Efficacy of Soft-Rot Disease Biocontrol Agents in the Inhibition of Production Field Pathogen Isolates.

The Dickeya and Pectobacterium bacterial species cause blackleg and soft-rot diseases on potato plants and tubers. Prophylactic actions are essential to conserve a high quality of seed potato tubers. Biocontrol approaches are emerging, but we need to know how efficient biocontrol agents are when facing the natural diversity of pathogens. In this work, we sampled 16 production fields, which were excluded from the seed tuber certification scheme, as well as seven experimental parcels, which were planted with seed tubers from those production fields. We collected and characterized 669 Dickeya and Pectobacterium isolates, all characterized using nucleotide sequence of the gapA gene. This deep sampling effort highlighted eleven Dickeya and Pectobacterium species, including four dominant species namely D. solani, D. dianthicola, P. atrosepticum and P. parmentieri. Variations in the relative abundance of pathogens revealed different diversity patterns at a field or parcel level. The Dickeya-enriched patterns were maintained in parcels planted with rejected seed tubers, suggesting a vertical transmission of the pathogen consortium. Then, we retained 41 isolates representing the observed species diversity of pathogens and we tested each of them against six biocontrol agents. From this work, we confirmed the importance of prophylactic actions to discard contaminated seed tubers. We also identified a couple of biocontrol agents of the Pseudomonas genus that were efficient against a wide range of pathogen species.

European Population of Pectobacterium punjabense: Genomic Diversity, Tuber Maceration Capacity and a Detection Tool for This Rarely Occurring Potato Pathogen.

Enterobacteria belonging to the Pectobacterium and Dickeya genera are responsible for soft rot and blackleg diseases occurring in many crops around the world. Since 2016, the number of described species has more than doubled. However, some new species, such as Pectobacterium punjabense, are often poorly characterized, and little is known about their genomic and phenotypic variation. Here, we explored several European culture collections and identified seven strains of P. punjabense. All were collected from potato blackleg symptoms, sometimes from a long time ago, i.e., the IFB5596 strain isolated almost 25 years ago. We showed that this species remains rare, with less than 0.24% of P. punjabense strains identified among pectinolytic bacteria present in the surveyed collections. The analysis of the genomic diversity revealed the non-clonal character of P. punjabense species. Furthermore, the strains showed aggressiveness differences. Finally, a qPCR Taqman assay was developed for rapid and specific strain characterization and for use in diagnostic programs.

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.

Elevation of Pectobacterium carotovorum subsp. odoriferum to species level as Pectobacterium odoriferum sp. nov., proposal of Pectobacterium brasiliense sp. nov. and Pectobacterium actinidiae sp. nov., emended description of Pectobacterium carotovorum and description of Pectobacterium versatile sp. nov., isolated from streams and symptoms on diverse plants.

The Pectobacteriumcarotovorum species corresponds to a complex, including two subspecies with validly published names, two proposed subspecies and two new species, Pectobacterium polaris and Pectobacterium aquaticum. Recent studies suggested that this complex needed revision. We examined the taxonomic status of 144 Pectobacterium strains isolated from a wide range of plant species, various geographical origins and waterways. Sequences of the leuS, dnaX and recA housekeeping genes clustered 114 of these Pectobacterium strains together within a not yet described clade. We sequenced eight strains of this clade and analysed them together with the 102 Pectobacterium genomes available in the NCBI database. Phylogenetic analysis, average nucleotide identity calculation and in silico DNA-DNA hybridization allowed us to differentiate seven clades. This led us to propose the elevation of Pectobacterium carotovorumsubsp. odoriferum to species level as Pectobacteriumodoriferum sp. nov. (type strain CFBP 1878T=LMG 5863T=NCPPB 3839T=ICMP 11533T), the proposal of Pectobacteriumactinidiae sp. nov. (type strain KKH3=LMG 26003 T=KCTC 23131T) and Pectobacteriumbrasiliense sp. nov. (type strain CFBP 6617T= LMG 21371T=NCPPB 4609T), to emend the description of Pectobacterium carotovorum (type strain CFBP 2046T=LMG 2404T=NCPPB 312T=ICMP 5702T), and to propose a novel species, Pectobacterium versatile sp. nov (type strain CFBP6051T= NCPPB 3387T=ICMP 9168T) which includes the strains previously described as 'Candidatus Pectobacterium maceratum'. Phenotypic analysis performed using Biolog GENIII plates on eight strains of P. versatile sp. nov. and related strains completed our analysis.

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.

Genome Sequence of the Potato Plant Pathogen Dickeya dianthicola Strain RNS04.9.

Dickeya dianthicola is one of the causative agents of soft rot and blackleg diseases, which are currently identified in European countries in a wide range of crops. Here, we report the draft genome sequence of D. dianthicola strain RNS04.9, which was isolated from a potato plant with blackleg symptoms in 2004.

Draft Genome Sequences of the Three Pectobacterium-Antagonistic Bacteria Pseudomonas brassicacearum PP1-210F and PA1G7 and Bacillus simplex BA2H3.

Pectobacterium spp. are bacterial pathogens causing soft rot diseases on a wide range of plants and crops. We present in this paper the draft genome sequences of three bacterial strains, Pseudomonas brassicacearum PP1-210F and PA1G7 and Bacillus simplex BA2H3, which exhibit antagonistic activities against the Pectobacterium plant pathogens.

Draft Genome Sequences of Pseudomonas fluorescens Strains PA4C2 and PA3G8 and Pseudomonas putida PA14H7, Three Biocontrol Bacteria against Dickeya Phytopathogens.

Pseudomonas fluorescens strains PA4C2 and PA3G8 and Pseudomonas putida strain PA14H7 were isolated from potato rhizosphere and show an ability to inhibit the growth of Dickeya phytopathogens. Here, we report their draft genome sequences, which provide a basis for understanding the molecular mechanisms involved in antibiosis against Dickeya.

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.

Genome Sequence of the Emerging Plant Pathogen Dickeya solani Strain RNS 08.23.3.1A.

Here we present the genome sequence of Dickeya solani strain RNS 08.23.3.1A (PRI3337), isolated from Solanum tuberosum. Dickeya solani, recently described on potato cultures in Europe, is a proposed new taxon closely related to the Dickeya dianthicola and Dickeya dadantii species.

Dickeya solani sp. nov., a pectinolytic plant-pathogenic bacterium isolated from potato (Solanum tuberosum).

Pectinolytic bacteria have been recently isolated from diseased potato plants exhibiting blackleg and slow wilt symptoms found in a number of European countries and Israel. These Gram-reaction-negative, motile, rods were identified as belonging to the genus Dickeya, previously the Pectobacterium chrysanthemi complex (Erwinia chrysanthemi), on the basis of production of a PCR product with the pelADE primers, 16S rRNA gene sequence analysis, fatty acid methyl esterase analysis, the production of phosphatases and the ability to produce indole and acids from α-methylglucoside. Differential physiological assays used previously to differentiate between strains of E. chrysanthemi, showed that these isolates belonged to biovar 3. Eight of the isolates, seven from potato and one from hyacinth, were analysed together with 21 reference strains representing all currently recognized taxa within the genus Dickeya. The novel isolates formed a distinct genetic clade in multilocus sequence analysis (MLSA) using concatenated sequences of the intergenic spacer (IGS), as well as dnaX, recA, dnaN, fusA, gapA, purA, rplB, rpoS and gyrA. Characterization by whole-cell MALDI-TOF mass spectrometry, pulsed field gel electrophoresis after digestion of whole-genome DNA with rare-cutting restriction enzymes, average nucleotide identity analysis and DNA-DNA hybridization studies, showed that although related to Dickeya dadantii, these isolates represent a novel species within the genus Dickeya, for which the name Dickeya solani sp. nov. (type strain IPO 2222(T) = LMG25993(T) = NCPPB4479(T)) is proposed.

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.

N-acyl homoserine lactones in diverse Pectobacterium and Dickeya plant pathogens: diversity, abundance, and involvement in virulence.

Soft-rot bacteria Pectobacterium and Dickeya use N-acyl homoserine lactones (NAHSLs) as diffusible signals for coordinating quorum sensing communication. The production of NAHSLs was investigated in a set of reference strains and recently-collected isolates, which belong to six species and share the ability to infect the potato host plant. All the pathogens produced different NAHSLs, among which the 3-oxo-hexanoyl- and the 3-oxo-octanoyl-L-homoserine lactones represent at least 90% of total produced NAHSL-amounts. The level of NAHSLs varied from 0.6 to 2 pg/cfu. The involvement of NAHSLs in tuber maceration was investigated by electroporating a quorum quenching vector in each of the bacterial pathogen strains. All the NAHSL-lactonase expressing strains produced a lower amount of NAHSLs as compared to those harboring the empty vector. Moreover, all except Dickeya dadantii 3937 induced a lower level of symptoms in potato tuber assay. Noticeably, aggressiveness appeared to be independent of both nature and amount of produced signals. This work highlights that quorum sensing similarly contributed to virulence in most of the tested Pectobacterium and Dickeya, even the strains had been isolated recently or during the past decades. Thus, these key regulatory-molecules appear as credible targets for developing anti-virulence strategies against these plant pathogens.

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.

Production of recombinant Potato mop-top virus coat protein in Escherichia coli and generation of antisera recognising native virus protein.

Potato mop-top virus (PMTV, Pomovirus) is difficult to detect because it is unevenly distributed and present at low concentration in infected tissues. The production of PMTV-free seed relies on sensitive and specific detection methods of virus detection, including serological methods. The possibility of using a PMTV recombinant coat protein (CP) as an antigen for antiserum production was investigated. The region encoding the PMTV CP was inserted into pET3A, expressed in Escherichia coli, and the recombinant PMTV CP produced was used to raise antibodies in rabbits. Three antisera were produced. All recognised efficiently the recombinant CP in Western blot analysis and the most sensitive antiserum (H5003) detected native CP on Western blots and in ELISA. Thus, recombinant CP can be used as an alternative to purified virus for the production of specific antibodies against PMTV.