Aphanomyces Euteiches Research Articles

Identification of Sources of Resistance to Aphanomyces Root Rot in Pisum.

Aphanomyces root rot (ARR), caused by Aphanomyces euteiches, is one of the most devastating diseases that affect the production of peas. Several control strategies such as crop rotation, biocontrol, and fungicides have been proposed, but none provides a complete solution. Therefore, the deployment of resistant cultivars is fundamental. ARR resistance breeding is hampered by the moderate levels of resistance identified so far. The available screening protocols require post-inoculation root assessment, which is destructive, time-consuming, and tedious. In an attempt to address these limitations, we developed a non-destructive screening protocol based on foliar symptoms and used it to identify new sources of resistance in a Pisum spp. germplasm collection. Accessions were root inoculated separately with two A. euteiches isolates, and leaf symptoms were assessed at 5, 10, 14, 17, and 20 days after inoculation (DAI). Although the majority of accessions exhibited high levels of susceptibility, thirty of them exhibited moderate resistance. These thirty accessions were selected for a second experiment, in which they were inoculated with both A. euteiches isolates at two inoculum doses. The objective of this second trial was to confirm the resistance of these accessions by evaluating root and biomass loss, as well as foliar symptoms, and to compare root and foliar evaluations. As a result, a high correlation (R2 = 0.75) between foliar and root evaluations was observed, validating the foliar evaluation method. Notably, accessions from P.s. subsp. humile exhibited the lowest symptomatology across all evaluation methods, representing valuable genetic resources for breeding programs aimed at developing pea varieties resistant to ARR.

Untargeted metabolomic analysis reveals a potential role of saponins in the partial resistance of pea (Pisum sativum) against a root rot pathogen, Aphanomyces euteiches.

In soil-borne diseases, the plant-pathogen interaction begins as soon as the seed germinates and develops into a seedling. Aphanomyces euteiches, an oomycete, stays dormant in soil and gets activated by sensing the host through chemical signals present in the root exudates. The composition of plant exudates may, thus, play an important role during the early phase of infection. To better understand the role of root exudates in plant resistance, we investigated the interaction between partially resistant lines (PI660736 and PI557500) and susceptible pea cultivars (CDC Meadow and AAC Chrome) against Aphanomyces euteiches during the pre-invasion phase. The root exudates of two sets of cultivars clearly distinguished from each other in inducing oospore germination. PI557500 root exudate not only had diminished induction but also inhibited the oospore germination. The contrast between the root exudates of resistance and susceptible cultivars was reflected in their metabolic profiles. Data from fractionation and oospore germination inhibitory experiments identified a group of saponins that accumulated differentially in susceptible and resistant cultivars. We detected 56 saponins and quantified 44 of them in pea root and 30 from root exudate; the majority of them, especially Soyasaponin I and dehydrosoyasaponin I with potent in vitro inhibitory activities, were present in significantly higher amounts in both roots and root exudates of PI660736 and PI557500 as compared to Meadow and Chrome. Our results provide evidence for saponins as deterrents against Aphanomyces euteiches, which might have contributed to the resistance against root rot in the studied pea cultivars.

Deciphering Aphanomyces euteiches-pea-biocontrol bacterium interactions through untargeted metabolomics

Aphanomyces euteiches causes root rot in pea, leading to significant yield losses. However, the metabolites involved in this pathosystem have not been thoroughly studied. This study aimed to fill this gap and explore mechanisms of bacterial suppression of A. euteiches via untargeted metabolomics using pea grown in a controlled environment. Chemical isotope labeling (CIL), followed by liquid chromatography-mass spectrometry (LC–MS), was used for metabolite separation and detection. Univariate and multivariate analyses showed clear separation of metabolites from pathogen-treated pea roots and roots from other treatments. A three-tier approach positively or putatively identified 5249 peak pairs or metabolites. Of these, 403 were positively identified in tier 1; 940 were putatively identified with high confidence in tier 2. There were substantial changes in amino acid pool, and fatty acid and phenylpropanoid pathway products. More metabolites, including salicylic and jasmonic acids, were upregulated than downregulated in A. euteiches-infected roots. 1-aminocyclopropane-1-carboxylic acid and 12-oxophytodienoic acid were upregulated in A. euteiches + bacterium-treated roots compared to A. euteiches-infected roots. A great number of metabolites were up- or down-regulated in response to A. euteiches infection compared with the control and A. euteiches + bacterium-treated plants. The results of this study could facilitate improved disease management.

Fine mapping of Ae-Ps4.5, a major locus for resistance to pathotype III of Aphanomyces euteiches in pea.

QTL mapping and recombinant screening confirmed the major effect of QTL Ae-Ps4.5 on pea resistance to pathotype III of Aphanomyces euteiches and fine-mapped the QTL to a 3.06-Mb interval. Aphanomyces root rot, caused by Aphanomyces euteiches, is the most important disease of pea (Pisum sativum L.) worldwide. The development of pea-resistant varieties is a major challenge to control the disease. Previous linkage studies identified seven main resistance quantitative trait loci (QTL), including the QTL Ae-Ps4.5 associated with partial resistance in US nurseries infested by the pea pathotype III of A. euteiches. This study aimed to confirm the major effect of Ae-Ps4.5 on A. euteiches pathotype III, refine its interval, and identify candidate genes underlying the QTL. QTL mapping on an updated genetic map from the Puget × 90-2079 pea recombinant inbred line population identified Ae-Ps4.5 in a 0.8-cM confidence interval with a high effect (R2 = 89%) for resistance to the Ae109 reference strain of A. euteiches (pathotype III) under controlled conditions. However, the QTL mapping did not detect Ae-Ps4.5 for resistance to the RB84 reference strain of A. euteiches (pathotype I). Screening 224-pea BC5F2 plant progeny derived from three near-isogenic lines (NILs) carrying the 90-2079 allele at Ae-Ps4.5 in the Puget genetic background with 26 SNP markers identified 15 NILs showing recombination in the QTL interval. Phenotyping of the recombinant lines for resistance to the Ae109 strain of A. euteiches reduced the QTL to a physical interval of 3.06Mb, containing 50 putative annotated genes on the Caméor pea genome V1a among which three candidate genes highlighted. This study provides closely linked SNP markers and putative candidate genes to accelerate pea breeding for resistant varieties to Aphanomyces root rot.

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 416bp with 65 contigs, 59.32% GC content, and 5653 predicted coding sequences (CDS). Genome size of Ps-S25 was 6 058 437bp with 66 contigs, a GC content of 60.08%, and 5575 predicted CDS. The genome size of TS-R2 was 5 282 152bp, 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-S66and 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.

The mycoparasite Pythium oligandrum induces legume pathogen resistance and shapes rhizosphere microbiota without impacting mutualistic interactions.

Pythium oligandrum is a soil-borne oomycete associated with rhizosphere and root tissues. Its ability to enhance plant growth, stimulate plant immunity and parasitize fungal and oomycete preys has led to the development of agricultural biocontrol products. Meanwhile, the effect of P. oligandrum on mutualistic interactions and more generally on root microbial communities has not been investigated. Here, we developed a biological system comprising P. oligandrum interacting with two legume plants, Medicago truncatula and Pisum sativum. P. oligandrum activity was investigated at the transcriptomics level through an RNAseq approach, metabolomics and finally metagenomics to investigate the impact of P. oligandrum on root microbiota. We found that P. oligandrum promotes plant growth in these two species and protects them against infection by the oomycete Aphanomyces euteiches, a devastating legume root pathogen. In addition, P. oligandrum up-regulated more than 1000 genes in M. truncatula roots including genes involved in plant defense and notably in the biosynthesis of antimicrobial compounds and validated the enhanced production of M. truncatula phytoalexins, medicarpin and formononetin. Despite this activation of plant immunity, we found that root colonization by P. oligandrum did not impaired symbiotic interactions, promoting the formation of large and multilobed symbiotic nodules with Ensifer meliloti and did not negatively affect the formation of arbuscular mycorrhizal symbiosis. Finally, metagenomic analyses showed the oomycete modifies the composition of fungal and bacterial communities. Together, our results provide novel insights regarding the involvement of P. oligandrum in the functioning of plant root microbiota.

Advanced backcross QTL analysis and comparative mapping with RIL QTL studies and GWAS provide an overview of QTL and marker haplotype diversity for resistance to Aphanomyces root rot in pea (Pisum sativum).

Aphanomyces euteiches is the most damaging soilborne pea pathogen in France. Breeding of pea resistant varieties combining a diversity of quantitative trait loci (QTL) is a promising strategy considering previous research achievements in dissecting polygenic resistance to A. euteiches. The objective of this study was to provide an overview of the diversity of QTL and marker haplotypes for resistance to A. euteiches, by integrating a novel QTL mapping study in advanced backcross (AB) populations with previous QTL analyses and genome-wide association study (GWAS) using common markers. QTL analysis was performed in two AB populations derived from the cross between the susceptible spring pea variety "Eden" and the two new sources of partial resistance "E11" and "LISA". The two AB populations were genotyped using 993 and 478 single nucleotide polymorphism (SNP) markers, respectively, and phenotyped for resistance to A. euteiches in controlled conditions and in infested fields at two locations. GWAS and QTL mapping previously reported in the pea-Aphanomyces collection and from four recombinant inbred line (RIL) populations, respectively, were updated using a total of 1,850 additional markers, including the markers used in the Eden x E11 and Eden x LISA populations analysis. A total of 29 resistance-associated SNPs and 171 resistance QTL were identified by GWAS and RIL or AB QTL analyses, respectively, which highlighted 10 consistent genetic regions confirming the previously reported QTL. No new consistent resistance QTL was detected from both Eden x E11 and Eden x LISA AB populations. However, a high diversity of resistance haplotypes was identified at 11 linkage disequilibrium (LD) blocks underlying consistent genetic regions, especially in 14 new sources of resistance from the pea-Aphanomyces collection. An accumulation of favorable haplotypes at these 11 blocks was confirmed in the most resistant pea lines of the collection. This study provides new SNP markers and rare haplotypes associated with the diversity of Aphanomyces root rot resistance QTL investigated, which will be useful for QTL pyramiding strategies to increase resistance levels in future pea varieties.

Effects of nitrogen fertilization and a commercial arbuscular mycorrhizal fungal inoculant on root rot and agronomic production of pea and lentil crops.

In the Canadian prairies, pulse crops such as field pea (Pisum sativum L.) and lentil (Lens culinaris L.) are economically important and widely grown. However, in recent years, root rot, caused by a variety of fungal and oomycete pathogens, including Aphanomyces euteiches, has become a limiting factor on yield. In this study, we examined the impacts of nitrogen (N) fertilization and a commercial arbuscular mycorrhizal fungal (AMF) inoculant on pea and lentil plant health and agronomic production at three locations in Saskatchewan: Swift Current, Indian Head and Melfort. The AMF inoculation had no impact on root rot severity, and therefore is not considered a reliable method to manage root rot in pea and lentil. In contrast, N fertilization led to reductions in root rot in Swift Current, but not the other two sites. However, N fertilization did reduce nodulation. When both pea and lentil are considered, the abundance of A. euteiches in soil increased from pre-seeding to mid-bloom. A negative correlation between soil pH and disease severity was also observed. The high between-site variability highlights the importance of testing root rot mitigation strategies under multiple soil conditions to develop site-specific recommendations. Use of N fertilizer as a root rot management strategy merits further exploration, including investigation into its interactions with other management strategies, soil properties, and costs and benefits.

Inoculum dose-disease response relationships for the pea root rot pathogen, Aphanomyces euteiches, are dependent on soil type and other pathogens.

The oomycete pathogen, Aphanomyces euteiches, was implicated for the first time in pea and lentil root rot in Saskatchewan and Alberta in 2012 and 2013. Subsequent surveys from 2014 to 2017 revealed that Aphanomyces root rot (ARR) was widespread across the Canadian prairies. The absence of effective chemical, biological, and cultural controls and lack of genetic resistance leave only one management option: avoidance. The objectives of this study were to relate oospore levels in autoclaved and non-autoclaved soils to ARR severity across soil types from the vast prairie landscape and to determine the relationship of measured DNA quantity of A. euteiches using droplet digital PCR or quantitative PCR to the initial oospore inoculum dose in soils. These objectives support a future end goal of creating a rapid assessment method capable of categorizing root rot risk in field soil samples to aid producers with pulse crop field selection decisions. The ARR severity to oospore dose relationship was statistically significantly affected by the soil type and location from which soils were collected and did not show a linear relationship. For most soil types, ARR did not develop at oospore levels below 100/g soil, but severity rose above this level, confirming a threshold level of 100 oospores/g soil for disease development. For most soil types, ARR severity was significantly higher in non-autoclaved compared to autoclaved treatments, demonstrating the role that other pathogens play in increasing disease severity. There was a significant linear relationship between DNA concentrations measured in soil and oospore inoculum concentration, although the strength of the relationship was better for some soil types, and in some soil types, DNA measurement results underestimated the number of oospores. This research is important for developing a root rot risk assessment system for the Canadian prairies based on soil inoculum quantification, following field validation of soil quantification and relationship to root rot disease severity.

Rhizosphere and Root Community Analysis of Oomycetes Associated with Poor Alfalfa (Medicago sativa) Seedling Establishment

Alfalfa ( Medicago sativa) seeds planted in cold, wet soil are prone to seed rot, pre- and postemergence damping-off, and seedling root rot. These diseases reduce initial stand density and affect forage yields and winter survival when root systems are stunted by infection. Successful management depends on identifying the causal agents but isolation in pure culture is time consuming and may fail to recover slow-growing organisms or those requiring special growth conditions. Here, the oomycetes in rhizosphere soil and roots from eight locations with a history of poor alfalfa establishment were identified by amplicon sequence analysis. Seedling bioassays were done with bulk soil and quantitative PCR (qPCR) assays were done using DNA from bulk soil, rhizosphere soil, and the root tissue for Aphanomyces euteiches, Phytophthora medicaginis, Pythium irregulare, P. sylvaticum, and P. ultimum var. ultimum. In the root tissue and rhizosphere, A. euteiches was the dominant taxon, followed by Phytophthora medicaginis and P. sansomeana, a broad-host-range pathogen not previously known to infect alfalfa. Pythium inflatum and Globisporangium perplexum were also significantly overrepresented in roots at five of the eight locations, indicating that these taxa are commonly enriched in alfalfa roots but had not previously been identified as alfalfa pathogens. The qPCR assays of bulk soil showed that A. euteiches and Phytophthora medicaginis were unevenly distributed in the plots and low concentrations of the pathogens led to high levels of root rot disease. These results support development of cultivars with high levels of resistance to the most common oomycete pathogens.

A greenhouse‐based high‐throughput phenotyping platform for identification and genetic dissection of resistance to Aphanomyces root rot in field pea

AbstractAphanomyces root rot (ARR) is a devastating disease in field pea (Pisum sativum L.) that can cause up to 100% crop failure. Assessment of ARR resistance can be a rigorous, costly, time‐demanding activity that is relatively low‐throughput and prone to human errors. These limits the ability to effectively and efficiently phenotype the disease symptoms arising from ARR infection which remains a perennial bottleneck to the successful evaluation and incorporation of disease resistance into new cultivars. In this study, we developed a greenhouse‐based high‐throughput phenotyping (HTP) platform that moves along the rails above the greenhouse benches and captures the visual symptoms caused by Aphanomyces euteiches in field pea. We pilot tested this platform alongside with conventional visual scoring in five experimental trials under greenhouse conditions, assaying over 12,600 single plants of advanced breeding lines developed by the North Dakota State University Pulse Breeding Program. Precision estimated through broad‐sense heritability (H2) was consistently higher for RGB‐derived indices (H2, Exg = 0.86) than the conventional visual scores (H2, disease severity index = 0.59). Prediction of disease severity using a random forest modeling of RGB‐derived indices achieved 0.69 accuracy on the test sets, with inaccurate classification partly attributed to the presence of tolerant lines (displaying root rot but no foliar symptoms) and within‐line genetic heterogeneity. We genetically dissected variation for ARR resistance from the population using RGB‐derived indices and visual scores through genome‐wide association mapping and identified a total of 260 associated single nucleotide polymorphism (SNP). The number of associated SNP for RGB‐derived indices was consistently higher than the number of associated SNP identified using visual scores, with the most significant SNP explaining about 5%–9% of variance per index. We identified previously mapped genes known to be involved in the biological pathways that trigger immunity against ARR and a few novel QTLs with small‐effect sizes that may be worthy of validation in the future. The newly identified QTLs and underlying genes, along with genotypes with promising resistance identified in this study, can be useful for improving a long‐term and durable resistance to ARR.

The root pathogen Aphanomyces euteiches secretes modular proteases in pea apoplast during host infection.

To successfully colonize the host, phytopathogens have developed a large repertoire of components to both combat the host plant defense mechanisms and to survive in adverse environmental conditions. Microbial proteases are predicted to be crucial components of these systems. In the present work, we aimed to identify active secreted proteases from the oomycete Aphanomyces euteiches, which causes root rot diseases on legumes. Genome mining and expression analysis highlighted an overrepresentation of microbial tandemly repeated proteases, which are upregulated during host infection. Activity Based Protein Profiling and mass spectrometry (ABPP-MS) on apoplastic fluids isolated from pea roots infected by the pathogen led to the identification of 35 active extracellular microbial proteases, which represents around 30% of the genes expressed encoding serine and cysteine proteases during infection. Notably, eight of the detected active secreted proteases carry an additional C-terminal domain. This study reveals novel active modular extracellular eukaryotic proteases as potential pathogenicity factors in Aphanomyces genus.

Evaluation of pea genotype PI180693 partial resistance towards aphanomyces root rot in commercial pea breeding.

The cultivation of vining pea (Pisum sativum) faces a major constraint with root rot diseases, caused by a complex of soil-borne pathogens including the oomycetes Aphanomyces euteiches and Phytophtora pisi. Disease resistant commercial varieties are lacking but the landrace PI180693 is used as a source of partial resistance in ongoing pea breeding programs. In this study, the level of resistance and their interaction with A. euteiches virulence levels of six new back-crossed pea breeding lines, deriving from the cross between the susceptible commercial cultivar Linnea and PI180693, were evaluated for their resistance towards aphanomyces root rot in growth chamber and green house tests. Resistance towards mixed infections by A. euteiches and P. pisi and commercial production traits were evaluated in field trials. In growth chamber trials, pathogen virulence levels had a significant effect on plant resistance, as resistance was more consistent against A. euteiches strains exhibiting high or intermediate virulence compared with lowly virulent strains. In fact, line Z1701-1 showed to be significantly more resistant than both parents when inoculated with a lowly virulent strain. In two separate field trials in 2020, all six breeding lines performed equally well as the resistant parent PI180693 at sites only containing A. euteiches, as there were no differences in disease index. In mixed infections, PI180693 exhibited significantly lower disease index scores than Linnea. However, breeding lines displayed higher disease index scores compared with PI180693, indicating higher susceptibility towards P. pisi. Data on seedling emergence from the same field trials suggested that PI180693 was particularly sensitive towards seed decay/damping off disease caused by P. pisi. Furthermore, the breeding lines performed equally well as Linnea in traits important for green pea production, again emphasizing the commercial potential. In summary, we show that the resistance from PI180693 interacts with virulence levels of the pathogen A. euteiches and is less effective towards root rot caused by P. pisi. Our results show the potential use of combining PI180693 partial resistance against aphanomyces root rot with commercially favorable breeding traits in commercial breeding programs.

A fine-tuned defense at the pea root caps: Involvement of border cells and arabinogalactan proteins against soilborne diseases.

Plants have to cope with a myriad of soilborne pathogens that affect crop production and food security. The complex interactions between the root system and microorganisms are determinant for the whole plant health. However, the knowledge regarding root defense responses is limited as compared to the aerial parts of the plant. Immune responses in roots appear to be tissue-specific suggesting a compartmentalization of defense mechanisms in these organs. The root cap releases cells termed root "associated cap-derived cells" (AC-DCs) or "border cells" embedded in a thick mucilage layer forming the root extracellular trap (RET) dedicated to root protection against soilborne pathogens. Pea (Pisum sativum) is the plant model used to characterize the composition of the RET and to unravel its function in root defense. The objective of this paper is to review modes of action of the RET from pea against diverse pathogens with a special focus on root rot disease caused by Aphanomyces euteiches, one of the most widely occurring and large-scale pea crop diseases. The RET, at the interface between the soil and the root, is enriched in antimicrobial compounds including defense-related proteins, secondary metabolites, and glycan-containing molecules. More especially arabinogalactan proteins (AGPs), a family of plant extracellular proteoglycans belonging to the hydroxyproline-rich glycoproteins were found to be particularly present in pea border cells and mucilage. Herein, we discuss the role of RET and AGPs in the interaction between roots and microorganisms and future potential developments for pea crop protection.

First Report of Root and Hypocotyl Rot of Dry Bean Caused by Aphanomyces euteiches in Alberta, Canada.

Dry bean (Phaseolus vulgaris L.) is a high-value crop grown under irrigation on 45,000 ha in southern Alberta, Canada. In 2019, one field of red beans showed premature yellowing and stunting of shoots in mid-July near Bow Island, AB. Roots had brown lesions along the length of hypocotyl and tap root, and decay and sloughing of the root cortex of lateral roots (Fig. 1). Roots were washed for 10 min under running tap water, and portions of the lesions were excised for DNA extractions using the Plant DNeasy kit (Qiagen) and multiplex PCR assays for an array of pathogens, which included Aphanomyces euteiches (Chatterton et al. 2019). Lesion pieces were also surface sterilized and plated onto PDA or cornmeal agar amended with metalaxyl, benomyl and vancomycin (CMBV) and visualized microscopically for oospores. The roots were PCR positive for A. euteiches, Rhizoctonia solani, and Pythium ultimum. Isolations yielded R. solani, and Fusarium spp. on PDA, but cultures on CMBV were over-grown with Rhizopus. Oospores measuring 24.7 +/- 1.08 m, consistent with the expected size of A. euteiches oospores of 18 - 25 m (Papavizas and Ayers, 1974), were visible in some of the root pieces. To obtain an A. euteiches isolate, soil was collected from three areas of the red bean field and used in a bait assay. Tests were performed using pea (cv. CDC Meadow) and dry bean (cv. Redbond) seeds treated with metalaxyl. Five seeds per pot were planted into field soils in 10-cm pots with 4 replicate pots/field. Soils were watered as needed until the 2nd node stage and then kept at saturation for 14 days, at which time roots were washed. Roots from the dry bean, but not the pea, plants showed severe root rot, including honey-brown discolouration of the lateral roots and extending to the hypocotyl, degradation of the root cortex, and presence of oospores. Diseased root pieces were plated onto CMBV without surface sterilization. Cultures with fast growing, white, aerial mycelia characteristic of A. euteiches on CMBV were recovered from dry bean roots from the three soil samples, considered as three isolates, and were transferred to PDATo confirm identity, total DNA was extracted from 7-day old cultures of the three isolates growing on PDA using the Qiagen DNeasy Plant Kit. The ribosomal DNA internal transcribed spacer (ITS) region was amplified using the primer pair ITS1 and ITS4, and sequenced (White et al. 1990). The sequences, deposited in GenBank with accession numbers OM976770, OM976771, and OM976772, were 100% identical to the ITS rDNA sequence of several isolates of A. euteiches (e.g. KM486066.1; 669/669 bp; 670/670 bp; 670/670 bp) using a BLASTn query and 99.7% identical to the CBS15773 voucher specimen (HQ643116.1, 661/663 bp; 662/664 bp; 662/664 bp). To confirm pathogenicity of these three isolates, cultures were used to produce zoospores for inoculating 14-day old red bean and pea seedlings (Chatterton et al. 2015). Control plants received minimal salt media solution and did not develop symptoms. Dry bean, but not pea, seedlings displayed root rot symptoms (Fig. 2). A. euteiches was re-isolated from dry bean seedlings by plating onto CMBV, and identity confirmed as described above. Although a previous survey detected A. euteiches in a small number of dry bean fields in southern Alberta (Chatterton et al. 2019), tests to confirm Koch's postulates were not performed. Aphanomyces root rot can be a devastating pathogen of pulse crops. Precautionary measures should be taken to prevent spread of this pathogen in the small dry bean growing area of southern Alberta by encouraging producers to use rotation intervals, monitoring movement of the pathogen, and evaluating cultivars for resistance to this pathogen.

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.

Identification and Characterization of Bacteria-Derived Antibiotics for the Biological Control of Pea Aphanomyces Root Rot.

Antibiosis has been proposed to contribute to the beneficial bacteria-mediated biocontrol against pea Aphanomyces root rot caused by the oomycete pathogen Aphanomyces euteiches. However, the antibiotics required for disease suppression remain unknown. In this study, we found that the wild type strains of Pseudomonas protegens Pf-5 and Pseudomonas fluorescens 2P24, but not their mutants that lack 2,4-diacetylphloroglucinol, strongly inhibited A. euteiches on culture plates. Purified 2,4-diacetylphloroglucinol compound caused extensive hyphal branching and stunted hyphal growth of A. euteiches. Using a GFP-based transcriptional reporter assay, we found that expression of the 2,4-diacetylphloroglucinol biosynthesis gene phlAPf-5 is activated by germinating pea seeds. The 2,4-diacetylphloroglucinol producing Pf-5 derivative, but not its 2,4-diacetylphloroglucinol non-producing mutant, reduced disease severity caused by A. euteiches on pea plants in greenhouse conditions. This is the first report that 2,4-diacetylphloroglucinol produced by strains of Pseudomonas species plays an important role in the biocontrol of pea Aphanomyces root rot.

Genetic diversity of the pea root pathogen Aphanomyces euteiches in Europe

AbstractThe oomycete pathogen Aphanomyces euteiches causes root rot in various legume species. In this study we focused on A. euteiches causing root rot in pea (Pisum sativum), thereby being responsible for severe yield losses in pea production. We aimed to understand the genetic diversity of A. euteiches in Europe, covering a north‐to‐south gradient spanning from Sweden, Norway and Finland to the UK, France and Italy. A collection of 85 European A. euteiches strains was obtained, all isolated from infected pea roots from commercial vining pea cultivation fields. The strains were genotyped using 22 simple‐sequence repeat markers. Multilocus genotypes were compiled and the genetic diversity between individual strains and population structure between countries was analysed. The population comprising strains from Italy was genetically different and did not share ancestry with any other population. Also, strains originating from Finland and the eastern parts of Sweden were found to be significantly different from the other populations, while strains from the rest of Europe were more closely related. A subset of 10 A. euteiches strains from four countries was further phenotyped on two susceptible pea genotypes, as well as on one genotype with partial resistance towards A. euteiches. All strains were pathogenic on all pea genotypes, but with varying levels of disease severity. No correlation between the genetic relatedness of strains and virulence levels was found. In summary, our study identified three genetically distinct groups of A. euteiches in Europe along a north‐to‐south gradient, indicating local pathogen differentiation.

Management strategies and distribution of Aphanomyces root rot of alfalfa (Medicago sativa), a continuing threat to forage production in the United States

AbstractAlfalfa (Medicago sativa) is one of several legumes that is affected by Aphanomyces root rot (ARR) caused by Aphanomyces euteiches. Symptoms of ARR on alfalfa seedlings include a yellow‐grey discolouration of roots, rotting and loss of lateral roots, stunted growth, chlorotic foliage and reduction of nitrogen‐producing nodules on roots. Infection can also occur on adult plants leading to loss of lateral roots and nodules. At the seedling stage, ARR decreases alfalfa stand establishment, and field longevity is reduced when adult plants are infected. A. euteiches is an oomycete pathogen that has motile zoospores and thick‐walled oospores that can survive for many years in soil. Two races are currently recognized by pathogenicity on differential alfalfa check cultivars. Most alfalfa cultivars contain race 1 resistance, but there is an increasing development of cultivars with resistance to race 2. Management strategies include planting resistant cultivars, avoiding planting in fields with poor drainage and rotating crops with nonhost plants.

Breeding and Evaluation of a New-Bred Semi-Leafless Pea (Pisum sativum L.) Cultivar Longwan No. 6

The pea (Pisum sativum L.) is an excellent protein source for livestock and human nutrition. However, its growth is hampered by several factors including powdery mildew (Erysiphe pisi DC) and lodging in irrigation areas. These limitations may be solved through combining a Canada powdery mildew-resistant pea (Mp1807) with green cotyledon cultivar (Graf) by means of sexual hybridization. A bred semi-leafless pea, cultivar Longwan No. 6, was selected and evaluated in multiple ecological zones to investigate and evaluate high-yield, adaptability, and resistance to root rot (Aphanomyces euteiches Drechs. f. sp. pisi) and powdery mildew from 2012 to 2014. The results revealed that Longwan No. 6 was characterized by lodging resistance and moderate powdery mildew resistance. The average grain yield of Longwan No. 6 is 2855.3 kg/hm2 in spring sowing and is increased by 18.4% compared to the control cultivar (Longwan No. 1). The average grain yield of Longwan No. 6 is 2349.9 kg/hm2 in winter sowing and is increased by 12.8% when compared to the control cultivar. The results indicated that Longwan No. 6 has a greater potential to increase yield and wide adaptability. Adopting the Longwan No. 6 pea cultivar contributes to improvements in pea production in irrigation areas.