Prevalence of the crayfish plague pathogen in red swamp crayfish populations in western France: How serious is the risk for the native white-clawed crayfish?

The crayfish plague pathogen Aphanomyces astaci has been implicated in a number of mass mortalities and irreversible population declines of native crayfish across Europe. At present, the reservoirs of the pathogen in Europe are mainly populations of invasive North American crayfish species. In southwestern Europe, including France, a particularly widespread invader is the red swamp crayfish Procambarus clarkii. Recent distribution data confirm that P. clarkii is present in at least 75 French departments, i.e. more than 78% of those in metropolitan France. We analysed the prevalence and pathogen load of A. astaci in 42 populations of this species in western France (Nouvelle Aquitaine region), where the species is most densely distributed, particularly in a wide range of environments around the Gironde estuary. The pathogen was detected by two different quantitative PCR assays in more than three quarters of the populations studied (34 out of 42); 163 out of 480 analysed crayfish individuals tested positive for the presence of A. astaci. In most cases, individual infection levels were very low, detectable with quantitative PCR but not sufficient for pathogen genotyping. In seven P. clarkii individuals from four populations, however, we were able to assess A. astaci variation by microsatellite markers and sequencing of mitochondrial markers. All these host specimens carried A. astaci genotype group D, haplotype d1, which has caused the majority of crayfish plague outbreaks in neighbouring Spain. In contrast, the French outbreaks genotyped to date (including eight newly analysed in this study) were mostly caused by strains of genotype group B, specific to the signal crayfish Pacifastacus leniusculus. Haplotype d1 found in P. clarkii was involved in one of the newly characterised outbreaks. Our study confirms that P. clarkii is a potentially important reservoir of the crayfish plague pathogen in France, but not the main source of the pathogen in mass mortalities of A. pallipes, probably due to different ecological requirements of the different invasive host crayfish. However, as P. clarkii continues to spread, the threat posed by this species to native crayfish is likely to increase.

Transcriptomic analysis identifies candidate genes for Aphanomyces root rot disease resistance in pea.

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.

Genomic characterization of three bacterial isolates antagonistic to the pea root rot pathogen Aphanomyces euteiches.

Microorganisms living in soil and rhizosphere or inside plants can promote plant growth and health. Genomic characterization of beneficial microbes could shed light on their special features. Through extensive field survey across Saskatchewan, Canada, followed by in vitro and greenhouse characterization, we identified several bacterial isolates antagonistic to pea root rot pathogen Aphanomyces euteiches. In this study, the genomes of three isolates-Pseudomonas sp. rhizo 66 (PD-S66), Pseudomonas synxantha rhizo 25 (Ps-S25), and Serratia sp. root 2 (TS-R2)-were sequenced, assembled, and annotated. Genome size of PD-S66 was 6 279 416 bp with 65 contigs, 59.32% GC content, and 5653 predicted coding sequences (CDS). Genome size of Ps-S25 was 6 058 437 bp with 66 contigs, a GC content of 60.08%, and 5575 predicted CDS. The genome size of TS-R2 was 5 282 152 bp, containing 26 contigs, a GC content of 56.17%, and 4956 predicted CDS. For the identification of the isolates, digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values were determined, which confirmed PD-S66 and TS-R2 as potential new species, belonging to Pseudomonas and Serratia genera, respectively, while Ps-S25 belongs to species Pseudomonas synxantha. Biosynthetic gene clusters were predicted using antiSMASH. The genomic data provided insight into the genetics and biochemical pathways supporting the antagonistic activity against A. euteiches of these isolates.

Diversity and distribution of Aphanomyces astaci in a European hotspot of ornamental crayfish introductions.

Ornamental trade has become an important introduction pathway of non-native aquatic species worldwide. Correspondingly, there has been an alarming increase in the number of established crayfish of aquarium origin in Europe over the previous decade. The oomycete Aphanomyces astaci, the pathogen causing crayfish plague responsible for serious declines of European crayfish populations, is dispersed with introduced North American crayfish. The role of ornamental taxa in introducing and spreading different genotypes of this pathogen in open waters remains unclear. We investigated the distribution, prevalence, and diversity of A. astaci in Budapest, Hungary, which became a hotspot of aquarium crayfish introductions. Their establishment in this area was facilitated by locally abundant thermal waters. We screened for A. astaci in six host taxa from 18 sites sampled between 2018 and 2021: five cambarids (Cambarellus patzcuarensis, Faxonius limosus, Procambarus alleni, P. clarkii, P. virginalis) and one native astacid (Pontastacus leptodactylus). The pathogen was confirmed at five sampled sites in four host taxa: P. virginalis, P. clarkii, F. limosus, and for the first time in European open waters also in P. alleni. Genotyping was successful only in individuals from two different brooks where multiple host species coexisted but revealed unexpected patterns. Mitochondrial B-haplogroup of A. astaci, previously usually reported from Pacifastacus leniusculus or infected European species, was detected in P. virginalis at both sites, and in both F. limosus and P. virginalis sampled from a thermally stable tributary of Barát brook in 2018. In contrast, A-haplogroup of A. astaci was detected in coexisting F. limosus, P. virginalis and P. clarkii sampled in the same watercourse just a few hundred meters downstream in 2020. Additional genotyping methods indicated that a previously unknown A. astaci strain was associated with the latter haplogroup. One P. virginalis individual from 2020 was apparently co-infected by strains representing both mitochondrial haplogroups. The results indicated multiple sources of A. astaci in Budapest, likely directly associated with the introduction of ornamental species, interspecific transmission of this pathogen among ornamental hosts, and potential for a quick spatial or temporal turnover of dominant A. astaci strains at a certain locality. This highlights that in regions with high richness of potential A. astaci hosts, host taxon/pathogen genotype combinations become unpredictable, which might prevent reliable genotyping of pathogen sources in local crayfish mass mortalities.

eDNA monitoring as a tool for evaluating the reintroduction of Austropotamobius pallipes after a crayfish plague outbreak.

The crayfish plague, a severe disease caused by the oomycete Aphanomyces astaci, is responsible for most population declines of susceptible crayfish in Europe. This pathogen has been devastating native populations of Austropotamobius pallipes since the 1970s in the Iberian Peninsula. In this study, we report a massive mortality event in one of the most important Spanish populations of A. pallipes. We aimed to: (i) identify the cause of the mortality, and (ii) evaluate the reintroduction viability of the species. Over the course of six months, we used environmental DNA (eDNA) and traditional trap-based methods to detect the presence of A. astaci or of native or invasive crayfish in order to evaluate the reintroduction viability of A. pallipes to the affected population. We did not capture any live crayfish or detect the presence of A. astaci in the reservoir water during the six months following the mass mortality event. Our analyses indicated that it was feasible to initiate a reintroduction program at the site, which will continue to be monitored for three to five years and will help improve the conservation status of A. pallipes.

Molecular detection of Aphanomyces astaci - An improved species specific qPCR assay.

The parasitic oomycete Aphanomyces astaci is the causative agent of crayfish plague, a devastating disease for European freshwater crayfish. Species specific quantitative real-time PCR (qPCR) can offer rapid detection of the pathogen. However, the well established A. astaci qPCR assay recommended by the World Organization for Animal Health (WOAH) amplifies the recently described Aphanomyces fennicus. Consequently, false-positive results may occur. This calls for the improvement of the established species specific A. astaci qPCR assay in order to avoid amplifying A. fennicus while screening for A. astaci. We developed an improved species specific A. astaci qPCR assay and validated the assay across three laboratories, using established procedures including different qPCR master mixes for each respective laboratory. Genomic DNA from A. astaci, A. fennicus and closely related Aphanomyces spp. was analysed and compared with both the improved and established assay. Additionally, DNA from crayfish tissue and environmental samples were analysed with both assays. The improved assay showed similar sensitivity with the established assay for all sample types, while proving highly specific for A. astaci avoiding amplification of A. fennicus and the other tested Aphanomyces spp. Environmental DNA (eDNA) samples collected at River Lierelva in Norway amplified with the established assay, but not with the improved assay indicating false positive. We were able to sequence a 530 bp fragment of the ITS region from these eDNA samples and the consensus sequence showed 99.9-100 % pairwise identity with A. fennicus and 97.2-98 % pairwise identity with A. astaci, suggesting that the occurrence of A. fennicus is not limited to Finland, where it was first discovered.

Comparison of exoskeleton microbial communities of co-occurring native and invasive crayfish species.

Host-associated microbial communities are an important determinant of individual fitness and have recently been highlighted as one of the factors influencing the success of invasive species. Invasive hosts introduce their microbes into the new environment, and then both the host and its associated microbes enter into a series of interactions with the native macroscopic and microscopic biota. As these processes are largely unexplored, we aimed to compare the exoskeletal microbial communities of co-occurring and phylogenetically related crayfish: the native narrow-clawed crayfish Pontastacus leptodactylus and the invasive signal crayfish Pacifastacus leniusculus from the recently invaded Korana River, Croatia. The results of high-throughput 16S rRNA sequencing showed that the exoskeletal microbiome of both species is very diverse, significantly influenced by the local environment and dominated by low abundance bacterial families from the phylum Proteobacteria. Furthermore, the exoskeletal microbiomes of the crayfish species differed significantly in the composition and abundance of Amplicon Sequence Variants (ASVs), suggesting that they are to some extent shaped by species-specific intrinsic factors, despite sharing a common habitat. However, over 95% of the bacterial genera associated with the exoskeleton were detected in the exoskeleton samples of both native and invasive crayfish. We paid particular attention to two known crayfish pathogens, Aphanomyces astaci and Saprolegnia parasitica, and find that both species carry low amounts of both pathogens. On the side, we find that a non-standard ddPCR protocol outperforms standard qPCR test for A. astaci under low concentration conditions. Taken together, our results indicate the possibility of bidirectional mixing and homogenisation of exoskeleton microbiome. As such, they can serve as a baseline in future detangling of the processes that act together to shape the microbiomes of co-occuring native and invasive congeners during biological invasions.

Aphanomyces astaci in Mexico: A new haplotype from dwarf crayfish Cambarellus montezumae.

The crayfish plague is an emerging infectious disease caused by the pathogen Aphanomyces astaci (Oomycota), which is responsible for the decimation of Eurasian freshwater crayfish. This pathogen can coexist with the North American crayfish. These are chronic carriers of the disease as consequence of an immune response that can contain the growth of the pathogen without killing it. The origin of A. astaci locates in the southeastern United States and coincides with the origin of the family Cambaridae. This diverse family of decapods is distributed in North America from southern Canada to Honduras. However, only the native crayfish species from Canada and the USA have been examined for the presence of A. astaci. In this study, we describe for the first time the presence of A. astaci in Mexico in a population of the native species Cambarellus montezumae. By analyzing the small (rrnS) and large (rrnL) mitochondrial ribosomal regions, we showed the presence of two haplotypes of A. astaci within the same population (d1-haplotype and, a novel haplotype that was named, mex1-haplotype). The finding of A. astaci in Mexico confirms the occurrence of this pathogen within the range of the family Cambaridae. The individuals of C. montezumae appear to be chronic carriers of A. astaci, indicated by the lack of documented crayfish plague outbreaks in this population, similar to the pattern observed in other North American species. Thus, the results are of special concern to susceptible species of southern regions of America, i.e., Parastacidae. Therefore, this work emphasizes the need to better understand the distribution and genetic diversity of A. astaci within the distribution range of the natural carriers, i.e., North American species, especially the unexplored area of the family Cambaridae.

Austropotamobius pallipes can be infected by two haplotypes of Aphanomyces astaci: A key example from an outbreak at an ex-situ conservation facility.

The crayfish plague, caused by the pathogen Aphanomyces astaci, is a pandemic disease endemic to North America that has been devastating susceptible crayfish populations in Europe since the 19th century. In Spain, this disease has decimated populations of the native crayfish species Austropotamobius pallipes due to introductions of North American crayfish, which act as vectors of the pathogen. To combat against these losses, several regional governments have established ex-situ breeding programs to restock wild populations of the species. In this study, we report on an outbreak of A. astaci that occurred in one of the most important A. pallipes aquaculture centers in Spain. Using a variety of detection methods, we analyzed affected crayfish and environmental samples from the facilities over a period of six months and determined that the outbreak was caused by two haplotypes of A. astaci, d1 and d2, which are both associated with the North American crayfish species Procambarus clarkii. To our knowledge, this is the first report of a two-haplotype coinfection of A. astaci outside the native range of this pathogen.

Aquatic Peptide: The Potential Anti-Cancer and Anti-Microbial Activity of GE18 Derived from Pathogenic Fungus Aphanomyces invadans.

Small molecules as well as peptide-based therapeutic approaches have attracted global interest due to their lower or no toxicity in nature, and their potential in addressing several health complications including immune diseases, cardiovascular diseases, metabolic disorders, osteoporosis and cancer. This study proposed a peptide, GE18 of subtilisin-like peptidase from the virulence factor of aquatic pathogenic fungus Aphanomyces invadans, which elicits anti-cancer and anti-microbial activities. To understand the potential GE18 peptide-induced biological effects, an in silico analysis, in vitro (L6 cells) and in vivo toxicity assays (using zebrafish embryo), in vitro anti-cancer assays and anti-microbial assays were performed. The outcomes of the in silico analyses demonstrated that the GE18 peptide has potent anti-cancer and anti-microbial activities. GE18 is non-toxic to in vitro non-cancerous cells and in vivo zebrafish larvae. However, the peptide showed significant anti-cancer properties against MCF-7 cells with an IC50 value of 35.34 µM, at 24 h. Besides the anti-proliferative effect on cancer cells, the peptide exposure does promote the ROS concentration, mitochondrial membrane potential and the subsequent upregulation of anti-cancer genes. On the other hand, GE18 elicits significant anti-microbial activity against P. aeruginosa, wherein GE18 significantly inhibits bacterial biofilm formation. Since the peptide has positively charged amino acid residues, it targets the cell membrane, as is evident in the FESEM analysis. Based on these outcomes, it is possible that the GE18 peptide is a significant anti-cancer and anti-microbial molecule.

Development of a rapid and sensitive real-time diagnostic assay to detect and quantify Aphanomyces invadans, the causative agent of epizootic ulcerative syndrome.

The oomycete Aphanomyces invadans causes epizootic ulcerative syndrome (EUS), a World Organization for Animal Health (WOAH)-listed disease that has seriously impacted a wide range of fish worldwide. Currently, only three conventional polymerase chain reaction (PCR) assays are recommended for the detection of A. invadans. The robust quantitative PCR (qPCR) assay has recently become more important due to its highly accurate nature and the applicability of qPCR-based environmental DNA (eDNA) detection in the monitoring of pathogens in aquatic environments. Therefore, in this study, we developed a novel TaqMan probe-based qPCR method to sensitively and quantitatively detect A. invadans. The assay limit of detection was determined using 10-fold serial dilutions of linearized A. invadans plasmid. Assay sensitivity was assessed in the presence of interfering substances and compared to three WOAH-listed primers using the mycelia and zoospores of A. invadans with and without fish muscle tissue. The assay specificity was also theoretically and experimentally assessed against other oomycetes, fish muscle tissue, and water samples. The assay's repeatability and reproducibility were determined. In this study, the limit of detection of the developed assay was 7.24 copies of A. invadans genomic DNA per reaction (95% confidence interval (CI): 2.75 to 19.05 copies/reaction). The assay showed the same sensitivity in the presence of other substances. Compared to the WOAH-recommended PCR assays, this assay had 10-times higher sensitivity for all tested samples. There were no cross-reactions with other closely related oomycetes, fish muscle, or water samples, indicating that the assay was highly specific for A. invadans. The repeatability and reproducibility tests showed little variation, ranging from 0.1-0.9% and 0.04-1.1%, respectively, indicating the high consistency, repeatability, and reliability of the developed assay. This highly rapid, sensitive, specific, and consistent EUS qPCR assay would be of importance in transboundary disease management and the monitoring of pathogens in aquatic environments.

Evaluation of Effective Class-Balancing Techniques for CNN-Based Assessment of Aphanomyces Root Rot Resistance in Pea (Pisum sativum L.).

Aphanomyces root rot (ARR) is a devastating disease that affects the production of pea. The plants are prone to infection at any growth stage, and there are no chemical or cultural controls. Thus, the development of resistant pea cultivars is important. Phenomics technologies to support the selection of resistant cultivars through phenotyping can be valuable. One such approach is to couple imaging technologies with deep learning algorithms that are considered efficient for the assessment of disease resistance across a large number of plant genotypes. In this study, the resistance to ARR was evaluated through a CNN-based assessment of pea root images. The proposed model, DeepARRNet, was designed to classify the pea root images into three classes based on ARR severity scores, namely, resistant, intermediate, and susceptible classes. The dataset consisted of 1581 pea root images with a skewed distribution. Hence, three effective data-balancing techniques were identified to solve the prevalent problem of unbalanced datasets. Random oversampling with image transformations, generative adversarial network (GAN)-based image synthesis, and loss function with class-weighted ratio were implemented during the training process. The result indicated that the classification F1-score was 0.92 ± 0.03 when GAN-synthesized images were added, 0.91 ± 0.04 for random resampling, and 0.88 ± 0.05 when class-weighted loss function was implemented, which was higher than when an unbalanced dataset without these techniques were used (0.83 ± 0.03). The systematic approaches evaluated in this study can be applied to other image-based phenotyping datasets, which can aid the development of deep-learning models with improved performance.

Detecting aquatic pathogens with field-compatible dried qPCR assays.

Field-ready qPCR assays with extended shelf-life support monitoring programs for emerging aquatic pathogens and enable quick conservation and management decisions. Here, we developed, validated, and tested the shelf-life of qPCR assays targeting Gyrodactylus salaris and Aphanomyces astaci with lyophilization and air-drying.

Identification of Novel Genes Associated with Partial Resistance to Aphanomyces Root Rot in Field Pea by BSR-Seq Analysis.

Aphanomyces root rot, caused by Aphanomyces euteiches, causes severe yield loss in field pea (Pisum sativum). The identification of a pea germplasm resistant to this disease is an important breeding objective. Polygenetic resistance has been reported in the field pea cultivar '00-2067'. To facilitate marker-assisted selection (MAS), bulked segregant RNA-seq (BSR-seq) analysis was conducted using an F8 RIL population derived from the cross of 'Carman' × '00-2067'. Root rot development was assessed under controlled conditions in replicated experiments. Resistant (R) and susceptible (S) bulks were constructed based on the root rot severity in a greenhouse study. The BSR-seq analysis of the R bulks generated 44,595,510~51,658,688 reads, of which the aligned sequences were linked to 44,757 genes in a reference genome. In total, 2356 differentially expressed genes were identified, of which 44 were used for gene annotation, including defense-related pathways (jasmonate, ethylene and salicylate) and the GO biological process. A total of 344.1 K SNPs were identified between the R and S bulks, of which 395 variants were located in 31 candidate genes. The identification of novel genes associated with partial resistance to Aphanomyces root rot in field pea by BSR-seq may facilitate efforts to improve management of this important disease.

Dataset of the de novo assembly and annotation of the marbled crayfish and the noble crayfish hepatopancreas transcriptomes.

OBJECTIVES: Crayfish plague disease, caused by the oomycete pathogen Aphanomyces astaci represents one of the greatest risks for the biodiversity of the freshwater crayfish. This data article covers the de novo transcriptome assembly and annotation data of the noble crayfish and the marbled crayfish challenged with Ap. astaci. Following the controlled infection experiment (Francesconi et al. in Front Ecol Evol, 2021, https://doi.org/10.3389/fevo.2021.647037 ), we conducted a differential gene expression analysis described in (Bostjancic et al. in BMC Genom, 2022, https://doi.org/10.1186/s12864-022-08571-z ) DATA DESCRIPTION: In total, 25 noble crayfish and 30 marbled crayfish were selected. Hepatopancreas tissue was isolated, followed by RNA sequencing using the Illumina NovaSeq 6000 platform. Raw data was checked for quality with FastQC, adapter and quality trimming were conducted using Trimmomatic followed by de novo assembly with Trinity. Assembly quality was assessed with BUSCO, at 93.30% and 93.98% completeness for the noble crayfish and the marbled crayfish, respectively. Transcripts were annotated using the Dammit! pipeline and assigned to KEGG pathways. Respective transcriptome and raw datasets may be reused as the reference transcriptome assemblies for future expression studies.

Host-pathogen coevolution drives innate immune response to Aphanomyces astaci infection in freshwater crayfish: transcriptomic evidence.

BACKGROUND: For over a century, scientists have studied host-pathogen interactions between the crayfish plague disease agent Aphanomyces astaci and freshwater crayfish. It has been hypothesised that North American crayfish hosts are disease-resistant due to the long-lasting coevolution with the pathogen. Similarly, the increasing number of latent infections reported in the historically sensitive European crayfish hosts seems to indicate that similar coevolutionary processes are occurring between European crayfish and A. astaci. Our current understanding of these host-pathogen interactions is largely focused on the innate immunity processes in the crayfish haemolymph and cuticle, but the molecular basis of the observed disease-resistance and susceptibility remain unclear. To understand how coevolution is shaping the host's molecular response to the pathogen, susceptible native European noble crayfish and invasive disease-resistant marbled crayfish were challenged with two A. astaci strains of different origin: a haplogroup A strain (introduced to Europe at least 50 years ago, low virulence) and a haplogroup B strain (signal crayfish in lake Tahoe, USA, high virulence). Here, we compare the gene expression profiles of the hepatopancreas, an integrated organ of crayfish immunity and metabolism. RESULTS: We characterised several novel innate immune-related gene groups in both crayfish species. Across all challenge groups, we detected 412 differentially expressed genes (DEGs) in the noble crayfish, and 257 DEGs in the marbled crayfish. In the noble crayfish, a clear immune response was detected to the haplogroup B strain, but not to the haplogroup A strain. In contrast, in the marbled crayfish we detected an immune response to the haplogroup A strain, but not to the haplogroup B strain. CONCLUSIONS: We highlight the hepatopancreas as an important hub for the synthesis of immune molecules in the response to A. astaci. A clear distinction between the innate immune response in the marbled crayfish and the noble crayfish is the capability of the marbled crayfish to mobilise a higher variety of innate immune response effectors. With this study we outline that the type and strength of the host immune response to the pathogen is strongly influenced by the coevolutionary history of the crayfish with specific A. astaci strains.

Antiproliferation of MP12 derived from a fungus, Aphanomyces invadans virulence factor, cysteine-rich trypsin inhibitor on human laryngeal epithelial cells, and in vivo zebrafish embryo model.

Peptide-based drug development is an emerging and promising approach in cancer therapeutics. The present study focuses on understanding the mechanism of MP12 peptide (MDNHVCIPLCPP) derived from cysteine-rich trypsin inhibitor protein of virulence factor of pathogenic fungus Aphanomyces invadans. MP12 is involved in antiproliferative activity against the human laryngeal epithelial cell (Hep-2), demonstrated in this study. MP12 sequence showed a significant binding score and has multiple hydrogen bond interactions with the proteins that play a vital role in apoptotic pathways such as Bcl-2, caspase-3, caspase-7, and XIAP. Based on the bioinformatics characterization and molecular docking result, further study was focused on MP12 antiproliferative activity. The peptide showed a dose-dependent inhibition against Hep-2 cell line proliferation, analyzed over MTT and neutral red uptake assays. The IC50 value of the MP12 peptide was calculated based on the antiproliferative property (24.7 ± 0.34 µM). MP12 treated Hep-2 cells showed significant shrinkage in cell morphology compared to untreated cells, inhibiting the cell cycle. The gene expression analysis validated that the MP12 significantly upregulates the caspase-3, caspase-7, and caspase-9 genes. The developmental toxicity study using zebrafish embryos as in vivo model proved that the MP12 is nontoxic. Based on the obtained results, we proposed that the peptide MP12 derived from cysteine-rich trypsin inhibitor protein of virulence molecule of pathogenic fungus have a potential antiproliferative activity. However, further clinical trials need to be focused on the mechanism and therapeutic application against laryngeal cancer.

Characterization of Aphanomyces euteiches Pathotypes Infecting Peas in Western Canada.

Aphanomyces root rot, caused by the soilborne oomycete Aphanomyces euteiches Drechs., has developed into a serious disease in the pea- and lentil-producing areas of the Great Plains of North America. Based on six pea differentials previously used to differentiate 11 pathotypes in France, pathotypes were identified among field isolates from Saskatchewan (14) and Alberta (18). Four isolates from the U.S.A. and standard isolates for pathotypes I and III designated in the French study were also included. Each isolate was tested twice in replicated experiments by inoculating French pea differentials 'Baccara', 'Capella', MN 313, 902131, 552, and PI 80693, along with the Canadian susceptible pea cultivar 'CDC Meadow' and partially resistant USDA line PI 660736 under controlled conditions. Pea plants grown in vermiculite were inoculated 10 days after seeding by pipetting 5 ml of a suspension containing 1 × 103 zoospores ml-1 to the base of each plant. Root discoloration was scored 10 days postinoculation using a 0 to 5 scale. Testing revealed that 38 of the isolates, including standard pathotype I isolate RB84, belonged to pathotype I; four isolates including standard pathotype III isolate Ae109 were pathotype III; and U.S.A. isolate Ae16-01 was a pathotype II isolate. An alfalfa isolate from Quebec was avirulent on all pea genotypes. These findings indicate that pathotype I is predominant on the Canadian prairies.

Tracing the origin of the crayfish plague pathogen, Aphanomyces astaci, to the Southeastern United States.

The oomycete Aphanomyces astaci is an emerging infectious pathogen affecting freshwater crayfish worldwide and is responsible for one of the most severe wildlife pandemics ever reported. The pathogen has caused mass mortalities of freshwater crayfish species in Europe and Asia, and threatens other susceptible species in Madagascar, Oceania and South America. The pathogen naturally coexists with some North American crayfish species that are its chronic carriers. Presumptions that A. astaci originated in North America are based on disease outbreaks that followed translocations of North American crayfish and on the identification of the pathogen mainly in Europe. We studied A. astaci in the southeastern US, a center of freshwater crayfish diversity. In order to decipher the origin of the pathogen, we investigated (1) the distribution and haplotype diversity of A. astaci, and (2) whether there are crayfish species-specificities and/or geographical restrictions for A. astaci haplotypes. A total of 132 individuals, corresponding to 19 crayfish species and one shrimp species from 23 locations, tested positive for A. astaci. Mitochondrial rnnS and rnnL sequences indicated that A. astaci from the southeastern US exhibited the highest genetic diversity so far described for the pathogen (eight haplotypes, six of which we newly describe). Our findings that A. astaci is widely distributed and genetically diverse in the region supports the hypothesis that the pathogen originated in the southeastern US. In contrast to previous assumptions, however, the pathogen exhibited no clear species-specificity or geographical patterns.