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Blackleg disease, caused by Leptosphaeria maculans, greatly affects the production of cabbage (Brassica oleracea). However, definitive R-gene(s) are yet to be identified in this crop. In contrast, a number of R-loci have been identified in A- or B-genome crops. Identification of few resistant cabbage genotypes indicates the presence of R-genes in this C-genome crop. High ancestral synteny between Brassica genomes suggests that the collinear regions of known A- or B-genome R-loci may also contain functional R-genes in the C-genome. Strong resistance was observed in the cotyledons of cabbage inbred line SCNU-98 against two L. maculans isolates, 03–02 s and 00–100 s. We investigated the collinear region of the Brassica napus blackleg resistance locus LepR2' in B. oleracea since both isolates of L. maculans contain corresponding avirulence genes. The locus was collinear to a 5.8 Mbp genomic segment of B. oleracea chromosome C09 containing 13 genes that have putative disease resistance-related domains. High expression of genes Bo9g117290 and Bo9g111510 against isolate 00–100 s, and high expression of genes Bo9g126150 and Bo9g111490 against both isolates in the resistant-line SCNU-98 indicate their putative roles in blackleg resistance, which remained to be functionally verified. This work enhances our understanding of R-gene-mediated resistance to blackleg in cabbage. [ABSTRACT FROM AUTHOR]

Potato blackleg is a seedborne disease that can cause significant economic losses for growers. Disease development depends mainly on two drivers, namely seed inoculum and local climatic conditions. To better establish the relationship between these two drivers, blackleg development was monitored in Swiss field trials at multiple locations from 2010 to 2013 involving three sets of naturally infected seed lots planted in each of three locations. The seed lot itself was thereby the most important factor explaining differences in disease development, rather than environmental factors. In a further on‐farm project conducted at various locations in Switzerland and southern Germany from 2013 to 2015, the implementation of a seed‐testing procedure was investigated. A total of 177 seed lots were tested for natural latent infection with soft rot Pectobacteriaceae and the corresponding blackleg incidence was tracked in 242 fields. The reliability of the relationship between latent infection and field incidence was found to be strongly linked to the bacterial species. Dickeya spp. field infection could be predicted with an acceptable reliability, whereas Pectobacterium carotovorum subsp. brasiliense, even when detected as latent tuber infection, was not consistently expressed as visual blackleg. Moreover, commonly found mixed latent infections with several bacterial species made it even harder to predict which bacteria would cause blackleg symptoms. Finally, variability in the reliability of seed testing may also be explained by differences in local farming practices. These trials over several years with naturally infected potato seed highlight the usefulness and limits of seed testing to manage blackleg. [ABSTRACT FROM AUTHOR]

Aims: The aim of this study was to isolate soilborne AHL degrading actinobacteria that can be used as biocontrol agents against the quorum sensing mediated virulence of Pectobacterium carotovorum subsp. carotovorum BR1 in in vitro and in planta studies. Methods: Actinobacterial strains capable of N-acyl homoserine lactone (AHL) degradation, Glutamicibacter nicotianae AI5a and Rhodococcus pyridinivorans AI4 were isolated and screened using Chromobacterium violaceum CV026 as a biosensor in an AHL degradation bioassay. The effect of AHL degradation by the potential biocontrol strains was studied by a co-culture assay. Reduction in plant cell wall degrading enzymes produced by PccBR1 due to the quorum quenching activity of the potential biocontrol strains was studied using colorimetric assays. The ability of the isolates to reduce soft rot symptoms was studied in in vitro cucumber and potato maceration assays. A cucumber plant infection model was developed to study blackleg in greenhouse experiments. Results: G. nicotianae AI5a, R. pyridinivorans AI4 and R. erythropolis CRD13.3C were able to reduce 3-oxo-C6- N-acyl homoserine lactone and production of plant cell wall degrading enzymes by PccBR1. The isolates were also able to prevent and attenuate soft rot caused by PccBR1 in vitro in potato and cucumber maceration assays. The isolates were also able to prevent blackleg in an in planta cucumber infection model. Conclusions: Among the three actinobacterial isolates, G. nicotianae AI5a showed best results and has immense potential as a biocontrol agent against the virulence of the quorum sensing phytopathogen PccBR1. [ABSTRACT FROM AUTHOR]

The commonly occurring blackleg is an economically important disease in oilseed rape cultivation. This disease is caused by a complex of two closely related species, Plenodomus lingam and P. biglobosus. To date, only P. lingam (syn.: Leptosphaeria maculans) has been known in Hungary as the cause of blackleg in oilseed rape crops. The present study aimed to determine if P. biglobosus (syn.: Leptosphaeria biglobosa) was present in Hungary. The two fungus pathogens are difficult to distinguish using conventional morphological criteria. Reliable detection and differentiation of the two species can only be achieved using molecular methods. This is the first report describing the pathogen, P. biglobosus, in Hungary. [ABSTRACT FROM AUTHOR]

In this study, potato plants displaying symptoms of blackleg and soft rot were collected in Guangdong Province, China, and the causative bacterial pathogens were isolated from symptomatic tissues. In total, five pathogenic isolates (Po-2, Po-3, Po-4, Po-10, and Po-11) were selected and preliminarily identified as Pectobacterium spp. based on 16S rRNA gene sequencing and detection of the pectate-lyase gene using species-specific primers. Physiological and biochemical tests revealed that the five isolates grew at 37 °C, utilized D-galactose, D-lactose, maltose, melibiose, raffinose, sodium citrate, sucrose and trehalose, but not inosine, inulin, malonate, sorbitol and were sensitive to erythromycin. All isolates were negative for acid production from α-methyl glucoside and production of reducing substances from sucrose. In addition, Po-2, Po-3, and Po-4 utilized Tween 80 and produced indole but did not utilize arabinose, while Po-10 and Po-11 did not use Tween 80 and did not produce indole but utilized arabinose. Pathogenicity assays showed that Po-2, Po-3, and Po-4 infected 32 plant species across 19 families, while Po-10 and Po-11 infected 24 species from 17 families. 16S rDNA and phylogenetic analysis based on eight housekeeping genes (acnA, gapA, icdA, mdh, mtlD, pgi, proA, and rpoS) showed that Po-2, Po-3, and Po-4 clustered with Pectobacterium carotovorum subsp. brasiliensis assignments confirmed using specific primer sets BR1f/L1r, while Po-10 and Po-11 clustered with Pectobacterium parmentieri. This is the first detailed report on P. carotovorum subsp. brasiliensis and P. parmentieri as causative agents of potato blackleg and soft rot in China. [ABSTRACT FROM AUTHOR]

Background: The fungal pathogen Leptosphaeria maculans (Lm). causes blackleg disease on canola/rapeseed in many parts of the world. It is important to use resistant cultivars to manage the disease and minimize yield losses. In this study, twenty-two Lm isolates were used to identify resistance genes in a collection of 243 canola/rapeseed (Brassica napus L.) accessions from Canada and China. These Lm isolates carry different compliments of avirulence genes, and the investigation was based on a genome-wide association study (GWAS) and genotype-by-sequencing (GBS). Results: Using the CROP-SNP pipeline, a total of 81,471 variants, including 78,632 SNPs and 2839 InDels, were identified. The GWAS was performed using TASSEL 5.0 with GLM + Q model. Thirty-two and 13 SNPs were identified from the Canadian and Chinese accessions, respectively, tightly associated with blackleg resistance with P values < 1 × 10− 4. These SNP loci were distributed on chromosomes A03, A05, A08, A09, C01, C04, C05, and C07, with the majority of them on A08 followed by A09 and A03. The significant SNPs identified on A08 were all located in a 2010-kb region and associated with resistance to 12 of the 22 Lm isolates. Furthermore, 25 resistance gene analogues (RGAs) were identified in these regions, including two nucleotide binding site (NBS) domain proteins, fourteen RLKs, three RLPs and six TM-CCs. These RGAs can be the potential candidate genes for blackleg resistance. Conclusion: This study provides insights into potentially new genomic regions for discovery of additional blackleg resistance genes. The identified regions associated with blackleg resistance in the germplasm collection may also contribute directly to the development of canola varieties with novel resistance genes against blackleg of canola. [ABSTRACT FROM AUTHOR]

Erwinia carotovora and E. chrysanthemi are the most important of the pectolytic bacteria that cause maceration of plant tissue and disease of many crop plants including potato. Several years ago it is revived the suggestion that the pectolytic bacteria be placed in a separate genus, Pectobacterium, on the basis of 16S rDNA sequences, but the generic epithet, Erwinia, remains the preferred designation in the scientific literature. [ABSTRACT FROM AUTHOR]

Introduction: There is consensus regarding the importance of blackleg vaccination as a preventive measure, and proper immunization protocols are available. However, few studies have evaluated the effectiveness of vaccine protection against Clostridium chauvoei and the treatment of the disease in calves exhibiting early or advanced clinical courses. This study describes twelve blackleg cases in unvaccinated calves and in calves that received a single dose of the vaccine. It also reports the recovery of some calves after antibiotic therapy. Methodology: Two necropsies of cattle dead from blackleg were performed. Fragments of skeletal muscle from these two cattle were immersed in paraffin for multiplex polymerase chain reaction (PCR) analysis. Results: Twelve calves up to nine months of age developed signs of blackleg and eight died. Ten of those 9-month-old calves had received only the first dose of a blackleg vaccine at 4 months of age, but no booster. The last two affected calves belonged to a herd that had never been vaccinated. Four out of five calves treated with penicillin for 6-7 days recovered from the disease. The diagnosis of blackleg was based on necropsy, histopathological findings and detection of C. chauvoei in skeletal muscle samples of two necropsied calves using PCR. Conclusions: The occurrence of cases only in calves that did not receive a booster dose or were not vaccinated indicated that the vaccine used was effective when performed as recommended by the manufacturer. However, neglecting the booster resulted in casualties due to blackleg. [ABSTRACT FROM AUTHOR]

Background: Dickeya solani is an important plant pathogenic bacterium causing severe losses in European potato production. This species draws a lot of attention due to its remarkable virulence, great devastating potential and easier spread in contrast to other Dickeya spp. In view of a high need for extensive studies on economically important soft rot Pectobacteriaceae, we performed a comparative genomics analysis on D. solani strains to search for genetic foundations that would explain the differences in the observed virulence levels within the D. solani population. Results: High quality assemblies of 8 de novo sequenced D. solani genomes have been obtained. Whole-sequence comparison, ANIb, ANIm, Tetra and pangenome-oriented analyses performed on these genomes and the sequences of 14 additional strains revealed an exceptionally high level of homogeneity among the studied genetic material of D. solani strains. With the use of 22 genomes, the pangenome of D. solani, comprising 84.7% core, 7.2% accessory and 8.1% unique genes, has been almost completely determined, suggesting the presence of a nearly closed pangenome structure. Attribution of the genes included in the D. solani pangenome fractions to functional COG categories showed that higher percentages of accessory and unique pangenome parts in contrast to the core section are encountered in phage/mobile elements- and transcription- associated groups with the genome of RNS 05.1.2A strain having the most significant impact. Also, the first D. solani large-scale genome-wide phylogeny computed on concatenated core gene alignments is herein reported. Conclusions: The almost closed status of D. solani pangenome achieved in this work points to the fact that the unique gene pool of this species should no longer expand. Such a feature is characteristic of taxa whose representatives either occupy isolated ecological niches or lack efficient mechanisms for gene exchange and recombination, which seems rational concerning a strictly pathogenic species with clonal population structure. Finally, no obvious correlations between the geographical origin of D. solani strains and their phylogeny were found, which might reflect the specificity of the international seed potato market. [ABSTRACT FROM AUTHOR]

Blackleg is a devastating disease of winter oil seed rape (WOSR) caused by the two species Leptosphaeria maculans and L. biglobosa "brassicae". In this study, Loop-Mediated Isothermal Amplification (LAMP) assays were developed using two primer sets, SirP and PKS5, to identify and monitor L. maculans "brassicae", and one primer set PKS21 to monitor L. biglobosa "brassicae". The three primer sets were tested in real-time LAMP with DNA templates from 38 Leptosphaeria spp. reference isolates. The SirP and PKS5 primer sets exclusively amplified DNA from L. maculans isolates whereas, the PKS21 primers amplified DNA solely from L. biglobosa. Further specificity tests with DNA templates from other ascomycetes showed that both PKS primers were highly specific to Leptosphaeria. The SirP primer set produced in a few cases non-specific amplifications, but they were easily differentiated from the positive reactions by the differences in melting curve temperatures. The detection limits of LAMP assays with PKS21, PKS5 and SirP primers were at 100 fg, 30 pg and 180 fg DNA, respectively. DNA from leaf segments with typical L. maculans lesions was successfully amplified using primer set SirP, as was visually indicated by the colour change of phenol red from pink to yellow. This feature makes the method suitable for simple screening of L. maculans in WOSR leaves and in spores collected by spore traps. [ABSTRACT FROM AUTHOR]

"The Great Five" (GF) is an artificial bacterial consortium developed to protect potato tubers from soft rot caused by Pectobacterium spp. and Dickeya spp. To investigate the commercialization potential of the GF, we developed liquid and powder formulations of the consortium and of each of the comprising strains (Serratia plymuthica strain A294, Enterobacter amnigenus strain A167, Rahnella aquatilis strain H145, Serratia rubidaea strain H440, and S. rubidaea strain H469). To form powders, the cells were lyophilized using a newly developed lyoprotectant: Reagent PS. The shelf life of the formulations stored at 8 and 22 °C was monitored for a period of 12 months. The longest shelf life was obtained for formulations stored at 8 °C; however, the viability of all formulations was negatively affected at 22 °C. For the consortium, a 2.5 log10 cfu (colony forming units) drop in cell number was recorded for the liquid formulation after 6 months, while in case of powders, the drop remained below 1 log10 cfu following 12 months. The ability of the powder formulations to preserve biocontrol activity of the consortium was tested on potato tubers treated with the formulations and a mixture of the soft rot pathogens. The inoculated tubers were stored for 6 months at 8 °C to mimic commercial storage conditions. Soft rot severity and incidence on potato tubers treated with formulations were significantly reduced (62–75% and 48–61%, respectively) in comparison to positive control with pathogens alone. The potential use of the newly developed formulations of "The Great Five" for the biocontrol of soft rot is discussed. Key Points: • An innovative reagent to protect bacterial cells during lyophilization was developed. • Powder formulations of "The Great Five" prolonged its shelf life. • The powder-formulated "The Great Five" was active against soft rot bacteria on potato tubers. [ABSTRACT FROM AUTHOR]

Recent advances in genomics have led to a greater understanding of blackleg disease of canola, caused by the fungus Leptosphaeria maculans. Genome sequences are available for several L. maculans isolates and different Brassica species, and several resistance and avirulence genes, which have a 'gene for gene' relationship, have been characterised. Although several pairs of complementary resistance and avirulence genes have been sequenced, the molecular basis of their recognition is unknown. Mechanisms responsible for conferring virulence include amino acid substitutions, gene deletion and Repeat Induced Point mutations of avirulence genes. In some cases, a single avirulence gene is recognised by two resistance genes, and virulence towards each resistance gene is conferred by separate mutations. Furthermore, some avirulence-resistance gene interactions are epistatic over others; for instance, the presence of AvrLm7 (avirulent towards Rlm7) masks the AvrLm3-Rlm3 interaction resulting in isolates phenotypically appearing virulent towards Rlm3 despite having the avirulent form of the AvrLm3 allele. Several high throughput methods, including the ascospore shower technique, are used to characterise cultivar specificity, frequency of avirulence genes, and to detect fungicide sensitivity of fungal populations from stubble. The frequency of virulence in field populations can change rapidly under selection pressure when cultivars with the same resistance genes are sown in subsequent seasons. If there is a high risk of breakdown of a particular source of resistance, canola growers can be advised to sow a different cultivar, thus avoiding an epidemic. [ABSTRACT FROM AUTHOR]

Blackleg, (stem canker, Leptosphaeria maculans), is the most economically important disease on oilseed rape (Brassica napus). Studies were undertaken to determine the effect of three different L. maculans isolates with different virulence/avirulence patterns on the expression of qualitative resistance and senescence on cotyledons of 11 B. napus genotypes. There were significant differences between genotypes (P < 0.01), isolates (P < 0.01) and a significant genotype by isolate interaction (P < 0.01) in terms of disease index and lesion diameter. Overall, B. napus LSF0530 was more resistant than Barrel > Scotia > Amber x Commanche DH Line. Westar, Drummonds Purple Top, and Parkside were the most susceptible. L. maculans isolates D5 and D13 were the most pathogenic, showing the greatest disease index and lesion diameters. Genotypes such as LSF0530, Scotia, Duell and Barrel responded with a distinct hypersensitive response, whereas Parkside, Drummonds Purple Top, Amber x Commanche DH Line and Westar all showed a distinct yellow halo around the lesions, indicative of cotyledon senescence likely involving programmed cell death (PCD). However, expression of the distinct yellow halo symptom was not observed in all B. napus genotypes infected by one specific L. maculans isolate nor in one specific B. napus genotype challenged with all the isolates. We believe that this is the first report, i), showing the involvement of a distinct yellow halo (senescence/PCD) associated with both 'typical' lesions and with an expression of HR in cotyledons of winter-type B. napus genotypes challenged with L. maculans isolates and, ii), that expression of the distinct yellow halo is clearly dependent on the interaction between particular isolate virulence(s) x particular winter-type B. napus genotype combinations. [ABSTRACT FROM AUTHOR]

Potato soft rot Pectobacteriaceae (SRP) cause large yield losses and are persistent in seed lots once established. In Norway, different Pectobacterium species are the predominant cause of soft rot and blackleg disease. This work aimed to evaluate the potential of real-time PCR for quantification of SRP in seed tubers, as well as investigating the status of potato seed health with respect to SRP in Norway. A total of 34 seed potato lots, including certified seeds, was grown and monitored over three consecutive years. All seed lots contained a quantifiable amount of SRP after enrichment, with very few subsamples being free of the pathogens. A high SRP prevalence based on a qPCR assay, as well as a high symptom incidence in certified seeds were observed, suggesting that current criteria for seed certification are insufficient to determine tuber health and predict field outcomes. Pectobacterium atrosepticum was the most abundant species in the examined seed lots and present in all lots. Consistently good performance of first generation seed lots with respect to blackleg and soft rot incidence, as well as low quantity of SRP in these seed lots demonstrated the importance of clean seed potatoes. Weather conditions during the growing season seemed to govern disease incidence and SRP prevalence more than seed grade. The impact of temperature, potato cultivar and Pectobacterium species on tuber soft rot development were further examined in tuber infection experiments, which showed that temperature was the most important factor in nearly all cultivars. Large-scale quantification of latent infection and predictive models that include contributing factors like weather, infecting bacterial species and cultivar are needed to reduce soft rot and blackleg. [ABSTRACT FROM AUTHOR]

Two TaqMan assays were developed for generic detection of Dickeya species in plant material. The assays enabled detection of all strains of D. chrysanthemi, D. dadantii, D. dianthicola, D. solani and D. zeae tested, but only weak reactions were found with strains of D. paradisiaca. The assays enabled detection of as low as 100 fg of target DNA in a background of potato DNA. The assays were evaluated with DNA extracted from potato tuber extracts, in which different Dickeya species can be present. Results of both assays were highly similar indicating that they can be used reliably in potato seed testing programs. [ABSTRACT FROM AUTHOR]

Blackleg and soft rot of potato cause economic loss through reduced yield and quality. The causal agents of bacterial blackleg and soft rot of potato were identified based on biological data and sequence analyses of the 16S rDNA gene. Between 2016 and 2018, diseased potato stems and tubers were collected in Chai Prakan District, Chiang Mai Province, and Chiang Khum District, Pa Yao Province. The symptoms included black stem lesions, soft rot on tubers, wilting, break down of the stem vascular ring and foliar yellowing. Of 13 bacterial isolates, five were identified as Pectobacterium carotovorum subsp. brasiliense, four‐Dickeya dadantii, two‐Pseudomonas putida and two‐Bacillus altitudinis. Pathogenicity tests of P. carotovorum subsp. brasiliense and D. dadantii resulted in lower leaves turning yellow and wilting followed by blackleg symptoms on lower stems and maceration of tuber tissue. Symptoms caused by P. putida were yellowing and wilting of leaves. B. altitudinis caused yellowing of the lower leaves and wilting followed by drying of leaf tissue. This is a first report of these bacterial pathogens causing blackleg and soft rot of potato in Thailand. [ABSTRACT FROM AUTHOR]

Understanding the transmission of plant pathogen inoculum during the periods when the host plants are not present is crucial for predicting the initiation of epidemics and optimizing mitigation strategies. However, inoculum production at the end of the cropping season, survival during the intercrop period, and the emergence or release of inoculum can be highly variable, difficult to assess, and generally inferred indirectly from symptom data. As a result, a lack of large datasets hampers the study of these epidemiological processes. Here, inoculum production was studied in Leptosphaeria maculans, the cause of phoma stem canker of oilseed rape. The fungus survives on stubble left in the field, from which ascospores are released at the beginning of the next cropping season. An image processing framework was developed to estimate the density of fruiting bodies produced on stem pieces following incubation in field conditions, and a quality assessment of the processing chain was performed. A total of 2540 standardized RGB digital images of stems were then analysed, collected from 27 oilseed rape fields in Brittany over four cropping seasons. Manual post‐processing removed 16% of the pictures, e.g. when moisture‐induced darkening of the oilseed rape stems caused overestimation of the area covered with fruiting bodies. The potential level of inoculum increased with increasing phoma stem canker severity at harvest, and depended on the source field and the cropping season. This work shows how image‐based phenotyping generates high‐throughput disease data, opening up the prospect of substantially increased precision in epidemiological studies. [ABSTRACT FROM AUTHOR]

Outbreaks of potato blackleg and soft rot caused by Pectobacterium species and more recently Dickeya species across the U.S. mid-Atlantic region have caused yield loss due to poor emergence as well as losses from stem and tuber rot. To develop management strategies for soft rot diseases, we must first identify which members of the soft rot Pectobacteriaceae are present in regional potato plantings. However, the rapidly expanding number of soft rot Pectobacteriaceae species and the lack of readily available comparative data for type strains of Pectobacterium and Dickeya hinder quick identification. This manuscript provides a comparative analysis of soft rot Pectobacteriaceae and a comprehensive comparison of type strains from this group using rep-PCR, MLSA and 16S sequence analysis, as well as phenotypic and physiological analyses using Biolog GEN III plates. These data were used to identify isolates cultured from symptomatic potato stems collected between 2016 and 2018. The isolates were characterized for phenotypic traits and by sequence analysis to identify the bacteria from potatoes with blackleg and soft rot symptoms in Pennsylvania potato fields. In this survey, P. actinidiae, P. brasiliense, P. polonicum, P. polaris, P. punjabense, P. parmentieri, and P. versatile were identified from Pennsylvania for the first time. Importantly, the presence of P. actinidiae in Pennsylvania represents the first report of this organism in the U.S. As expected, P. carotorvorum and D. dianthicola were also isolated. In addition to a resource for future work studying the Dickeya and Pectobacterium associated with potato blackleg and soft rot, we provide recommendations for future surveys to monitor for quarantine or emerging soft rot Pectobacteriace regionally. [ABSTRACT FROM AUTHOR]

Pectobacterium and Dickeya species are the causal agents of blackleg and soft rot diseases in potatoes. The main pathogenic species identified so far on potatoes are Dickeya dianthicola, Dickeya solani, Pectobacterium atrosepticum, Pectobacterium brasiliense, Pectobacterium carotovorum, and Pectobacterium parmentieri. Ten years ago, the most prevalent Soft Rot Pectobacteriaceae in Europe were the Dickeya species, P. atrosepticum and P. carotovorum, with some variations among countries. Since then, a drastic increase in the abundance of P. brasiliense has been observed in most European countries. This shift is difficult to explain without comparing the pathogenicity of all Dickeya and Pectobacterium species. The pathogenicity of all the above-mentioned bacterial species was assessed in field trials and in vitro tuber slice trials in Switzerland. Two isolates of each species were inoculated by soaking tubers of cv. Desiree in a suspension of 105 CFU/mL, before planting in the field. For all trials, the Dickeya species were the most virulent ones, but long-term strain surveys performed in Switzerland indicate that P. brasiliense is currently the most frequent species detected. Our results show that the pathogenicity of the species is not the main factor explaining the high prevalence of P. brasiliense and P. parmentieri in the Swiss potato fields.

An outbreak of bacterial soft rot and blackleg of potato has occurred since 2014 with the epicenter being in the northeastern region of the United States. Multiple species of Pectobacterium and Dickeya are causal agents, resulting in losses to commercial and seed potato production over the past decade in the Northeastern and North Central United States. To clarify the pathogen present at the outset of the epidemic in 2015 and 2016, a phylogenetic study was made of 121 pectolytic soft rot bacteria isolated from symptomatic potato; also included were 27 type strains of Dickeya and Pectobacterium species, and 47 historic reference strains. Phylogenetic trees constructed based on multilocus sequence alignments of concatenated dnaJ, dnaX and gyrB fragments revealed the epidemic isolates to cluster with type strains of D. chrysanthemi, D. dianthicola, D. dadantii, P. atrosepticum, P. brasiliense, P. carotovorum, P. parmentieri, P. polaris, P. punjabense, and P. versatile. Genetic diversity within D. dianthicola strains was low, with one sequence type (ST1) identified in 17 of 19 strains. Pectobacterium parmentieri was more diverse, with ten sequence types detected among 37 of the 2015–2016 strains. This study can aid in monitoring future shifts in potato soft rot pathogens within the U.S. and inform strategies for disease management.

Blackleg is an acute and frequently fatal infection that mainly affects cattle and is caused by Clostridium chauvoei. Formalin-killed, whole-cell vaccines are commonly used to control blackleg. The aim of this study was to verify the protective efficacy of two commercial vaccines against the infection of guinea pigs with two strains of C. chauvoei, a virulent field strain (SBP 07/09) and the reference strain used in official tests (Manguinhos-Teixeira or MT). The strains used in the challenge were characterized by whole genome sequencing, and the minimal inhibitory concentrations of 15 antimicrobials were determined. To assess the protective efficacy, guinea pigs were vaccinated and subsequently challenged with C. chauvoei. All four vaccinated and challenged groups seroconverted after vaccination, while the control group remained seronegative, as determined by indirect ELISA. The identical performance of the two C. chauvoei strains in terms of virulence after challenge and their inability to infect vaccinated animals was correlated with their high genetic homology. Both commercial vaccines showed good protective efficacy against both the reference and field strains. Although C. chauvoei vaccination failures have been reported, the results from our study and others reported high similarity among C. chauvoei strains from all over the world, which suggests that the vaccine failures are not due to antigenic variability but inadequate vaccine management.

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.

Pectobacterium atrosepticum is a species of plant pathogenic bacteria responsible for significant losses in potato production worldwide. Pectobacterium atrosepticum can cause blackleg disease on potato stems as well as the tuber disease termed potato soft rot. Methods for the effective control of these diseases are limited and are primarily based on good agricultural practices. Bacteriophages, viruses of bacteria, could be used as an alternative, environmentally friendly, control measure. Here, we describe the isolation and characterization of 29 phages virulent to P. atrosepticum. The phages belong to 12 different species based on a 95% sequence identity cut-off. Furthermore, based on sequence diversity and propagation results, we selected six of these phages to form a phage cocktail. The phages in the cocktail was tested on a number of P. atrosepticum strains in order to determine their host range. The phages was found to lyse 93% of the tested strains. The cocktail was subsequently tested for its effectiveness in combatting potato soft rot under simulated storage conditions. Use of the phage cocktail reduced both disease incidence and disease severity by 61% and 64%, respectively, strongly indicating that phage biocontrol has the potential to reduce the economic impact of soft rot in potato production. A phage cocktail consisting of six bacteriophages can significantly reduce soft rot symptoms on potato tubers infected with Pectobacterium atrosepticum under simulated storage conditions. [ABSTRACT FROM AUTHOR]

Outbreaks of light leaf spot (Pyrenopeziza brassicae), a disease new to North America, occurred along with the diseases blackleg (Leptosphaeria spp.) and white leaf spot (Mycosphaerella capsellae) on Brassica crops in western Oregon. Disease symptoms in seed fields planted in the fall of 2013 were observed in the spring of 2014. Field surveys were conducted for disease during 2014 and 2015 to determine the geographic range of light leaf spot, blackleg, and white leaf spot within the Willamette Valley of western Oregon. Infected seed crops included turnip and oilseed rape in Benton, Lane, Linn, Marion, Polk, and Yamhill counties. New protocols that are more sensitive for detection of light leaf spot were developed for the pathogen population in the Pacific Northwest of the USA. Pyrenopeziza brassicae was detected when samples also contained L. maculans or M. capsellae. The test did not react with a range of other fungi evaluated. Sensitivity of the PCR protocol reported is robust at 10 pg of genomic DNA. Standard and real-time PCR screenings developed in this research increase sensitivity for detection of P. brassicae in affected Brassica hosts, which is critical for timely diagnosis and management of this newly-introduced disease in Brassica crops in the Pacific Northwestern region of the USA. [ABSTRACT FROM AUTHOR]

Clostridium chauvoei toxin A (CctA), neuraminidase (NanA), and flagellin (FliC) proteins contribute to the pathogenicity of Clostridium chauvoei, the causative agent of blackleg in cattle. The aim of this study was to analyze the genetic variability of cctA, nanA, and fliC genes in C. chauvoei isolates from the Rio Grande do Sul and São Paulo state-Brazil, during different sampling periods. The presence of these genes was verified through PCR amplification and partial gene sequencing of 17 strains. Alignment of PCR amplicons combined with bioinformatics analysis was used in an attempt to study the variability across C. chauvoei solates. The similarity among the partial sequences of cctA and nanA genes was 100%. The sequencing of fliC revealed three different paralog alleles of flagellin, and two strains were seen to be polymorphic, with amino acid alterations in the predicted protein. Overall, this study indicates that strains of C. chauvoei isolated in Brazil are highly conserved with respect to the virulence factors evaluated. [ABSTRACT FROM AUTHOR]

Clostridium chauvoei is the etiologic agent of blackleg in cattle, inducing fever, severe myonecrosis, oedemic lesions and ultimately death of infected animals. The pathogen often results in such rapid death that antibiotic therapy is futile and thus vaccination is the only efficient strategy in order to control the disease. The β-barrel pore forming leucocidin Clostridium chauvoei toxin A (CctA) is one of the best characterised toxins of C. chauvoei and has been shown to be an important virulence factor. It has been reported to induce protective immunity and is conserved across C. chauvoei strains collected from diverse geographical locations for more than 50 years. The aim of this study was to identify the location of the CctA toxin during liquid culture fermentation and to use CctA to develop an in vitro assay to replace the current guinea pig challenge assay for vaccine potency in standard batch release procedures. We report that CctA is fully secreted in C. chauvoei culture and show that it is found abundantly in the supernatant of liquid cultures. Sera from cattle vaccinated with a commercial blackleg vaccine revealed strong haemolysin-neutralizing activity against recombinant CctA which reached titres of 1000 times 28 days post-vaccination. Similarly, guinea pig sera from an official potency control test reached titres of 600 times 14 days post-vaccination. In contrast, ELISA was not able to specifically measure anti-CctA antibodies in cattle serum due to strong cross-reactions with antibodies against other proteins present pre-vaccination. We conclude that haemolysin-neutralizing antibodies are a valuable measurement for protective immunity against blackleg and have the potential to be a suitable replacement of the guinea pig challenge potency test, which would forego the unnecessary challenge of laboratory animals. • Leucocidin CctA of C. chauvoei is fully secreted during growth in liquid medium. • Elongation factor Tu of C. chauvoei has been found on the cell surface. • The haemolytic activity of C. chauvoei is specific to leucocidin CctA. • Blackleg vaccination induces neutralizing antibodies against CctA in cattle and guinea pigs. • Development of a CctA-neutralization assay to potentially replace the guinea pig challenge potency test. [ABSTRACT FROM AUTHOR]

Dickeya and Pectobacterium are responsible for causing blackleg of plants and soft rot of tubers in storage and in the field, giving rise to losses in seed potato production. In an attempt to improve potato health, biocontrol activity of known and putative antagonists was screened using in vitro and in planta assays, followed by analysis of their persistence at various storage temperatures. Most antagonists had low survival on potato tuber surfaces at 4 °C. The population dynamics of the best low‐temperature tolerant strain and also the most efficient antagonist, Serratia plymuthica A30, along with Dickeya solani as target pathogen, was studied with TaqMan real‐time PCR throughout the storage period. Tubers of three potato cultivars were treated in the autumn with the antagonist and then inoculated with D. solani. Although the cell densities of both strains decreased during the storage period in inoculated tubers, the pathogen population was always lower in the presence of the antagonist. The treated tubers were planted in the field the following growing season to evaluate the efficiency of the bacterial antagonist for controlling disease incidence. The potato endophyte S. plymuthica A30 protected potato plants by reducing blackleg development on average by 58.5% and transmission to tuber progeny as latent infection by 47–75%. These results suggest that treatment of potato tubers with biocontrol agents after harvest can reduce the severity of soft rot disease during storage and affect the transmission of soft rot bacteria from mother tubers to progeny tubers during field cultivation. [ABSTRACT FROM AUTHOR]

Abstract Four hundred and fifty bacteria were evaluated for antagonistic activity against bacterial soft rot of potato caused by Pectobacterium carotovorum sp strain II16. A strain Ar10 exhibiting potent antagonist activity has been identified as Bacillus amyloliquefaciens on the basis of biochemical and molecular characterization. Cell free supernatant showed a broad spectrum of antibacterial activity against human and phytopathogenic bacteria in the range of 10-60 AU/mL. Incubation of P. carotovorum cells with increasing concentrations of the antibacterial compound showed a killing rate of 94.8 and 96% at MIC and 2xMIC respectively. In addition, the antibacterial agent did not exert haemolytic activity at the active concentration and has been preliminary characterized by TLC and GC–MS as a glycolipid compound. Treatment of potato tubers with strain Ar10 for 72 h significantly reduced the severity of disease symptoms (100 and 85.05% reduction of necrosis deep / area and weight loss respectively). The same levels in disease symptoms severity was also recorded following treatment of potato tubers with cell free supernatant for 1 h. Data suggest that protection against potato soft rot disease may be related to glycolipid production by strain Ar10. The present study affords new alternatives for anti- Pectobacterium carotovorum bioactive compounds against the soft rot disease of potato. [ABSTRACT FROM AUTHOR]

Pectobacterium brasiliense (Pbr) is considered as one of the most virulent species among the Pectobacteriaceae. This species has a broad host range within horticulture crops and is well distributed elsewhere. It has been found to be pathogenic not only in the field causing blackleg and soft rot of potato, but it is also transmitted via storage causing soft rot of other vegetables. Genomic analysis and other cost-effective molecular detection methods such as a quantitative polymerase chain reaction (qPCR) are essential to investigate the ecology and pathogenesis of the Pbr. The lack of fast, field deployable point-of-care testing (POCT) methods, specific control strategies and current limited genomic knowledge make management of this species difficult. Thus far, no comprehensive review exists about Pbr, however there is an intense need to research the biology, detection, pathogenicity and management of Pbr, not only because of its fast distribution across Europe and other countries but also due to its increased survival to various climatic conditions. This review outlines the information available in peer-reviewed literature regarding host range, detection methods, genomics, geographical distribution, nomenclature and taxonomical evolution along with some of the possible management and control strategies. In summary, the conclusions and a further directions highlight the management of this species.

• Fungicide resistant Leptosphaeria maculans strains were isolated. • All resistance is due to insertion of DNA into the erg11 promoter. • A new pipeline for estimating frequencies of insertions was developed. • Frequency of azole resistance in populations is as high as 33%. Continued use of fungicides provides a strong selection pressure towards strains with mutations to render these chemicals less effective. Previous research has shown that resistance to the demethylation inhibitor (DMI) fungicides, which target ergosterol synthesis, in the canola pathogen Leptosphaeria maculans has emerged in Australia and Europe. The change in fungicide sensitivity of individual isolates was found to be due to DNA insertions into the promoter of the erg11 / CYP51 DMI target gene. Whether or not these were the only types of mutations and how prevalent they were in Australian populations was explored in the current study. New isolates with reduced DMI sensitivity were obtained from screens on DMI-treated plants, revealing eight independent insertions in the erg11 promoter. A novel deep amplicon sequencing approach applied to populations of ascospores fired from stubble identified an additional undetected insertion allele and quantified the frequencies of all known insertions, suggesting that, at least in the samples processed, the combined frequency of resistant alleles is between 0.0376% and 32.6%. Combined insertion allele frequencies positively correlated with population-level measures of in planta resistance to four different DMI treatments. Additionally, there was no evidence for erg11 coding mutations playing a role in conferring resistance in Australian populations. This research provides a key method for assessing fungicide resistance frequency in stubble-borne populations of plant pathogens and a baseline from which additional surveillance can be conducted in L. maculans. Whether or not the observed resistance allele frequencies are associated with loss of effective disease control in the field remains to be established. [ABSTRACT FROM AUTHOR]

Canola (Brassica napus) is an important crop species grown worldwide for its seeds, which are crushed for edible oil. Canola production is threatened by blackleg disease, caused by the fungal pathogen Leptosphaeria maculans, which can lead up to 100% yield loss. A plant’s disease resistance response depends highly on the regulation of the expression of defence genes. DNA methylation, an epigenetic mark, is one of the most important regulatory mechanisms in a plant’s defence system. Here, the DNA methylation pattern of promoters of defence genes has been investigated between leaves from control and infected plants with L. maculans of resistant and susceptible cultivars of B. napus. In total, over 12,000 promoters were found to be differentially methylated between leaves from control and infected plants in the two cultivars, of which 225 promoters were related to defence genes and differentially methylated between the resistant and susceptible cultivars. The majority of defence gene promoters were hypo- or hyper-methylated in the first and second true leaves, but not in the third and fourth leaves. The outcomes will assist in developing an insight into genome-wide DNA methylation patterns in canola cultivars, and ultimately help breeders to optimise the breeding programmes for enhancing resistance against blackleg disease.

The hemibiotrophic fungus, Leptosphaeria maculans is the most devastating pathogen, causing blackleg disease in canola (Brassica napus L). To study the genomic regions involved in quantitative resistance (QR), 259–276 DH lines from Darmor-bzh/Yudal (DYDH) population were assessed for resistance to blackleg under shade house and field conditions across 3 years. In different experiments, the broad sense heritability varied from 43 to 95%. A total of 27 significant quantitative trait loci (QTL) for QR were detected on 12 chromosomes and explained between 2.14 and 10.13% of the genotypic variance. Of the significant QTL, at least seven were repeatedly detected across different experiments on chromosomes A02, A07, A09, A10, C01, and C09. Resistance alleles were mainly contributed by ‘Darmor-bzh’ but ‘Yudal’ also contributed few of them. Our results suggest that plant maturity and plant height may have a pleiotropic effect on QR in our conditions. We confirmed that Rlm9 which is present in ‘Darmor-bzh’ is not effective to confer resistance in our Australian field conditions. Comparative mapping showed that several R genes coding for nucleotide-binding leucine-rich repeat (LRR) receptors map in close proximity (within 200 Kb) of the significant trait-marker associations on the reference ‘Darmor-bzh’ genome assembly. More importantly, eight significant QTL regions were detected across diverse growing environments: Australia, France, and United Kingdom. These stable QTL identified herein can be utilized for enhancing QR in elite canola germplasm via marker- assisted or genomic selection strategies.

Pectobacterium atrosepticum is a pectolytic enterobacterium that causes blackleg in plants. Bacteriophage Peat2 was isolated from P. atrosepticum infected potato tissue and its genome sequenced. Analysis of the 48,659 bp genome revealed that Peat2 resembles the Myoviridae family Klebsiella pneumoniae bacteriophage JD001 and includes a CRISPR Cas 4 RecB-like nuclease. A lytic bacteriophage, Peat2 details provide important information for application as a biocontrol agent of disease caused by phytopathogenic P. atrosepticum. [ABSTRACT FROM AUTHOR]

Phoma stem canker (blackleg), caused by the fungi Leptosphaeria maculans (anamorph Phoma lingam ) and L. biglobosa , is one of the most devastating diseases in oilseed rape ( Brassica napus L.) production worldwide. However, the population structure and genetic variation of L. biglobosa populations in China have rarely been investigated. Here, a collection of 214 fungal strains of blackleg from China (including Shaanxi, Jiangxi, Hubei, Jiangsu, Chongqing, Sichuan, Guangxi, Guizhou, Hunan, and Henan) and Europe (France and Ukraine) was identified as L. biglobosa . Three simple sequence repeat (SSR) markers were developed to characterize their population structure. The results showed that the Nei's average gene diversity ranged from 0.6771 for the population from Jiangsu to 0.3009 for the population from Hunan. In addition, most of the genetic variability (96%) occurred within groups and there were only relatively small amounts among groups (4.0%) ( F ST = 0.043, p = 0.042 < 0.05). Pairwise population differentiation ( F ST ) suggested that significant genetic differentiation was observed between different L. biglobosa populations. Bayesian and unweighted average method analysis revealed that these L. biglobosa strains were clustered into three branches, and three European strains were similar to those from eastern China. The pathogenicity assay showed that those in Group III were significantly more virulent than those in Group I (t = 2.69, p = 0.016). The study also showed that Group III was dominant in Chinese L. biglobosa populations, which provides new insights for the further study of population evolution and the management of this pathogen.

Blackleg disease is devastating for wasabi ( Eutrema japonicum ) production, occurring at any time and everywhere within the main production area of the Sichuan Province, China. There have been very few studies on the chemical control of this disease. In this study, we isolated and identified a local popular strain of the pathogen Plenodomus wasabiae . The isolated fungus strain caused typical disease spots on the leaves and rhizomes upon inoculation back to wasabi seedlings. The symptoms of blackleg disease developed very quickly, becaming visible on the second day after exposure to P. wasabiae and leading to death within one week. We then evaluated the efficacy of ten widely used fungicides to screen out effective fungicides. The efficacy of the tested fungicides was determined through mycelial growth inhibition on medium plates. As a result, tebuconazole and pyraclostrobin were able to inhibit the mycelial growth of P. wasabiae , and the most widely used dimethomorph in local production areas produced the lowest inhibition activity (13.8%). Nevertheless, the highest control efficacy of tebuconazole and pyraclostrobin on wasabi seedlings was only 47.48% and 39.03%, respectively. Generally, the control efficacy of spraying the fungicide before inoculation was better than that after inoculation. An increase in the application concentration of the two fungicides did not proportionately result in improved performance. We cloned the full-length sequence of sterol 14-demethylase ( CYP51 ) and cytochrome B ( CYTB ) of which the mutations may contribute to the possible antifungalresistance. These two genes of the isolated fungus do not possess any reported mutations that lead to fungicide resistance. Previous studies indicate that there is a significant difference between fungicides in terms of the effectiveness of controlling blackleg disease; however, the control efficacy of fungicides is limited in blackleg control. Therefore, field management to prevent wound infection and unfavorable environmental conditions are more important than pesticide management.