S4.16.03.3I; 13, P. odoriferum BCS7; 14, D. dianthicola NCPPB 453T, 15, D. dianthicola S4.16.03.P2.4; 16, D. dianthicola S4.16.03.LID.

The genome map of the D. dianthicola LAR.16.03.LID was generated using CGView Server [44]. Synteny analysis of the complete genomes of D. dianthicola LAR.16.03.LID, D. dianthicola ME23 and D. dianthicola RNS049 was performed by using the MAUVE software [45]. Paired end reads for the strain LAR.16.03.LID was mapped against the two complete genome sequences of the D. dianthicola strains ME23 and RNS049 with threshold (0.8 of identity on 0.5 of read length) using CLC Genomics Workbench version 10.1.1 software. The mappings were used for detection of variations (SNPs and InDels) using basic variant calling tool from CLC genomic workbench version 10.1.1.

The presence of clustered regularly interspaced short palindromic repeats (CRISPRs) was done using CRISPRfinder (http://crispr.i2bc.paris-saclay.fr/Server/) [46]. The prophage identification tool PHAge Search Tool – Enhanced Release (PHASTER) was used to check for the region containing prophage-like elements in bacterial genomes (http://phaster.ca/) [47]. The Predicted resistome was checked using Resistance Gene Identifier tool (https://card.mcmaster.ca/analyze/rgi). Finally the presence of genomic regions containing secondary metabolite biosynthesis gene cluster were identified using AntiSMASH server (version 4.1.0, https://doi.org/10.1093/nar/gkv437).

To investigate the phylogenitical position of the Moroccan D. dianthicola against the available genomes of this specie in NCBI. An MLSA was generated using fifteen housekeeping gene (fusA, rpoD, leuS, rpoS, purA, infB, gyrB, recA, groEL, secY, glyA, gapA, rplB, dnaX, gyrA) using MEGA7 software.

ANI: Average nucleotide identity; isDDH: In-silico DNA-DNA hybridization; CDS: Coding DNA sequence; InDel: Insertion deletion; MLSA: Multi-locus sequence analysis; NCBI: National center for biotechnology information; SNP: Single nucleotide polymorphism; ONT: Oxford Nanopore Technologies

Ethics approval and consent to participate: NA
Consent for publication: All authors approved the ﬁnal manuscript.
Availability of data and materials: The datasets supporting the conclusions of this article are included within the article and its additional files.
Competing interests: The authors declare no conflict of interest.
Funding: This work was supported by national agency for research (ANR-15-CE21-0003). And cooperative project between France and Morocco (PRAD 14-02, Campus France n°30229ZK), I2BC was supported by the LabExSaclay Plant Sciences-SPS (ANR-10-LABX-0040-SPS).
Authors’ contributions: DF designed the experiment, DF and MM supervised the data production and analysis, SO carried out isolation, taxonomical characterization and genome assembly and analysis, SK participate partially in sanger sequence analysis, SO and SS conducting potato maceration assays, KR and SK conducted statistical analysis, CLR and CV carried out the ONT sequencing and assembly of the complete genome, SO and DF write the manuscript.
Acknowledgements: NA
Authors' information (optional): NA

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FigS1: Phylogenetic analysis of the gapA barcode.

Table S1: Primers used in this study

Table S2: Strains of Pectobacterium and Dickeya isolated from different regions in Morocco

Table S3-1: Strain-specific region number 1 identified by Mauve

Table S3-2: Strain-specific region number 2 identified by Mauve

Table S3-3: Strain-specific region number 3 identified by Mauve

Table S3-4: Strain-specific region number 4 identified by Mauve

Table S3-5: Strain-specific region number 5 identified by Mauve

Table S3-6: Strain-specific region number 6 identified by Mauve

Table S4: Secondary metabolite gene clusters identified with antismath

Table S5: Prophage in Dickeya dianthicola identified using Phaster

Table S6: CRISPR identification in Dickeya dianthicola using CRISPER Finder

Table S7: Resistome genes identified in Dickeya dianthicola genomes

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Dickeya dianthicola emerged from a potato culture area exhibiting a wide diversity of pectinolytic pathogens in northern Morocco

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