Manipulating multi-level selection in a fungal entomopathogen reveals social conflicts and a method for improving biocontrol traits.

Changes in parasite virulence are commonly expected to lead to trade-offs in other life history traits that can affect fitness. Understanding these trade-offs is particularly important if we want to manipulate the virulence of microbial biological control agents. Theoretically, selection across different spatial scales, i.e. between- and within-hosts, shapes these trade-offs. However, trade-offs are also dependent on parasite biology. Despite their applied importance the evolution of virulence in fungal parasites is poorly understood: virulence can be unstable in culture and commonly fails to increase in simple passage experiments. We hypothesized that manipulating selection intensity at different scales would reveal virulence trade-offs in a fungal pathogen of aphids, Akanthomyces muscarius. Starting with a genetically diverse stock we selected for speed of kill, parasite yield or infectivity by manipulating competition within and between hosts and between-populations of hosts over 7 rounds of infection. We characterized ancestral and evolved lineages by whole genome sequencing and by measuring virulence, growth rate, sporulation and fitness. While several lineages showed increases in virulence, we saw none of the trade-offs commonly found in obligately-killing parasites. Phenotypically similar lineages within treatments often shared multiple single-nucleotide variants, indicating strong convergent evolution. The most dramatic phenotypic changes were in timing of sporulation and spore production in vitro. We found that early sporulation led to reduced competitive fitness but could increase yield of spores on media, a trade-off characteristic of social conflict. Notably, the selection regime with strongest between-population competition and lowest genetic diversity produced the most consistent shift to early sporulation, as predicted by social evolution theory. Multi-level selection therefore revealed social interactions novel to fungi and showed that these biocontrol agents have the genomic flexibility to improve multiple traits-virulence and spore production-that are often in conflict in other parasites.

Two-speed genome evolution drives pathogenicity in fungal pathogens of animals.

The origins and evolution of virulence in amphibian-infecting chytrids Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal) are largely unknown. Here, we use deep nanopore sequencing of Bsal and comparative genomics against 21 high-quality genome assemblies that span the fungal Chytridiomycota. We discover that Bsal has the most repeat-rich genome of the Chytridiomycota, comprising 40.9% repetitive elements; this genome has expanded to more than 3× the length of its conspecific Bd, with autonomous and fully functional LTR/Gypsy elements contributing significantly to the expansion. The M36 metalloprotease virulence factors are highly expanded (n = 177) in Bsal, most of which (53%) are flanked by transposable elements, suggesting they have a repeat-associated expansion. We find enrichment upstream of M36 metalloprotease genes of three novel repeat families belonging to the repeat superfamily of LINEs that are implicated with gene copy number variations. Additionally, Bsal has a highly compartmentalized genome architecture, with virulence factors enriched in gene-sparse/repeat-rich compartments, while core conserved genes are enriched in gene-rich/repeat-poor compartments. Genes upregulated during infection are primarily found in the gene-sparse/repeat-rich compartment in both Bd and Bsal. Furthermore, genes with signatures of positive selection in Bd are enriched in repeat-rich regions, suggesting these regions are a cradle for the evolution of chytrid pathogenicity. These are the hallmarks of two-speed genome evolution, and this study provides evidence of two-speed genomes in an animal pathogen, shedding light on the evolution of fungal pathogens of vertebrates driving global declines and extinctions.

Genome sequence and genetic diversity of European ash trees.

Ash trees (genus Fraxinus, family Oleaceae) are widespread throughout the Northern Hemisphere, but are being devastated in Europe by the fungus Hymenoscyphus fraxineus, causing ash dieback, and in North America by the herbivorous beetle Agrilus planipennis. Here we sequence the genome of a low-heterozygosity Fraxinus excelsior tree from Gloucestershire, UK, annotating 38,852 protein-coding genes of which 25% appear ash specific when compared with the genomes of ten other plant species. Analyses of paralogous genes suggest a whole-genome duplication shared with olive (Olea europaea, Oleaceae). We also re-sequence 37 F. excelsior trees from Europe, finding evidence for apparent long-term decline in effective population size. Using our reference sequence, we re-analyse association transcriptomic data, yielding improved markers for reduced susceptibility to ash dieback. Surveys of these markers in British populations suggest that reduced susceptibility to ash dieback may be more widespread in Great Britain than in Denmark. We also present evidence that susceptibility of trees to H. fraxineus is associated with their iridoid glycoside levels. This rapid, integrated, multidisciplinary research response to an emerging health threat in a non-model organism opens the way for mitigation of the epidemic.

Genome Sequence Data Reveal at Least Two Distinct Incursions of the Tropical Race 4 Variant of Fusarium Wilt into South America.

The global banana industry is threatened by one of the most devastating diseases: Fusarium wilt of banana. Fusarium wilt of banana is caused by the soilborne fungus Fusarium oxysporum f. sp. cubense (Foc), which almost annihilated the banana production in the late 1950s. A new strain of Foc, known as tropical race 4 (TR4), attacks a wide range of banana varieties, including Cavendish clones, which are the source of 99% of banana exports. In 2019, Foc TR4 was reported in Colombia, and more recently (2021) in Peru. In this study, we sequenced three fungal isolates identified as Foc TR4 from La Guajira (Colombia) and compared them against 19 whole-genome sequences of Foc TR4 publicly available, including four genome sequences recently released from Peru. To understand the genetic relatedness of the Colombian Foc TR4 isolates and those from Peru, we conducted a phylogenetic analysis based on a genome-wide set of single nucleotide polymorphisms (SNPs). Additionally, we compared the genomes of the 22 available Foc TR4 isolates, looking for the presence-absence of gene polymorphisms and genomic regions. Our results reveal that (i) the Colombian and Peruvian isolates are genetically distant, which could be better explained by independent incursions of the pathogen to the continent, and (ii) there is a high correspondence between the genetic relatedness and geographic origin of Foc TR4. The profile of present/absent genes and the distribution of missing genomic regions showed a high correspondence to the clades recovered in the phylogenetic analysis, supporting the results obtained by SNP-based phylogeny.

Chitosan induces differential transcript usage of chitosanase 3 encoding gene (csn3) in the biocontrol fungus Pochonia chlamydosporia 123.

BACKGROUND: Pochonia chlamydosporia is an endophytic fungus used for nematode biocontrol that employs its cellular and molecular machinery to degrade the nematode egg-shell. Chitosanases, among other enzymes, are involved in this process. In this study, we improve the genome sequence assembly of P. chlamydosporia 123, by utilizing long Pacific Biosciences (PacBio) sequence reads. Combining this improved genome assembly with previous RNA-seq data revealed alternative isoforms of a chitosanase in the presence of chitosan. This study could open new insights into understanding fungal resistance to chitosan and root-knot nematode (RKN) egg infection processes. RESULTS: The P. chlamydosporia 123 genome sequence assembly has been updated using long-read PacBio sequencing and now includes 12,810 predicted protein-coding genes. Compared with the previous assembly based on short reads, there are 701 newly annotated genes, and 69 previous genes are now split. Eight of the new genes were differentially expressed in fungus interactions with Meloidogyne javanica eggs or chitosan. A survey of the RNA-seq data revealed alternative splicing in the csn3 gene that encodes a chitosanase, with four putative splicing variants: csn3_v1, csn3_v2, csn3_v3 and csn3_v4. When P. chlamydosporia is treated with 0.1 mg·mL- 1 chitosan for 4 days, csn3 is expressed 10-fold compared with untreated controls. Furthermore, the relative abundances of each of the four transcripts are different in chitosan treatment compared with controls. In controls, the abundances of each transcript are nil, 32, 55, and 12% for isoforms csn3_v1, csn3_v2, csn3_v3 and csn3_v4 respectively. Conversely, in chitosan-treated P. chlamydosporia, the abundances are respectively 80, 15%, 2-3%, 2-3%. Since isoform csn3_v1 is expressed with chitosan only, the putatively encoded enzyme is probably induced and likely important for chitosan degradation. CONCLUSIONS: Alternative splicing events have been discovered and described in the chitosanase 3 encoding gene from P. chlamydosporia 123. Gene csn3 takes part in RKN parasitism process and chitosan enhances its expression. The isoform csn3_v1 would be related to the degradation of this polymer in bulk form, while other isoforms may be related to the degradation of chitosan in the nematode egg-shell.

Identification and Characterization of Fusarium nirenbergiae Associated with Saffron Corm Rot Disease.

Saffron (Crocus sativus L.) is the most expensive spice plant and is distributed widely around the world. However, its production is limited by corm rot, a disastrous disease, attributed to Fusarium oxysporum in many regions of the world. In 2020, extensive surveys were carried out in Zhejiang, Shanghai, Anhui, and Guizhou provinces as saffron growing areas of China. Fourteen single-spore isolates were obtained from rotted corms and identified as F. nirenbergiae according to morphological appearance and multilocus phylogenetic analysis with translation elongation factor 1-α (tef1), DNA-directed RNA polymerase II largest subunit (rpb2), and ß-tubulin (tub2). Results of the pathogenicity assay supported the conclusion that F. nirenbergiae is the pathogen responsible for corm rot. In this study, we obtained the whole genome sequence of two highly virulent F. nirenbergiae strains via the Illumina HiSeq platform. Genome sequence assemblies of approximately 52.7 and 52.2 Mb were generated for isolates WY5 and SH1, respectively. To the best of our knowledge, this is the first report of F. nirenbergiae causing C. sativus corm rot in China and indeed worldwide. Results from this research contribute to our understanding of genetic diversity, genomic information, and host determination, which will enable researchers to design appropriate management measures for this hazardous disease.

Chitosan modulates Pochonia chlamydosporia gene expression during nematode egg parasitism.

Climate change makes plant-parasitic nematodes (PPN) an increasing threat to commercial crops. PPN can be managed sustainably by the biocontrol fungus Pochonia chlamydosporia (Pc). Chitosan generated from chitin deacetylation enhances PPN parasitism by Pc. In this work, we investigate the molecular mechanisms of Pc for chitosan resistance and root-knot nematode (RKN) parasitism, using transcriptomics. Chitosan and RKN modify the expression of Pc genes, mainly those involved in oxidation-reduction processes. Both agents significantly modify the expression of genes associated to 113 GO terms and 180 Pc genes. Genes encoding putative glycoproteins (Pc adhesives) to nematode eggshell, as well as genes involved in redox, carbohydrate and lipid metabolism trigger the response to chitosan. We identify genes expressed in both the parasitic and endophytic phases of the Pc lifecycle; these include proteases, chitosanases and transcription factors. Using the Pathogen-Host Interaction database (PHI-base), our previous RNA-seq data and RT-PCR of Pc colonizing banana we have investigated genes expressed both in the parasitic and endophytic phases of Pc lifecycle.

Taxonomic Revision of the Banana Fusarium Wilt TR4 Pathogen Is Premature.

Taxonomic revisions for pathogens of crops should be based on robust underpinning evidence. Recently, a substantial revision was proposed for the taxonomy of the causative agent of Fusarium wilt on banana. We reanalyzed the data on which this revision was based and discovered that the data do not robustly support the proposals. Several apparent discrepancies and errors in the published phylogenies cast further doubt on the conclusions drawn from them. Although we do not assert that the authors' conclusions are incorrect, we posit that the taxonomic changes are premature, given the data currently in the public domain.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

The Genome of Peronospora belbahrii Reveals High Heterozygosity, a Low Number of Canonical Effectors, and TC-Rich Promoters.

Along with Plasmopara destructor, Peronosopora belbahrii has arguably been the economically most important newly emerging downy mildew pathogen of the past two decades. Originating from Africa, it has started devastating basil production throughout the world, most likely due to the distribution of infested seed material. Here, we present the genome of this pathogen and results from comparisons of its genomic features to other oomycetes. The assembly of the nuclear genome was around 35.4 Mbp in length, with an N50 scaffold length of around 248 kbp and an L50 scaffold count of 46. The circular mitochondrial genome consisted of around 40.1 kbp. From the repeat-masked genome, 9,049 protein-coding genes were predicted, out of which 335 were predicted to have extracellular functions, representing the smallest secretome so far found in peronosporalean oomycetes. About 16% of the genome consists of repetitive sequences, and, based on simple sequence repeat regions, we provide a set of microsatellites that could be used for population genetic studies of P. belbahrii. P. belbahrii has undergone a high degree of convergent evolution with other obligate parasitic pathogen groups, reflecting its obligate biotrophic lifestyle. Features of its secretome, signaling networks, and promoters are presented, and some patterns are hypothesized to reflect the high degree of host specificity in Peronospora species. In addition, we suggest the presence of additional virulence factors apart from classical effector classes that are promising candidates for future functional studies.

Intraspecific diversification of the crop wild relative Brassica cretica Lam. using demographic model selection.

BACKGROUND: Crop wild relatives (CWRs) contain genetic diversity, representing an invaluable resource for crop improvement. Many of their traits have the potential to help crops to adapt to changing conditions that they experience due to climate change. An impressive global effort for the conservation of various CWR will facilitate their use in crop breeding for food security. The genus Brassica is listed in Annex I of the International Treaty on Plant Genetic Resources for Food and Agriculture. Brassica oleracea (or wild cabbage), a species native to southern and western Europe, has become established as an important human food crop plant because of its large reserves stored over the winter in its leaves. Brassica cretica Lam. (Bc) is a CWR in the brassica group and B. cretica subsp. nivea (Bcn) has been suggested as a separate subspecies. The species Bc has been proposed as a potential gene donor to brassica crops, including broccoli, cabbage, cauliflower, oilseed rape, etc. RESULTS: We sequenced genomes of four Bc individuals, including two Bcn and two Bc. Demographic analysis based on our whole-genome sequence data suggests that populations of Bc are not isolated. Classification of the Bc into distinct subspecies is not supported by the data. Using only the non-coding part of the data (thus, the parts of the genome that has evolved nearly neutrally), we find the gene flow between different Bc population is recent and its genomic diversity is high. CONCLUSIONS: Despite predictions on the disruptive effect of gene flow in adaptation, when selection is not strong enough to prevent the loss of locally adapted alleles, studies show that gene flow can promote adaptation, that local adaptations can be maintained despite high gene flow, and that genetic architecture plays a fundamental role in the origin and maintenance of local adaptation with gene flow. Thus, in the genomic era it is important to link the selected demographic models with the underlying processes of genomic variation because, if this variation is largely selectively neutral, we cannot assume that a diverse population of crop wild relatives will necessarily exhibit the wide-ranging adaptive diversity required for further crop improvement.

AzeR, a transcriptional regulator that responds to azelaic acid in Pseudomonas nitroreducens.

Azelaic acid is a dicarboxylic acid that has recently been shown to play a role in plant-bacteria signalling and also occurs naturally in several cereals. Several bacteria have been reported to be able to utilize azelaic acid as a unique source of carbon and energy, including Pseudomonas nitroreducens. In this study, we utilize P. nitroreducens as a model organism to study bacterial degradation of and response to azelaic acid. We report genetic evidence of azelaic acid degradation and the identification of a transcriptional regulator that responds to azelaic acid in P. nitroreducens DSM 9128. Three mutants possessing transposons in genes of an acyl-CoA ligase, an acyl-CoA dehydrogenase and an isocitrate lyase display a deficient ability in growing in azelaic acid. Studies on transcriptional regulation of these genes resulted in the identification of an IclR family repressor that we designated as AzeR, which specifically responds to azelaic acid. A bioinformatics survey reveals that AzeR is confined to a few proteobacterial genera that are likely to be able to degrade and utilize azelaic acid as the sole source of carbon and energy.

Investigation into Adaptation in Genes Associated with Response to Estrogenic Pollution in Populations of Roach (Rutilus rutilus) Living in English Rivers.

Exposure of male fish to estrogenic substances from wastewater treatment works (WwTWs) results in feminization and reduced reproductive fitness. Nevertheless, self-sustaining populations of roach (Rutilus rutilus) inhabit river stretches polluted with estrogenic WwTW effluents. In this study, we examine whether such roach populations have evolved adaptations to tolerate estrogenic pollution by comparing frequency differences in single-nucleotide polymorphisms (SNPs) between populations sampled from rivers receiving either high- or low-level WwTW discharges. SNPs within 36 "candidate" genes, selected for their involvement in estrogenic responses, and 120 SNPs in reference genes were genotyped in 465 roaches. There was no evidence for selection in highly estrogen-dependent candidate genes, including those for the estrogen receptors, aromatases, and vitellogenins. The androgen receptor (ar) and cytochrome P450 1A genes were associated with large shifts in allele frequencies between catchments and in individual populations, but there is no clear link to estrogen pollution. Selection at ar in the effluent-dominated River Lee may have resulted from historical contamination with endocrine-disrupting pesticides. Critically, although our results suggest population-specific selection including at genes related to endocrine disruption, there was no strong evidence that the selection resulted from exposure to estrogen pollution.

Genomics-Informed Molecular Detection of Xanthomonas vasicola pv. vasculorum Strains Causing Severe Bacterial Leaf Streak of Corn.

Xanthomonas vasicola pv. vasculorum (syn. X. campestris pv. vasculorum) was initially identified as the causal agent of bacterial leaf streak of corn in South Africa. The pathovar vasculorum causes disease on sugarcane and corn, but a subset of these strains was noted for its increased disease severity in corn. This subset was reclassified as X. campestris pv. zeae in the early 1990s and was found to have slightly different biochemical and genetic properties than isolates from sugarcane. There has been an emergence of X. campestris pv. zeae-like strains of X. vasicola pv. vasculorum in both the United States and Argentina since 2010. We performed whole genome sequencing on U.S. isolates to confirm their identity. Informed by comparative genomics, we then developed specific TaqMan qPCR and loop-mediated isothermal amplification (LAMP) assays for the detection of this specific subset of X. vasicola pv. vasculorum strains. The qPCR 4909 assay was tested against 27 xanthomonads (diverse representation), 32 DNA extractions from corn leaves confirmed as positive or negative for the bacterium, 41 X. vasicola pv. vasculorum isolates from corn in the United States and Argentina, and 31 additional bacteria associated with corn, sugarcane, or sorghum. In all cases the assay was shown to be specific for the X. vasicola pv. vasculorum isolates that cause more severe disease on corn. We then tested the LAMP 166 assay against the 27 xanthomonads and 32 corn leaf DNA samples, and we found this assay was also specific for this subset of X. vasicola pv. vasculorum isolates. We also developed a live/dead cells distinction protocol using propidium monoazide prior to DNA extraction for analyzing seed washes using these assays. These two detection assays can be useful for both diagnosticians and researchers to specifically identify the X. vasicola pv. vasculorum isolates that cause more severe symptoms on corn.

Transfer of Xanthomonas campestris pv. arecae and X. campestris pv. musacearum to X. vasicola (Vauterin) as X. vasicola pv. arecae comb. nov. and X. vasicola pv. musacearum comb. nov. and Description of X. vasicola pv. vasculorum pv. nov.

We present an amended description of the bacterial species Xanthomonas vasicola to include the causative agent of banana Xanthomonas wilt, as well as strains that cause disease on Areca palm, Tripsacum grass, sugarcane, and maize. Genome-sequence data reveal that these strains all share more than 98% average nucleotide with each other and with the type strain. Our analyses and proposals should help to resolve the taxonomic confusion that surrounds some of these pathogens and help to prevent future use of invalid names.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Shotgun metagenomics reveals differences in antibiotic resistance genes among bacterial communities in Western Balkans glacial lakes sediments.

Long-term overuse of antibiotics has driven the propagation and spreading of antibiotic resistance genes (ARGs) such as efflux pumps in the environment, which can be transferred to clinically relevant pathogens. This study explored the abundance and diversity of ARGs and mobile genetic elements within bacterial communities from sediments of three Western Balkans glacial lakes: Plav Lake (high impact of human population), Black Lake (medium impact of human population) and Donje Bare Lake (remote lake, minimal impact of human population) via shotgun metagenomics. Assembled metagenomic sequences revealed that Resistance-Nodulation-Division (RND) efflux pumps genes were most abundant in metagenome from the Plav Lake. The Integron Finder bioinformatics tool detected 38 clusters of attC sites lacking integron-integrases (CALIN) elements: 20 from Plav Lake, four from Black Lake and 14 from Donje Bare Lake. A complete integron sequence was recovered only from the assembled metagenome from Plav Lake. Plasmid contents within the metagenomes were similar, with proportions of contigs being plasmid-related: 1.73% for Plav Lake, 1.59% for Black Lake and 1.64% for Donje Bare Lake. The investigation showed that RNDs and mobile genetic elements content correlated with human population impact.

A Grain Rot of Rice in Iran Caused by a Xanthomonas Strain Closely Related to X. sacchari.

Rice grain rot disease was detected for the first time in Mazandaran Province, Iran. The bacteria isolated from infected rice plants showed grains rotted and darkening. A Xanthomonas strain closely connected to X. sacchari was identified using molecular and whole genome sequencing approaches confirmed as the causal agent by fulfilling Koch's postulates.

The Zymoseptoria tritici ORFeome: A Functional Genomics Community Resource.

Libraries of protein-encoding sequences can be generated by identification of open reading frames (ORFs) from a genome of choice that are then assembled into collections of plasmids termed ORFeome libraries. These represent powerful resources to facilitate functional genomic characterization of genes and their encoded products. Here, we report the generation of an ORFeome for Zymoseptoria tritici, which causes the most serious disease of wheat in temperate regions of the world. We screened the genome of strain IP0323 for high confidence gene models, identifying 4,075 candidates from 10,933 predicted genes. These were amplified from genomic DNA, were cloned into the Gateway entry vector pDONR207, and were sequenced, providing a total of 3,022 quality-controlled plasmids. The ORFeome includes genes predicted to encode effectors (n = 410) and secondary metabolite biosynthetic proteins (n = 171) in addition to genes residing at dispensable chromosomes (n = 122) or those that are preferentially expressed during plant infection (n = 527). The ORFeome plasmid library is compatible with our previously developed suite of Gateway destination vectors, which have various combinations of promoters, selection markers, and epitope tags. The Z. tritici ORFeome constitutes a powerful resource for functional genomics and offers unparalleled opportunities to understand the biology of Z. tritici.[Formula: see text] Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Tpc1 is an important Zn(II)2Cys6 transcriptional regulator required for polarized growth and virulence in the rice blast fungus.

The establishment of polarity is a critical process in pathogenic fungi, mediating infection-related morphogenesis and host tissue invasion. Here, we report the identification of TPC1 (Transcription factor for Polarity Control 1), which regulates invasive polarized growth in the rice blast fungus Magnaporthe oryzae. TPC1 encodes a putative transcription factor of the fungal Zn(II)2Cys6 family, exclusive to filamentous fungi. Tpc1-deficient mutants show severe defects in conidiogenesis, infection-associated autophagy, glycogen and lipid metabolism, and plant tissue colonisation. By tracking actin-binding proteins, septin-5 and autophagosome components, we show that Tpc1 regulates cytoskeletal dynamics and infection-associated autophagy during appressorium-mediated plant penetration. We found that Tpc1 interacts with Mst12 and modulates its DNA-binding activity, while Tpc1 nuclear localisation also depends on the MAP kinase Pmk1, consistent with the involvement of Tpc1 in this signalling pathway, which is critical for appressorium development. Importantly, Tpc1 directly regulates NOXD expression, the p22phox subunit of the fungal NADPH oxidase complex via an interaction with Mst12. Tpc1 therefore controls spatial and temporal regulation of cortical F-actin through regulation of the NADPH oxidase complex during appressorium re-polarisation. Consequently, Tpc1 is a core developmental regulator in filamentous fungi, linking the regulated synthesis of reactive oxygen species and the Pmk1 pathway, with polarity control during host invasion.

Improved method for genotyping the causative agent of crayfish plague (Aphanomyces astaci) based on mitochondrial DNA.

Aphanomyces astaci causes crayfish plague, which is a devastating disease of European freshwater crayfish. The likely first introduction of A. astaci into Europe was in the mid-19th century in Italy, presumably with the introduction of North American crayfish. These crayfish can carry A. astaci in their cuticle as a benign infection. Aphanomyces astaci rapidly spread across Europe causing the decline of the highly susceptible indigenous crayfish species. Random amplified polymorphic DNA-PCR analysis of A. astaci pure cultures characterized five genotype groups (A, B, C, D and E). Current A. astaci genotyping techniques (microsatellites and genotype-specific regions, both targeting nuclear DNA) can be applied directly to DNA extracted from infected cuticles but require high infection levels. Therefore, they are not suitable for genotyping benign infections in North American crayfish (carriers). In the present study, we combine bioinformatics and molecular biology techniques to develop A. astaci genotyping molecular markers that target the mitochondrial DNA, increasing the sensitivity of the genotyping tools. The assays were validated on DNA extracts of A. astaci pure cultures, crayfish tissue extractions from crayfish plague outbreaks and tissue extractions from North American carriers. We demonstrate the presence of A. astaci genotype groups A and B in UK waters.