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The crayfish plague pathogen Aphanomyces astaci (Oomycota: Saprolegniales) is native to North America but expanded with its crayfish hosts to other regions. In most of its invaded range, A. astaci haplotypes are associated with specific American crayfish, probably due to introduction bottlenecks, but haplotype diversity is higher and clear host-specific associations are lacking in its native range. However, little is known about the infection rate and load of this pathogen in North America. We investigated the distribution, prevalence and genetic variation of A. astaci in Pennsylvania (eastern USA), where multiple native and introduced crayfish species (family Cambaridae) occur. We used A. astaci -specific quantitative PCR to screen 533 individuals representing 8 crayfish species (2 Cambarus and 6 Faxonius ) from 49 sites. Faxonius limosus , an American species first introduced to Europe and carrier of A. astaci genotype group E, was of particular interest. We confirmed A. astaci infections in 76% of sites in all but 1 host taxon, with the pathogen infection rate and load comparable to established populations of North American crayfish studied in Europe and Japan. Despite the absence of highly infected hosts, we genotyped A. astaci from 14 sites. We only detected 2 mitochondrial haplotypes, but nuclear markers indicated the presence of at least 4 distinct pathogen genotypes, none documented from invaded areas in Europe or Asia. Genotype group E was not detected in F. limosus , possibly due to limited spatial distribution of the original strain. Our results highlight both benefits and limitations of combining multiple pathogen genotyping methods.

Freshwater crayfish immunity has received great attention due to the need for urgent conservation. This concern has increased the understanding of the cellular and humoral defense systems, although the regulatory mechanisms involved in these processes need updating. There are, however, aspects of the immune response that require clarification and integration. The particular issues addressed in this review include an overall description of the oomycete Aphanomyces astaci , the causative agent of the pandemic plague disease, which affects freshwater crayfish, and an overview of crustaceans’ immunity with a focus on freshwater crayfish. It includes a classification system of hemocyte sub-types, the molecular factors involved in hematopoiesis and the differential role of the hemocyte subpopulations in cell-mediated responses, including hemocyte infiltration, inflammation, encapsulation and the link with the extracellular trap cell death pathway (ETosis). In addition, other topics discussed include the identity and functions of hyaline cells, the generation of neoplasia, and the emerging topic of the role of sessile hemocytes in peripheral immunity. Finally, attention is paid to the molecular execution of the immune response, from recognition by the pattern recognition receptors (PRRs), the role of the signaling network in propagating and maintaining the immune signals, to the effector elements such as the putative function of the Down syndrome adhesion molecules (Dscam) in innate immune memory.

Root tips of many plant species release a number of border, or border-like, cells that are thought to play a major role in the protection of root meristem. However, little is currently known on the structure and function of the cell wall components of such root cells. Here, we investigate the sugar composition of the cell wall of the root cap in two species: pea (Pisum sativum), which makes border cells, and Brassica napus, which makes border-like cells. We find that the cell walls are highly enriched in arabinose and galactose, two major residues of arabinogalactan proteins. We confirm the presence of arabinogalactan protein epitopes on root cap cell walls using immunofluorescence microscopy. We then focused on these proteoglycans by analyzing their carbohydrate moieties, linkages, and electrophoretic characteristics. The data reveal (1) significant structural differences between B. napus and pea root cap arabinogalactan proteins and (2) a cross-link between these proteoglycans and pectic polysaccharides. Finally, we assessed the impact of root cap arabinogalactan proteins on the behavior of zoospores of Aphanomyces euteiches, an oomycetous pathogen of pea roots. We find that although the arabinogalactan proteins of both species induce encystment and prevent germination, the effects of both species are similar. However, the arabinogalactan protein fraction from pea attracts zoospores far more effectively than that from B. napus. This suggests that root arabinogalactan proteins are involved in the control of early infection of roots and highlights a novel role for these proteoglycans in root-microbe interactions.

Background Aphanomyces euteiches is a soil-borne oomycete that causes root rot in pea and other legume species. Symptoms of Aphanomyces root rot (ARR) include root discoloration and wilting, leading to significant yield losses in pea production. Resistance to ARR is known to be polygenic but the roles of single genes in the pea immune response are still poorly understood. This study uses transcriptomics to elucidate the immune response of two pea genotypes varying in their levels of resistance to A. euteiches . Results In this study, we inoculated roots of the pea ( P. sativum L. ) genotypes ‘Linnea’ (susceptible) and ‘PI180693’ (resistant) with two different A. euteiches strains varying in levels of virulence. The roots were harvested at 6 h post-inoculation (hpi), 20 hpi and 48 hpi, followed by differential gene expression analysis. Our results showed a time- and genotype-dependent immune response towards A. euteiches infection, involving several WRKY and MYB-like transcription factors, along with genes associated with jasmonic acid (JA) and abscisic acid (ABA) signaling. By cross-referencing with genes segregating with partial resistance to ARR, we identified 39 candidate disease resistance genes at the later stage of infection. Among the genes solely upregulated in the resistant genotype ‘PI180693’, Psat7g091800.1 was polymorphic between the pea genotypes and encoded a Leucine-rich repeat receptor-like kinase reminiscent of the Arabidopsis thaliana FLAGELLIN-SENSITIVE 2 receptor. Conclusions This study provides new insights into the gene expression dynamics controlling the immune response of resistant and susceptible pea genotypes to A. euteiches infection. We present a set of 39 candidate disease resistance genes for ARR in pea, including the putative immune receptor Psat7g091800.1, for future functional validation.

Aphanomyces astaci, the crayfish plague pathogen, first appeared in Europe in the mid-19(th) century and is still responsible for mass mortalities of native European crayfish. The spread of this parasite across the continent is especially facilitated by invasive North American crayfish species that serve as its reservoir. In France, multiple cases of native crayfish mortalities have been suggested to be connected with the presence of the signal crayfish Pacifastacus leniusculus, which is highly abundant in the country. It shares similar habitats as the native white-clawed crayfish Austropotamobius pallipes and, when infected, the signal crayfish might therefore easily transmit the pathogen to the native species. We investigated the prevalence of A. astaci in French signal crayfish populations to evaluate the danger they represent to local populations of native crayfish. Over 500 individuals of Pacifastacus leniusculus from 45 French populations were analysed, plus several additional individuals of other non-indigenous crayfish species Orconectes limosus, O. immunis and Procambarus clarkii. Altogether, 20% of analysed signal crayfish tested positive for Aphanomyces astaci, and the pathogen was detected in more than half of the studied populations. Local prevalence varied significantly, ranging from 0% up to 80%, but wide confidence intervals suggest that the number of populations infected by A. astaci may be even higher than our results show. Analysis of several individuals of other introduced species revealed infections among two of these, O. immunis and P. clarkii. Our results confirm that the widespread signal crayfish serves as a key reservoir of Aphanomyces astaci in France and therefore represents a serious danger to native crayfish species, especially the white-clawed crayfish. The prevalence in other non-indigenous crayfish should also be investigated as they likely contribute to pathogen transmission in the country.

The oomycete Aphanomyces astaci, the causative agent of crayfish plague, is listed as one of the 100 worst invasive species in the world, destroying the native crayfish populations throughout Eurasia. The aim of this study was to examine the potential of selected mitochondrial (mt) genes to track the diversity of the crayfish plague pathogen A. astaci. Two sets of primers were developed to amplify the mtDNA of ribosomal rnnS and rnnL subunits. We confirmed two main lineages, with four different haplogroups and five haplotypes among 27 studied A. astaci strains. The haplogroups detected were (1) the A-haplogroup with the a-haplotype strains originating from Orconectes sp., Pacifastacus leniusculus and Astacus astacus; (2) the B-haplogroup with the b-haplotype strains originating from the P. leniusculus; (3) the D-haplogroup with the d1 and d2-haplotypes strains originating from Procambarus clarkii; and (4) the E-haplogroup with the e-haplotype strains originating from the Orconectes limosus. The described markers are stable and reliable and the results are easily repeatable in different laboratories. The present method has high applicability as it allows the detection and characterization of the A. astaci haplotype in acute disease outbreaks in the wild, directly from the infected crayfish tissue samples.

Oomycete plant pathogens secrete effector proteins to promote disease. The damaging soilborne legume pathogen Aphanomyces euteiches harbors a specific repertoire of Small Secreted Protein effectors (AeSSPs), but their biological functions remain unknown. Here we characterize AeSSP1256. The function of AeSSP1256 is investigated by physiological and molecular characterization of Medicago truncatula roots expressing the effector. A potential protein target of AeSSP1256 is identified by yeast-two hybrid, co-immunoprecipitation, and fluorescent resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM) assays, as well as promoter studies and mutant characterization. AeSSP1256 impairs M. truncatula root development and promotes pathogen infection. The effector is localized to the nucleoli rim, triggers nucleoli enlargement and downregulates expression of M. truncatula ribosome-related genes. AeSSP1256 interacts with a functional nucleocytoplasmic plant RNA helicase (MtRH10). AeSSP1256 relocates MtRH10 to the perinucleolar space and hinders its binding to plant RNA. MtRH10 is associated with ribosome-related genes, root development and defense. This work reveals that an oomycete effector targets a plant RNA helicase, possibly to trigger nucleolar stress and thereby promote pathogen infection.

Freshwater crayfish Pontastacus leptodactylus is an indigenous European species of considerable ecological and commercial significance. However, it is highly susceptible to the oomycete Aphanomyces astaci , the causative agent of crayfish plague. Although its importance is widely recognized, little information exists on how seasonality affects the physiology and welfare of this species. The present study was designed to (i) investigate seasonal effects on the physiological responses of P. leptodactylus in relation to abiotic factors and crayfish plague outbreak, and (ii) identify the most suitable period for restocking in temperate regions. To achieve this, we characterized for the first time the mRNA expression of hsp70, hsp90, hif-1α, ubiquitin, and gapdh (as endogenous control gene), alongside protein levels of Hsp70, Hsp90, hydroxylated Hif-1α, ubiquitin conjugates, cleaved caspases, Bax/Bcl-2 ratio, and interleukin-6 (IL-6). Our findings revealed that cold stress triggered both the heat shock response and apoptosis. Interestingly, despite the activation of apoptosis, which generally suppresses inflammation, IL-6 levels increased, suggesting a possible association with A. astaci infection. Moreover, elevated Hif-1α levels indicated hypoxic stress during warmer months, likely linked to reduced oxygen availability and turbidity. Overall results suggest that crayfish plague epidemics may intensify with rising temperatures, whereas winter appears to be the optimal period for restocking. Interestingly, in addition, freshwater crayfish in temperate latitudes are organisms that face both cold and heat stress seasonally in the same ecosystem.

Development of durable plant genetic resistance to pathogens through strategies of QTL pyramiding and diversification requires in depth knowledge of polygenic resistance within the available germplasm. Polygenic partial resistance to Aphanomyces root rot, caused by Aphanomyces euteiches, one of the most damaging pathogens of pea worldwide, was previously dissected in individual mapping populations. However, there are no data available regarding the diversity of the resistance QTL across a broader collection of pea germplasm. In this study, we performed a meta-analysis of Aphanomyces root rot resistance QTL in the four main sources of resistance in pea and compared their genomic localization with genes/QTL controlling morphological or phenological traits and with putative candidate genes. Meta-analysis, conducted using 244 individual QTL reported previously in three mapping populations (Puget x 90-2079, Baccara x PI180693 and Baccara x 552) and in a fourth mapping population in this study (DSP x 90-2131), resulted in the identification of 27 meta-QTL for resistance to A. euteiches. Confidence intervals of meta-QTL were, on average, reduced four-fold compared to mean confidence intervals of individual QTL. Eleven consistent meta-QTL, which highlight seven highly consistent genomic regions, were identified. Few meta-QTL specificities were observed among mapping populations, suggesting that sources of resistance are not independent. Seven resistance meta-QTL, including six of the highly consistent genomic regions, co-localized with six of the meta-QTL identified in this study for earliness and plant height and with three morphological genes (Af, A, R). Alleles contributing to the resistance were often associated with undesirable alleles for dry pea breeding. Candidate genes underlying six main meta-QTL regions were identified using colinearity between the pea and Medicago truncatula genomes. QTL meta-analysis provided an overview of the moderately low diversity of loci controlling partial resistance to A. euteiches in four main sources of resistance in pea. Seven highly consistent genomic regions with potential use in marker-assisted-selection were identified. Confidence intervals at several main QTL regions were reduced and co-segregation among resistance and morphological/phenological alleles was identified. Further work will be required to identify the best combinations of QTL for durably increasing partial resistance to A. euteiches.

Key messageA stable and major QTL, which mapped to an approximately 20.0 cM region on pea chromosome 4, was identified as the most consistent region conferring partial resistance to Aphanomyces euteiches.Aphanomyces root rot (ARR), caused by Aphanomyces euteiches Drechs., is a destructive soilborne disease of field pea (Pisum Sativum L.). No completely resistant pea germplasm is available, and current ARR management strategies rely on partial resistance and fungicidal seed treatments. In this study, an F8 recombinant inbred line population of 135 individuals from the cross ‘Reward’ (susceptible) × ‘00-2067’ (tolerant) was evaluated for reaction to ARR under greenhouse conditions with the A. euteiches isolate Ae-MDCR1 and over 2 years in a field nursery in Morden, Manitoba. Root rot severity, foliar weight, plant vigor and height were used as estimates of tolerance to ARR. Genotyping was conducted with a 13.2 K single-nucleotide polymorphism (SNP) array and 222 simple sequence repeat (SSR) markers. Statistical analyses of the phenotypic data indicated significant (P < 0.001) genotypic effects and significant G × E interactions (P < 0.05) in all experiments. After filtering, 3050 (23.1%) of the SNP and 30 (13.5%) of the SSR markers were retained for linkage analysis, which distributed 2999 (2978 SNP + 21 SSR) of the markers onto nine linkage groups representing the seven chromosomes of pea. Mapping of quantitative trait loci (QTL) identified 8 major-effect (R2 > 20%), 13 moderate-effect (10% < R2 < 20%) effect and 6 minor-effect (R2 < 10%) QTL. A genomic region on chromosome 4, delimited by the SNP markers PsCam037549_22628_1642 and PsCam026054_14999_2864, was identified as the most consistent region responsible for partial resistance to A. euteiches isolate Ae-MDCR1. Other genomic regions important for resistance were of the order chromosome 5, 6 and 7.