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Potato wart disease is caused by the obligate fungal pathogen Synchytrium endobioticum. DNA extraction from compost, purified spores and crude wart tissue derived from tuber galls of infected potatoes often results in low S. endobioticum DNA concentration or highly contaminated with DNA coming from other microorganisms and the potato host. Therefore, Illumina sequencing of these samples generally results in suboptimal recovery of the nuclear genome sequences of S. endobioticum. A hybridization-based target enrichment protocol was developed to strongly enhance the recovery of S. endobioticum DNA while off-target organisms DNA remains uncaptured. The design strategy involved creating a set of 180,000 molecular baits targeting both gene and non-gene regions of S. endobioticum. The baits were applied to whole genome amplified DNA samples of various S. endobioticum pathotypes (races) in compost, from purified spores and crude wart tissue samples. This was followed by Illumina sequencing and bioinformatic analyses. Compared to non-enriched samples, target enriched samples: 1) showed a significant increase in the proportion of sequenced bases mapped to the S. endobioticum nuclear genome, especially for crude wart tissue samples; 2) yielded sequencing data with higher and better nuclear genome coverage; 3) biased genome assembly towards S. endobioticum sequences, yielding smaller assembly sizes but higher representation of putative S. endobioticum contigs; 4) showed an increase in the number of S. endobioticum genes detected in the genome assemblies. Our hybridization-based target enrichment protocol offers a valuable tool for enhancing genome sequencing and NGS-based molecular detection of S. endobioticum, especially in difficult samples.

Potato wart disease caused by the obligate biotrophic fungus Synchytrium endobioticum is a devastating disease that can result in significant crop losses. Resting spores of this pathogen can remain viable and infectious in soil for decades. The detection of viable resting spores using conventional methods such as bioassays and direct microscopic examination are challenging and time-consuming and require specific expertise and facilities. Molecular methods, such as real-time PCR, have been shown to be effective in detecting the presence of S. endobioticum DNA in soil samples but cannot differentiate between viable and nonviable spores. In this paper, we present three novel mRNA-based molecular tests to potentially detect viable S. endobioticum resting spores. The tests are specific to the transcribed mRNA and do not detect the genomic DNA of the target genes. We demonstrate the analytical sensitivity using synthetic constructs of the target mRNAs. The tests were found to be able to repeatedly detect 10 target copies per reaction. Soils and waste of potato processing industries free from S. endobioticum were used to assess the exclusivity of the tests. The biological relevance of mRNA detection was determined in the context of replicated bioassays. Applications of the tests to facilitate collection management, assessment of the effects of treatments on presumed viability of S. endobioticum resting spores, and the potential use in descheduling of previously infested plots are discussed.

BACKGROUND: Resistance to rodenticides has been reported globally and poses a considerable problem for efficacy in pest control. The most-documented resistance to rodenticides in commensal rodents is associated with mutations in the Vkorc1 gene, in particular in codon 139. Resistance to anticoagulant rodenticides has been reported in the Netherlands since 1989. A study from 2013 showed that 25% of 169 Norway rats (Rattus norvegicus) had a mutation at codon 139 of the Vkorc1 gene. To gain insight in the current status of rodenticide resistance amongst R. norvegicus and house mice Mus musculus in the Netherlands, we tested these rodents for mutations in codon 139 of the Vkorc1 gene. In addition, we collected data from pest controllers on their use of rodenticides and experience with rodenticide resistance. RESULTS: A total of 1801 rodent samples were collected throughout the country consisting of 1404 R. norvegicus and 397 M. musculus. In total, 15% of R. norvegicus [95% confidence interval (CI): 13–17%] and 38% of M. musculus (95% CI: 33–43%) carried a genetic mutation at codon 139 of the Vkorc1 gene. CONCLUSION: This study demonstrates genetic mutations at codon 139 of the Vkorc1 gene in M. musculus in the Netherlands. Resistance to anticoagulant rodenticides is present in R. norvegicus and M. musculus in multiple regions in the Netherlands. The results of this comprehensive study provide a baseline and facilitate trend analyses of Vkorc1 codon 139 mutations and evaluation of integrated pest management (IPM) strategies as these are enrolled in the Netherlands.

P. brasiliense is an important bacterial pathogen causing blackleg (BL) in potatoes. Nevertheless, P. brasiliense is often detected in seed lots that do not develop any of the typical blackleg symptoms in the potato crop when planted. Field bioassays identified that P. brasiliense strains can be categorized into two distinct classes, some able to cause blackleg symptoms and some unable to do it. A comparative pangenomic approach was performed on 116 P. brasiliense strains, of which 15 were characterized as BL-causing strains and 25 as non-causative. In a genetically homogeneous clade comprising all BL-causing P. brasiliense strains, two genes only present in the BL-causing strains were identified, one encoding a predicted lysozyme inhibitor Lprl (LZI) and one encoding a putative Toll/interleukin-1 receptor (TIR) domain-containing protein. TaqMan assays for the specific detection of BL-causing P. brasiliense were developed and integrated with the previously developed generic P. brasiliense assay into a triplex TaqMan assay. This simultaneous detection makes the scoring more efficient as only a single tube is needed, and it is more robust as BL-causing strains of P. brasiliense should be positive for all three assays. Individual P. brasiliense strains were found to be either positive for all three assays or only for the P. brasiliense assay. In potato samples, the mixed presence of BL-causing and not BL-causing P. brasiliense strains was observed as shown by the difference in Ct value of the TaqMan assays. However, upon extension of the number of strains, it became clear that in recent years additional BL-causing lineages of P. brasiliense were detected for which additional assays must be developed.

Synchytrium endobioticum (Schilb.) Perc. is a chytrid fungus causing potato wart disease and is one of the most important quarantine diseases on cultivated potato. Infected host tissues develop warts rendering the crop unmarketable. Resting spores, that can remain viable and infectious for decades, are formed in warted tissues and are released into the surrounding soil when host tissue decays. To better understand the pathogen’s diversity and to potentially uncover pathways of migrations and introduction events, molecular characterization was performed on the historical S. endobioticum resting spore collection of the Dutch National Plant Protection Organization. Mitochondrial genomes were assembled and annotated, and four novel structural variants were identified from these materials with intronic presence-absence variation in cox1 or cob genes and structural variation in the dpoB – TIR region. Several fungal isolates were shown to contain mixtures of structural variants. We analyzed the mitogenomic sequences obtained from recent potato wart disease findings in Canada and the Netherlands in the context of the historical materials and found that fungal isolates from the new Dutch outbreak contained a specific mixture of mitogenomic variants previously not observed in the Netherlands. Based on the mitogenomic profile, pathotype 38(Nevşehir) was suspected which was later verified with the Spieckermann bioassay. To further facilitate dissemination of data and interactive visual analytics we created a public Nextstrain webpage with S. endobioticum mitogenomic sequences and associated metadata on their geographic origin, pathotype identity and (mixture) of mitogenomic variants (https://nextstrain.nrcnvwa.nl/Sendo).

As the honey bee and bumble bee may suffer from the same or related microbial pathogens, cross contamination from commercially reared Bombus spp. to honey bees and wild bumble bees and vice versa is a major concern. Honey bee-collected pollen to feed commercially reared Bombus spp. is a potential risk. Nosema spp. is a fungal pathogen in bees. In this study, we developed new quantitative detection tools based on the detection of RNA using a TaqMan-based RT-qPCR for Nosema ceranae and Nosema apis, with extraction controls based on the actin gene of honey bees and bumble bees, respectively. These tools were subsequently applied to study the epidemiology of N. ceranae, a main disease in honey bees. We screened gamma radiation and cold treatment sterilisation for their efficacy to kill N. ceranae spores fed in sugar water and in pollen to honey bees and bumble bees, respectively. N. ceranae infection in adult bumble bees was checked. Spores passing the inter-alimentary track were found but no infection was observed. N. ceranae spores were fed to honey bees. Their presence and multiplication were demonstrated, showing the spores were both viable and infectious. Our results indicate that N. ceranae found in honey bees cannot infect commercially reared bumble bees (Bombus terrestris) and, that gamma radiation effectively kills N. ceranae. The highly specific and sensitive molecular assays developed, were exploited to detect N. ceranae in pollen and faeces, which would allow more comprehensive epidemiological studies on this important pathogen.

Potato wart disease is considered one of the most important quarantine pests for cultivated potato and is caused by the obligate biotrophic chytrid fungus Synchytrium endobioticum. This review integrates observations from early potato wart research and recent molecular, genetic, and genomic studies of the pathogen and its host potato. Taxonomy, epidemiology, pathology, and formation of new pathotypes are discussed, and a model for molecular S. endobioticum–potato interaction is proposed. Taxonomy: Currently classified as kingdom: Fungi, phylum: Chytridiomycota, class: Chytridiomycetes, order: Chytridiales, family: Synchytriaceae, genus: Synchytrium, species: Synchytrium endobioticum, there is strong molecular support for Synchytriaceae to be transferred to the order Synchytriales. Hosts and disease symptoms: Solanum tuberosum is the main host for S. endobioticum but other solanaceous species have been reported as alternative hosts. It is not known if these alternative hosts play a role in the survival of the pathogen in (borders of) infested fields. Disease symptoms on potato tubers are characterized by the warty cauliflower-like malformations that are the result of cell enlargement and cell multiplication induced by the pathogen. Meristematic tissue on tubers, stolons, eyes, sprouts, and inflorescences can be infected while the potato root system seems to be immune. Pathotypes: For S. endobioticum over 40 pathotypes, which are defined as groups of isolates with a similar response to a set of differential potato varieties, are described. Pathotypes 1(D1), 2(G1), 6(O1), and 18(T1) are currently regarded to be most widespread. However, with the current differential set other pathogen diversity largely remains undetected. Pathogen–host interaction: A single effector has been described for S. endobioticum (AvrSen1), which is recognized by the potato Sen1 resistance gene product. This is also the first effector that has been described in Chytridiomycota, showing that in

Background: Bacterial plant pathogens of the Pectobacterium genus are responsible for a wide spectrum of diseases in plants, including important crops such as potato, tomato, lettuce, and banana. Investigation of the genetic diversity underlying virulence and host specificity can be performed at genome level by using a comprehensive comparative approach called pangenomics. A pangenomic approach, using newly developed functionalities in PanTools, was applied to analyze the complex phylogeny of the Pectobacterium genus. We specifically used the pangenome to investigate genetic differences between virulent and avirulent strains of P. brasiliense, a potato blackleg causing species dominantly present in Western Europe. Results: Here we generated a multilevel pangenome for Pectobacterium, comprising 197 strains across 19 species, including type strains, with a focus on P. brasiliense. The extensive phylogenetic analysis of the Pectobacterium genus showed robust distinct clades, with most detail provided by 452,388 parsimony-informative single-nucleotide polymorphisms identified in single-copy orthologs. The average Pectobacterium genome consists of 47% core genes, 1% unique genes, and 52% accessory genes. Using the pangenome, we zoomed in on differences between virulent and avirulent P. brasiliense strains and identified 86 genes associated to virulent strains. We found that the organization of genes is highly structured and linked with gene conservation, function, and transcriptional orientation. Conclusion: The pangenome analysis demonstrates that evolution in Pectobacteria is a highly dynamic process, including gene acquisitions partly in clusters, genome rearrangements, and loss of genes. Pectobacterium species are typically not characterized by a set of species-specific genes, but instead present themselves using new gene combinations from the shared gene pool. A multilevel pangenomic approach, fusing DNA, protein, biological function, taxonomic group, and phenotypes

The Fusarium fujikuroi species complex (FFSC) and F. oxysporum species complex (FOSC) are two related groups of plant pathogens causing a wide diversity of diseases in agricultural crops world wide. The aims of this study are (1) to clarify the phylogeny of the FFSC, (2) to identify potential deviation from tree-like evolution, (3) to explore the value of using mitogenomes for these kinds of analyses, and (4) to better understand mitogenome evolution. In total, we have sequenced 24 species from the FFSC and a representative set of recently analyzed FOSC strains was chosen, while F. redolens was used as outgroup for the two species complexes. A species tree was constructed based on the concatenated alignment of seven nuclear genes and the mitogenome, which was contrasted to individual gene trees to identify potential conflicts. These comparisons indicated conflicts especially within the previously described African clade of the FFSC. Furthermore, the analysis of the mitogenomes revealed the presence of a variant of the large variable (LV) region in FFSC which was previously only reported for FOSC. The distribution of this variant and the results of sequence comparisons indicate horizontal genetic transfer between members of the two species complexes, most probably through introgression. In addition, a duplication of atp9 was found inside an intron of cob, which suggests that even highly conserved mitochondrial genes can have paralogs. Paralogization in turn may lead to inaccurate single gene phylogenies. In conclusion, mitochondrial genomes provide a robust basis for phylogeny. Comparative phylogenetic analysis indicated that gene flow among and between members of FFSC and FOSC has played an important role in the evolutionary history of these two groups. Since mitogenomes show greater levels of conservation and synteny than nuclear regions, they are more likely to be compatible for recombination than nuclear regions. Therefore, mitogenomes can be used as indicators t

Synchytrium endobioticum is an obligate biotrophic soilborne Chytridiomycota (chytrid) species that causes potato wart disease, and represents the most basal lineage among the fungal plant pathogens. We have chosen a functional genomics approach exploiting knowledge acquired from other fungal taxa and compared this to several saprobic and pathogenic chytrid species. Observations linked to obligate biotrophy, genome plasticity and pathogenicity are reported. Essential purine pathway genes were found uniquely absent in S. endobioticum, suggesting that it relies on scavenging guanine from its host for survival. The small gene-dense and intron-rich chytrid genomes were not protected for genome duplications by repeat-induced point mutation. Both pathogenic chytrids Batrachochytrium dendrobatidis and S. endobioticum contained the largest amounts of repeats, and we identified S. endobioticum specific candidate effectors that are associated with repeat-rich regions. These candidate effectors share a highly conserved motif, and show isolate specific duplications. A reduced set of cell wall degrading enzymes, and LysM protein expansions were found in S. endobioticum, which may prevent triggering plant defense responses. Our study underlines the high diversity in chytrids compared to the well-studied Ascomycota and Basidiomycota, reflects characteristic biological differences between the phyla, and shows commonalities in genomic features among pathogenic fungi.

Plant pathogenic fungi in the Fusarium genus cause severe damage to crops, resulting in great financial losses and health hazards. Specialized metabolites synthesized by these fungi are known to play key roles in the infection process, and to provide survival advantages inside and outside the host. However, systematic studies of the evolution of specialized metabolite-coding potential across Fusarium have been scarce. Here, we apply a combination of bioinformatic approaches to identify biosynthetic gene clusters (BGCs) across publicly available genomes from Fusarium, to group them into annotated families and to study gain/loss events of BGC families throughout the history of the genus. Comparison with MIBiG reference BGCs allowed assignment of 29 gene cluster families (GCFs) to pathways responsible for the production of known compounds, while for 57 GCFs, the molecular products remain unknown. Comparative analysis of BGC repertoires using ancestral state reconstruction raised several new hypotheses on how BGCs contribute to Fusarium pathogenicity or host specificity, sometimes surprisingly so: for example, a gene cluster for the biosynthesis of hexadehydro-astechrome was identified in the genome of the biocontrol strain Fusarium oxysporum Fo47, while being absent in that of the tomato pathogen F. oxysporum f.sp. lycopersici. Several BGCs were also identified on supernumerary chromosomes; heterologous expression of genes for three terpene synthases encoded on the Fusarium poae supernumerary chromosome and subsequent GC/MS analysis showed that these genes are functional and encode enzymes that each are able to synthesize koraiol; this observed functional redundancy supports the hypothesis that localization of copies of BGCs on supernumerary chromosomes provides freedom for evolutionary innovations to occur, while the original function remains conserved. Altogether, this systematic overview of biosynthetic diversity in Fusarium paves the way for targeted natural produc

Background: The Fusarium oxysporum species complex (FOSC) contains several phylogenetic lineages. Phylogenetic studies identified two to three major clades within the FOSC. The mitochondrial sequences are highly informative phylogenetic markers, but have been mostly neglected due to technical difficulties. Results: A total of 61 complete mitogenomes of FOSC strains were de novo assembled and annotated. Length variations and intron patterns support the separation of three phylogenetic species. The variable region of the mitogenome that is typical for the genus Fusarium shows two new variants in the FOSC. The variant typical for Fusarium is found in members of all three clades, while variant 2 is found in clades 2 and 3 and variant 3 only in clade 2. The extended set of loci analyzed using a new implementation of the genealogical concordance species recognition method support the identification of three phylogenetic species within the FOSC. Comparative analysis of the mitogenomes in the FOSC revealed ongoing mitochondrial recombination within, but not between phylogenetic species. Conclusions: The recombination indicates the presence of a parasexual cycle in F. oxysporum. The obstacles hindering the usage of the mitogenomes are resolved by using next generation sequencing and selective genome assemblers, such as GRAbB. Complete mitogenome sequences offer a stable basis and reference point for phylogenetic and population genetic studies.

Black Sigatoka or black leaf streak disease, caused by the Dothideomycete fungus Pseudocercospora fijiensis (previously: Mycosphaerella fijiensis), is the most significant foliar disease of banana worldwide. Due to the lack of effective host resistance, management of this disease requires frequent fungicide applications, which greatly increase the economic and environmental costs to produce banana. Weekly applications in most banana plantations lead to rapid evolution of fungicide-resistant strains within populations causing disease-control failures throughout the world. Given its extremely high economic importance, two strains of P. fijiensis were sequenced and assembled with the aid of a new genetic linkage map. The 74-Mb genome of P. fijiensis is massively expanded by LTR retrotransposons, making it the largest genome within the Dothideomycetes. Melting-curve assays suggest that the genomes of two closely related members of the Sigatoka disease complex, P. eumusae and P. musae, also are expanded. Electrophoretic karyotyping and analyses of molecular markers in P. fijiensis field populations showed chromosome-length polymorphisms and high genetic diversity. Genetic differentiation was also detected using neutral markers, suggesting strong selection with limited gene flow at the studied geographic scale. Frequencies of fungicide resistance in fungicide-treated plantations were much higher than those in untreated wild-type P. fijiensis populations. A homologue of the Cladosporium fulvum Avr4 effector, PfAvr4, was identified in the P. fijiensis genome. Infiltration of the purified PfAVR4 protein into leaves of the resistant banana variety Calcutta 4 resulted in a hypersensitive-like response. This result suggests that Calcutta 4 could carry an unknown resistance gene recognizing PfAVR4. Besides adding to our understanding of the overall Dothideomycete genome structures, the P. fijiensis genome will aid in developing fungicide treatment schedules to combat this pathog

GRAbB (Genomic Region Assembly by Baiting) is a new program that is dedicated to assemble specific genomic regions from NGS data. This approach is especially useful when dealing with multi copy regions, such as mitochondrial genome and the rDNA repeat region, parts of the genome that are often neglected or poorly assembled, although they contain interesting information from phylogenetic or epidemiologic perspectives, but also single copy regions can be assembled. The program is capable of targeting multiple regions within a single run. Furthermore, GRAbB can be used to extract specific loci from NGS data, based on homology, like sequences that are used for barcoding. To make the assembly specific, a known part of the region, such as the sequence of a PCR amplicon or a homologous sequence from a related species must be specified. By assembling only the region of interest, the assembly process is computationally much less demanding and may lead to assemblies of better quality. In this study the different applications and functionalities of the program are demonstrated such as: exhaustive assembly (rDNA region and mitochondrial genome), extracting homologous regions or genes (IGS, RPB1, RPB2 and TEF1a), as well as extracting multiple regions within a single run. The program is also compared with MITObim, which is meant for the exhaustive assembly of a single target based on a similar query sequence. GRAbB is shown to be more efficient than MITObim in terms of speed, memory and disk usage. The other functionalities (handling multiple targets simultaneously and extracting homologous regions) of the new program are not matched by other programs. The program is available with explanatory documentation at https://github.com/b-brankovics/grabb. GRAbB has been tested on Ubuntu (12.04 and 14.04), Fedora (23), CentOS (7.1.1503) and Mac OS X (10.7). Furthermore, GRAbB is available as a docker repository: brankovics/grabb (https://hub.docker.com/r/brankovics/grabb/).

The occurrence resistance to methyl benzimidazole carbamates (MBC)-fungicides in the Fusarium graminearum species complex (FGSC) is becoming a serious problem in the control of Fusarium head blight in China. The resistance is caused by point mutations in the ß2-tubulingene. So far, five resistant genotypes (F167Y, E198Q, E198L, E198K and F200Y) have been reported in the field. To establish a high-throughput method for rapid detection of all the five mutations simultaneously, an efficient single-nucleotide-polymorphism-based genotyping method was developed based on the Luminex xMAP system. One pair of amplification primers and five allele specific primer extension probes were designed and optimized to specially distinguish the different genotypes within one single reaction. This method has good extensibility and can be combined with previous reported probes to form a highly integrated tool for species, trichothecene chemotype and MBC resistance detection. Using this method, carbendazim resistant FGSC isolates from Jiangsu, Anhui and Sichuan Province in China were identified. High and moderate frequencies of resistance were observed in Jiangsu and Anhui Province, respectively. Carbendazim resistance in F. asiaticum is only observed in the 3ADON genotype. Overall, our method proved to be useful for early detection of MBC resistance in the field and the result aids in the choice of fungicide type.

This pathogen causes serious losses in more than 200 crop species worldwide. Although there are fungicides for its control, many classes of fungicides have failed due to its genetic plasticity. It has become an important model for molecular study of necrotrophic fungi. The aim of the genome sequencing is to provide the community with the tools to analyse and dissect the genetic basis of pathogenicity., This pathogen causes serious losses in more than 200 crop species worldwide. Although there are fungicides for its control, many classes of fungicides have failed due to its genetic plasticity. It has become an important model for molecular study of necrotrophic fungi. The aim of the genome sequencing is to provide the community with the tools to analyse and dissect the genetic basis of pathogenicity.

We searched the genome of Mycosphaerella fijiensis for molecular markers that would allow population genetics analysis of this plant pathogen. M. fijiensis, the causal agent of banana leaf streak disease, also known as black Sigatoka, is the most devastating pathogen attacking bananas (Musa spp). Recently, the entire genome sequence of M. fijiensis became available. We screened this database for VNTR markers. Forty-two primer pairs were selected for validation, based on repeat type and length and the number of repeat units. Five VNTR markers showing multiple alleles were validated with a reference set of isolates from different parts of the world and a population from a banana plantation in Costa Rica. Polymorphism information content values varied from 0.6414 to 0.7544 for the reference set and from 0.0400 and 0.7373 for the population set. Eighty percent of the polymorphism information content values were above 0.60, indicating that the markers are highly informative. These markers allowed robust scoring of agarose gels and proved to be useful for variability and population genetics studies. In conclusion, the strategy we developed to identify and validate VNTR markers is an efficient means to incorporate markers that can be used for fungicide resistance management and to develop breeding strategies to control banana black leaf streak disease. This is the first report of VNTR-minisatellites from the M. fijiensis genome sequence.