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

Resistance of Plasmopara viticola to QoI fungicides: variability and competitiveness

Depuis son introduction en Europe Plasmopara viticola (mildiou de la vigne) développe régulièrement des résistances aux produits phytosanitaires, y compris à des molécules récentes telles que les inhibiteurs de la respiration mitochondriale de type QoI (Quinone outside Inhibitor). L’analyse du gène codant pour l’enzyme cible (cytochrome b) révèle la présence d’une mutation majeure en position G143A, impliquée dans l’acquisition de cette résistance. L’étude de la variabilité de cette zone du génome mitochondrial démontre l’existence d’au moins deux phénomènes d’apparition de la résistance indépendants chez le mildiou de la vigne en Europe. À partir de 1015 isolats de mildiou collectés dans le vignoble français en 2003 et 2004, nous montrons la prévalence de 4 haplotypes majeurs que nous avons appelés IS, IR, IIS et IIR. L’haplotype I est le plus fréquent (77,44%). La distribution des allèles de résistance en début de saison représente 23,25% en France. L’évaluation de différents paramètres impliqués dans le développement de l’agent pathogène durant son cycle de reproduction asexuée et la mise au point d’un indice de fitness (IA) ont permis d’estimer le coût de la résistance. Des tests de compétitivité réalisés entre une souche sensible et deux résistantes aux QoIs complètent cette étude qui montre que les souches résistantes possèdent un aussi bon potentiel de survie que les souches sensibles durant la phase asexuée en conditions de laboratoire, lequel dépend essentiellement de leur fitness.The efficacy of QoI fungicides against grape downy mildews in European vineyards has decreased significantly in the last years. A single nucleotide polymorphism, G143A in the cytochrome b gene of Plasmopara viticola was detected to confer QoI resistance. Polymorphism analyses on the mitochondrial genome showed that 4 major haplotypes (IR, IS, IIR, IIS) coexisted in French vineyards. In France, the most frequent haplotype (IR, IS) reached 77.44% in P. viticola populations. The resistant alleles (IR, IIR) frequencies ranged from 0 to 75% with an average of 23.25%. Based on determination of the fitness index (Fi), QoI-resistant strains did not exhibit a cost and often tended to have good fitness. To assess these results, competition assays with different mixtures of sensitive and resistant strains using biological and molecular (Q-PCR) tests were done. The competitiveness of resistant isolates varied according to their aggressiveness index, suggesting that there is no noticeable cost of QoI resistance in controlled conditions in P. viticola

Resistance of Plasmopara viticola: diversity and fitness of resistant isolates to QoI fungicides

International audienc

Diversity and fitness of Plasmopara viticola isolates resistant to QoI fungicides

The effectiveness of Quinone outside Inhibitor (QoI) fungicides against grape downy mildew in European vineyards has significantly decreased in the last decade. One nucleotide polymorphism, G143A in the cytochrome b gene of Plasmopara viticola, is involved in resistance to QoIs. Previous genetic examination on the mitochondrial genomes showed four major haplotypes (IR, IS, IIR, IIS) coexisting in European vineyards. A resistant allele (G143A) was present in IR and IIR haplotypes. The purpose of the present study was to estimate the diversity of the different mitochondrial haplotypes and their distribution in QoI-resistant populations before evaluating the potential cost of the resistant mutation G143A in P. viticola population. From 2000 to 2004, the frequencies of resistant isolates ranged from 0% to 23.25% with an average of 4.64 % among the populations examined. To evaluate the fitness of sensitive and resistant isolates, a comparison of different biological parameters including latent period, spore production and infection frequency was performed, enabling a fitness index (FI) to be determined. Resistant isolates exhibited greater infection frequency than sensitive isolates, whereas no significant difference was found in sporulation ability and latent period between sensitive and resistant isolates. To further investigate competitiveness among isolates, an assay including two resistant isolates in different proportion with a sensitive isolate was conducted on eight asexual growing cycles in the absence of a QoI fungicide. The competitiveness of resistant isolates varied according to their fitness parameters, suggesting that there is no noticeable cost of QoI resistance in controlled conditions in Plasmopara viticola

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. © 2019 IUMS

A natural single nucleotide mutation in the small regulatory RNA ArcZ of Dickeya solani switches off the antimicrobial activities against yeast and bacteria

International audienceThe necrotrophic plant pathogenic bacterium Dickeya solani emerged in the potato agrosystem in Europe. All isolated strains of D . solani contain several large polyketide synthase/non-ribosomal peptide synthetase (PKS/NRPS) gene clusters. Analogy with genes described in other bacteria suggests that the clusters ooc and zms are involved in the production of secondary metabolites of the oocydin and zeamine families, respectively. A third cluster named sol was recently shown to produce an antifungal molecule. In this study, we constructed mutants impaired in each of the three secondary metabolite clusters sol , ooc , and zms to compare first the phenotype of the D . solani wild-type strain D s0432-1 with its associated mutants. We demonstrated the antimicrobial functions of these three PKS/NRPS clusters against bacteria, yeasts or fungi. The cluster sol , conserved in several other Dickeya species, produces a secondary metabolite inhibiting yeasts. Phenotyping and comparative genomics of different D . solani wild-type isolates revealed that the small regulatory RNA ArcZ plays a major role in the control of the clusters sol and zms . A single-point mutation, conserved in some Dickeya wild-type strains, including the D . solani type strain IPO 2222, impairs the ArcZ function by affecting its processing into an active form

Complete Genome Sequence of the Type Strain Pectobacterium punjabense SS95, Isolated from a Potato Plant with Blackleg Symptoms.

Pectobacterium punjabense is a newly described species causing blackleg disease in potato plants. Therefore, by the combination of long (Oxford Nanopore Technologies, MinION) and short (Illumina MiSeq) reads, we sequenced the complete genome of P. punjabense SS95T, which contains a circular chromosome of 4.793 Mb with a GC content of 50.7%

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

International audienceInvasive 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