Untargeted profiling of secondary metabolites and phytotoxins associated with stemphylium blight of lentil.

MAIN CONCLUSION: Stemphylium botryosum alters lentil secondary metabolism and differentially affects resistant and susceptible genotypes. Untargeted metabolomics identifies metabolites and their potential biosynthetic pathways that play a crucial role in resistance to S. botryosum. The molecular and metabolic processes that mediate resistance to stemphylium blight caused by Stemphylium botryosum Wallr. in lentil are largely unknown. Identifying metabolites and pathways associated with Stemphylium infection may provide valuable insights and novel targets to breed for enhanced resistance. The metabolic changes following infection of four lentil genotypes by S. botryosum were investigated by comprehensive untargeted metabolic profiling employing reversed-phase or hydrophilic interaction liquid chromatography (HILIC) coupled to a Q-Exactive mass spectrometer. At the pre-flowering stage, plants were inoculated with S. botryosum isolate SB19 spore suspension and leaf samples were collected at 24, 96 and 144 h post-inoculation (hpi). Mock-inoculated plants were used as negative controls. After analyte separation, high-resolution mass spectrometry data was acquired in positive and negative ionization modes. Multivariate modeling revealed significant treatment, genotype and hpi effects on metabolic profile changes that reflect lentil response to Stemphylium infection. In addition, univariate analyses highlighted numerous differentially accumulated metabolites. By contrasting the metabolic profiles of SB19-inoculated and mock-inoculated plants and among lentil genotypes, 840 pathogenesis-related metabolites were detected including seven S. botryosum phytotoxins. These metabolites included amino acids, sugars, fatty acids and flavonoids in primary and secondary metabolism. Metabolic pathway analysis revealed 11 significant pathways including flavonoid and phenylpropanoid biosynthesis, which were affected upon S. botryosum infection. This research contributes to ongoing efforts toward a comprehensive understanding of the regulation and reprogramming of lentil metabolism under biotic stress, which will provide targets for potential applications in breeding for enhanced disease resistance.

Cross-Kingdom Gene Coexpression Analysis Using a Stemphylium botryosum-Lens ervoides System Revealed Plasticity of Intercommunication Between the Pathogen Secretome and the Host Immune Systems.

Necrotrophic pathogens are responsible for significant declines in crop yield and quality worldwide. During the infection process, a pathogen releases a series of secretory proteins to counteract the plant immune system, and this interaction of pathogen and host molecules determines whether the pathogen will successfully invade the host plant tissues. In this study, we adopted co-transcriptomic approaches to analyze the Lens ervoides-Stemphylium botryosum system, with a focus on 1,216 fungal genes coding for secretory proteins and 8,810 disease-responsive genes of the host 48, 96, and 144 h postinoculation, captured in two F9 recombinant inbred lines (RILs) displaying contrasting disease responses. By constructing in planta gene coexpression networks (GCNs) for S. botryosum, we found that the pathogen tended to co-upregulate genes regulating cell wall degradation enzymes, effectors, oxidoreductases, and peptidases to a much higher degree in the susceptible host LR-66-577 than in the resistant RIL LR-66-637, indicating that the promotion of these digestive enzymes and toxins increased S. botryosum virulence. Construction of cross-kingdom GCNs between pathogen and plant for the two RILs revealed that the co-upregulation of these fungal digestive enzymes and toxins simultaneously promoted a series of defense responses such as redox change, expression of membrane-related genes and serine/threonine kinase, and stress and disease responses in the susceptible RIL which was not observed in the resistant RIL, indicating that these activities exacerbated susceptibility to S. botryosum.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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.

Using a transcriptome sequencing approach to explore candidate resistance genes against stemphylium blight in the wild lentil species Lens ervoides.

BACKGROUND: Stemphylium blight (SB), caused by Stemphylium botryosum, is a devastating disease in lentil production. Although it is known that accessions of Lens ervoides possess superior SB resistance at much higher frequency than the cultivated lentil species, very little is known about the molecular basis regulating SB resistance in L. ervoides. Therefore, a comprehensive molecular study of SB resistance in L. ervoides was needed to exploit this wild resource available at genebanks for use by plant breeders in resistance breeding. RESULTS: Microscopic and qPCR quantification of fungal growth revealed that 48, 96, and 144 h post-inoculation (hpi) were interesting time points for disease development in L. ervoides recombinant inbred lines (RILs) LR-66-637 (resistant to SB) and LR-66-577 (susceptible to SB). Results of transcriptome sequencing at 0, 48, 96 and 144 hpi showed that 8810 genes were disease-responsive genes after challenge by S. botryosum. Among them, 7526 genes displayed a similar expression trend in both RILs, and some of them were likely involved in non-host resistance. The remaining 1284 genes were differentially expressed genes (DEGs) between RILs. Of those, 712 DEGs upregulated in LR-66-637 were mostly enriched in 'carbohydrate metabolic process', 'cell wall organization or biogenesis', and 'polysaccharide metabolic process'. In contrast, there were another 572 DEGs that were upregulated in LR-66-577, and some of them were enriched in 'oxidation-reduction process', 'asparagine metabolic process' and 'asparagine biosynthetic process'. After comparing DEGs to genes identified in previously described quantitative trait loci (QTLs) for resistance to SB, nine genes were common and three of them showed differential gene expression between a resistant and a susceptible bulk consisting of five RILs each. Results showed that two genes encoding calcium-transporting ATPase and glutamate receptor3.2 were candidate resistance genes, whereas one gene with unknown function was a candidate susceptibility gene. CONCLUSION: This study provides new insights into the mechanisms of resistance and susceptibility in L. ervoides RILs responding to S. botryosum infection. Furthermore, we identified candidate resistance or susceptibility genes which warrant further gene function analyses, and which could be valuable for resistance breeding, if their role in resistance or susceptibility can be confirmed.

Genetic map-guided genome assembly reveals a virulence-governing minichromosome in the lentil anthracnose pathogen Colletotrichum lentis.

Colletotrichum lentis causes anthracnose, which is a serious disease on lentil and can account for up to 70% crop loss. Two pathogenic races, 0 and 1, have been described in the C. lentis population from lentil. To unravel the genetic control of virulence, an isolate of the virulent race 0 was sequenced at 1481-fold genomic coverage. The 56.10-Mb genome assembly consists of 50 scaffolds with N50 scaffold length of 4.89 Mb. A total of 11 436 protein-coding gene models was predicted in the genome with 237 coding candidate effectors, 43 secondary metabolite biosynthetic enzymes and 229 carbohydrate-active enzymes (CAZymes), suggesting a contraction of the virulence gene repertoire in C. lentis. Scaffolds were assigned to 10 core and two minichromosomes using a population (race 0 × race 1, n = 94 progeny isolates) sequencing-based, high-density (14 312 single nucleotide polymorphisms) genetic map. Composite interval mapping revealed a single quantitative trait locus (QTL), qClVIR-11, located on minichromosome 11, explaining 85% of the variability in virulence of the C. lentis population. The QTL covers a physical distance of 0.84 Mb with 98 genes, including seven candidate effector and two secondary metabolite genes. Taken together, the study provides genetic and physical evidence for the existence of a minichromosome controlling the C. lentis virulence on lentil.

Wild Help for Enhancing Genetic Resistance in Lentil Against Fungal Diseases.

Lentil (Lens culinaris) is one of the cool season grain legume crops and an important source of dietary proteins and fibre. Fungal diseases are main constraints to lentil production and account for significant yield and quality losses. Lentil has a narrow genetic base presumably due to a bottleneck during domestication and as a result, any resistance to fungal diseases in the cultivated genepool is gradually eroded and overcome by pathogens. New sources of resistance have been identified in wild lentil (Lens ervoides). This article provides an overview of harnessing resistance potential of wild germplasm to enhance genetic resistance in lentil cultivars using next-generation sequencing-based genotyping, comparative genomics and marker-assisted selection breeding.

QTL mapping of early flowering and resistance to ascochyta blight in chickpea.

In western Canada, chickpea (Cicer arietinum L.) production is challenged by short growing seasons and infestations with ascochyta blight. Research was conducted to determine the genetic basis of the association between flowering time and reaction to ascochyta blight in chickpea. Ninety-two chickpea recombinant inbred lines (RILs) developed from a cross between ICCV 96029 and CDC Frontier were evaluated for flowering responses and ascochyta blight reactions in growth chambers and fields at multiple locations and during several years. A wide range of variation was exhibited by the RILs for days to flower, days to maturity, node of first flowering, plant height, and ascochyta blight resistance. Moderate to high broad sense heritability was estimated for ascochyta blight reaction (H(2) = 0.14-0.34) and for days to flowering (H(2) = 0.45-0.87) depending on the environments. Negative correlations were observed among the RILs for days to flowering and ascochyta blight resistance, ranging from r = -0.21 (P < 0.05) to -0.58 (P < 0.0001). A genetic linkage map consisting of eight linkage groups was developed using 349 SNP markers. Seven QTLs for days to flowering were identified that individually explained 9%-44% of the phenotypic variation. Eight QTLs were identified for ascochyta blight resistance that explained phenotypic variation ranging from 10% to 19%. Clusters of QTLs for days to flowering and ascochyta blight resistances were found on chromosome 3 at the interval of 8.6-23.11 cM and on chromosome 8 at the interval of 53.88-62.33 cM.

Candidate effectors contribute to race differentiation and virulence of the lentil anthracnose pathogen Colletotrichum lentis.

BACKGROUND: The hemibiotroph Colletotrichum lentis, causative agent of anthracnose on Lens culinaris (lentil) was recently described as a new species. During its interaction with the host plant, C. lentis likely secretes numerous effector proteins, including toxins to alter the plant's innate immunity, thereby gaining access to the host tissues for nutrition and reproduction. RESULTS: In silico analysis of 2000 ESTs generated from C. lentis-infected lentil leaf tissues identified 15 candidate effectors. In planta infection stage-specific gene expression waves among candidate effectors were revealed for the appressorial penetration phase, biotrophic phase and necrotrophic phase. No sign of positive selection pressure [ω (dN/dS) < 1] in effectors was detected at the intraspecific level. A single nucleotide polymorphism in the ORF of candidate effector ClCE6, used to develop a KASPar marker, differentiated perfectly between pathogenic race 0 and race 1 isolates when tested on 52 isolates arbitrarily selected from a large culture collection representing the western Canadian population of C. lentis. Furthermore, an EST encoding argininosuccinate lyase (Arg) was identified as a bacterial gene. A toxin protein ClToxB was further characterized as a potential host-specific toxin through heterologous in planta expression. The knock-down of ClToxB transcripts by RNAi resulted in reduced virulence, suggesting that ClToxB is a virulence factor. In silico analysis of the ClToxB sequence and comparative genomics revealed that ToxB is unlikely a foreign gene in the C. lentis genome. Incongruency between established species relationships and that established based on gene sequence data confirmed ToxB arose through evolution from a common ancestor, whereas the bacterial gene Arg identified in C. lentis was horizontally transferred from bacteria. CONCLUSIONS: EST mining and expression profiling revealed a set of in planta expressed candidate effectors. We developed a KASPar assay using effector polymorphism to differentiate C. lentis races. Comparative genomics revealed a foreign gene encoding a potential virulence factor Arg, which was horizontally transferred from bacteria into the genus Colletotrichum. ClToxB is further characterized as a host-specific toxin that is likely to contribute to quantitative differences in virulence between the races 0 and 1.

Overexpression of a novel biotrophy-specific Colletotrichum truncatum effector, CtNUDIX, in hemibiotrophic fungal phytopathogens causes incompatibility with their host plants.

The hemibiotrophic fungus Colletotrichum truncatum causes anthracnose disease on lentils and a few other grain legumes. It shows initial symptomless intracellular growth, where colonized host cells remain viable (biotrophy), and then switches to necrotrophic growth, killing the colonized host plant tissues. Here, we report a novel effector gene, CtNUDIX, from C. truncatum that is exclusively expressed during the late biotrophic phase (before the switch to necrotrophy) and elicits a hypersensitive response (HR)-like cell death in tobacco leaves transiently expressing the effector. CtNUDIX homologs, which contain a signal peptide and a Nudix hydrolase domain, may be unique to hemibiotrophic fungal and fungus-like plant pathogens. CtNUDIX lacking a signal peptide or a Nudix motif failed to induce cell death in tobacco. Expression of CtNUDIX:eGFP in tobacco suggested that the fusion protein might act on the host cell plasma membrane. Overexpression of CtNUDIX in C. truncatum and the rice blast pathogen, Magnaporthe oryzae, resulted in incompatibility with the hosts lentil and barley, respectively, by causing an HR-like response in infected host cells associated with the biotrophic invasive hyphae. These results suggest that C. truncatum and possibly M. oryzae elicit cell death to signal the transition from biotrophy to necrotrophy.

Identification of Lens culinaris defense genes responsive to the anthracnose pathogen Colletotrichum truncatum.

BACKGROUND: Anthracnose of lentil, caused by the hemibiotrophic fungal pathogen Colletotrichum truncatum is a serious threat to lentil production in western Canada. Colletotrichum truncatum employs a bi-phasic infection strategy characterized by initial symptomless biotrophic and subsequent destructive necrotrophic colonization of its host. The transition from biotrophy to necrotrophy (known as the biotrophy-necrotrophy switch [BNS]) is critical in anthracnose development. Understanding plant responses during the BNS is the key to designing a strategy for incorporating resistance against hemibiotrophic pathogens either via introgression of resistance genes or quantitative trait loci contributing to host defense into elite cultivars, or via incorporation of resistance by biotechnological means. RESULTS: The in planta BNS of C. truncatum was determined by histochemical analysis of infected lentil leaf tissues in time-course experiments. A total of 2852 lentil expressed sequence tags (ESTs) derived from C. truncatum-infected leaf tissues were analyzed to catalogue defense related genes. These ESTs could be assembled into 1682 unigenes. Of these, 101 unigenes encoded membrane and transport associated proteins, 159 encoded proteins implicated in signal transduction and 387 were predicted to be stress and defense related proteins (GenBank accessions: JG293480 to JG293479). The most abundant class of defense related proteins contained pathogenesis related proteins (encoded by 125 ESTs) followed by heat shock proteins, glutathione S-transferase, protein kinases, protein phosphatase, zinc finger proteins, peroxidase, GTP binding proteins, resistance proteins and syringolide-induced proteins. Quantitative RT-PCR was conducted to compare the expression of two resistance genes of the NBS-LRR class in susceptible and partially resistant genotypes. One (contig186) was induced 6 days post-inoculation (dpi) in a susceptible host genotype (Eston) whereas the mRNA level of another ( LT21-1990) peaked 4 dpi in a partially resistant host genotype (Robin), suggesting roles in conditioning the susceptibility and conferring tolerance to the pathogen, respectively. CONCLUSIONS: Data obtained in this study suggest that lentil cells recognize C. truncatum at the BNS and in response, mount an inducible defense as evident by a high number of transcripts (23% of the total pathogen-responsive lentil transcriptome) encoding defense related proteins. Temporal expression polymorphism of defense related genes could be used to distinguish the response of a lentil genotype as susceptible or resistant.

The chickpea root rot complex in Saskatchewan, Canada- detection of emerging pathogens and their relative pathogenicity.

Chickpea fields in Saskatchewan, one of the three Canadian prairie provinces, have suffered from major health issues since 2019, but no definitive cause has been determined. Field surveys were conducted in Saskatchewan in 2020 and 2021 in order to develop a better understanding of root rot pathogens associated with chickpea. Root samples were analyzed for the presence of 11 potential chickpea root rot pathogens using end-point PCR. Fusarium redolens, F. solani and F. avenaceum were the most prevalent pathogen species detected in both survey years. The cause of Fusarium wilt in chickpea, F. oxysporum f. sp. ciceris, was not detected in either year, nor were Phytophthora spp. and Verticillium albo-atrum. Berkeleyomyces sp. was detected in one field in each year, and Verticillium dahliae was detected in several fields sampled in 2021. These two pathogens have not been reported previously on chickpea in Saskatchewan. The prevalence of Fusarium species obtained from 2021 root isolations was similar to that determined by molecular tests, with frequent isolation of F. redolens, F. oxysporum, F. avenaceum and F. solani. A series of indoor pathogenicity testing compared root disease severity caused by a selection of 16 isolates of six Fusarium species and single isolates of V. dahliae, Berkeleyomyces sp. and Macrophomina phaseolina. Results showed that select isolates of F. avenaceum were the most aggressive of the Fusarium isolates on chickpea. Despite relatively low inoculum density, a highly aggressive isolate of F. avenaceum caused severe stunting and more root rot symptoms than single isolates of V. dahliae, Berkeleyomyces sp. and M. phaseolina under the test conditions.

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.

Glomerella truncata: another Glomerella species with an atypical mating system.

In the genus Glomerella all species studied to date do not fit the usual mating system of heterothallic ascomycetes. This study investigated the mating system of G. truncata (anamorph Colletotrichum truncatum), a pathogen responsible for lentil anthracnose. Twenty-two field isolates from the Canadian prairies were crossed in all possible combinations, including selfings. All isolates also were screened for the presence of the MAT1-1 and MAT1-2 idiomorphs by targeting small conserved areas of the MAT genes (the alpha domain and the high mobility group HMG box) with degenerate primers, and a pair of G. truncata-specific HMG primers (CT21HMG) were designed. The results of the classical mating study suggested that G. truncata is heterothallic. Isolates fell into two incompatibility groups, which is consistent with a bipolar mating system but different from what has been described in other Glomerella species. Molecular screening showed that the HMG box used as a marker for the MAT1-2 idiomorph was present in both partners of fertile crosses in G. truncata, unlike in the typical ascomycete system, but as previously described for two other Glomerella species. G. truncata therefore appears to share unusual mating system characteristics with the other Glomerella species studied to date.

EST mining identifies proteins putatively secreted by the anthracnose pathogen Colletotrichum truncatum.

BACKGROUND: Colletotrichum truncatum is a haploid, hemibiotrophic, ascomycete fungal pathogen that causes anthracnose disease on many economically important leguminous crops. This pathogen exploits sequential biotrophic- and necrotrophic- infection strategies to colonize the host. Transition from biotrophy to a destructive necrotrophic phase called the biotrophy-necrotrophy switch is critical in symptom development. C. truncatum likely secretes an arsenal of proteins that are implicated in maintaining a compatible interaction with its host. Some of them might be transition specific. RESULTS: A directional cDNA library was constructed from mRNA isolated from infected Lens culinaris leaflet tissues displaying the biotrophy-necrotrophy switch of C. truncatum and 5000 expressed sequence tags (ESTs) with an average read of > 600 bp from the 5-prime end were generated. Nearly 39% of the ESTs were predicted to encode proteins of fungal origin and among these, 162 ESTs were predicted to contain N-terminal signal peptides (SPs) in their deduced open reading frames (ORFs). The 162 sequences could be assembled into 122 tentative unigenes comprising 32 contigs and 90 singletons. Sequence analyses of unigenes revealed four potential groups: hydrolases, cell envelope associated proteins (CEAPs), candidate effectors and other proteins. Eleven candidate effector genes were identified based on features common to characterized fungal effectors, i.e. they encode small, soluble (lack of transmembrane domain), cysteine-rich proteins with a putative SP. For a selected subset of CEAPs and candidate effectors, semiquantitative RT-PCR showed that these transcripts were either expressed constitutively in both in vitro and in planta or induced during plant infection. Using potato virus X (PVX) based transient expression assays, we showed that one of the candidate effectors, i. e. contig 8 that encodes a cerato-platanin (CP) domain containing protein, unlike CP proteins from other fungal pathogens was unable to elicit a hypersensitive response (HR). CONCLUSIONS: The current study catalogues proteins putatively secreted at the in planta biotrophy-necrotrophy transition of C. truncatum. Some of these proteins may have a role in establishing compatible interaction with the host plant.

Interactive Gene Expression Patterns of Susceptible and Resistant Lens ervoides Recombinant Inbred Lines and the Necrotroph Ascochyta lentis.

Ascochyta lentis is a foliar pathogen of Lens species and is of worldwide importance in cultivated lentil production. High levels of resistance were identified in the wild species Lens ervoides. This resistance was explored through histopathology, qPCR estimation of fungal biomass and transcriptome sequencing in a susceptible and a resistant recombinant inbred line (RIL) of L. ervoides infected with an aggressive isolate of A. lentis. Necrotrophic growth was delayed in the resistant RIL compared to accelerated necrotrophy of A. lentis in the susceptible RIL. Analysis of the fungal secretome indicated that the early activation of cell wall-degrading enzymes contributed to increased virulence of A. lentis. On the host side, gene co-expression analysis revealed that the invasion by A. lentis caused mRNA, DNA and protein decay in infected plants regardless of the level of resistance in the host. The resistant RIL exhibited a stronger gene co-expression in lipid localization and sulfur processes, and cellular responses to nutrients and stimuli than the susceptible RIL. In addition, differential gene analysis revealed that the repression of both, gibberellin signaling and cell death associated with the hypersensitive response (HR), were associated with enhanced A. lentis resistance.

Defense responses of lentil (Lens culinaris) genotypes carrying non-allelic ascochyta blight resistance genes to Ascochyta lentis infection.

Ascochyta blight of lentil is an important fungal disease in many lentil-producing regions of the world causing major yield and grain quality losses. Quick shifts in aggressiveness of the population of the causal agent Ascochyta lentis mandates developing germplasm with novel and durable resistance. In the absence of complete resistance, lentil genotypes CDC Robin and 964a-46 have frequently been used as sources of partial resistance to ascochyta blight and carry non-allelic ascochyta blight resistance genes. RNA-seq analysis was conducted to identify differences in the transcriptome of CDC Robin, 964a-46 and the susceptible check Eston after inoculation with A. lentis. Candidate defense genes differentially expressed among the genotypes had hypothetical functions in various layers of plant defense, including pathogen recognition, phytohormone signaling pathways and downstream defense responses. CDC Robin and 964a-46 activated cell surface receptors (e.g. receptor like kinases) tentatively associated with pathogen-associated molecular patterns (PAMP) recognition and nucleotide-binding site leucine-rich repeat (NBS-LRR) receptors associated with intracellular effector recognition upon A. lentis infection, and differed in their activation of salicylic acid, abscisic acid and jasmonic acid / ethylene signal transduction pathways. These differences were reflected in the differential expression of downstream defense responses such as pathogenesis-related proteins, and genes associated with the induction of cell death and cell-wall reinforcement. A significant correlation between expression levels of a selection of genes based on quantitative real-time PCR and their expression levels estimated through RNA-seq demonstrated the technical and analytical accuracy of RNA-seq for identification of genes differentially expressed among genotypes. The presence of different resistance mechanisms in 964a-46 and CDC Robin indicates their value for pyramiding gene leading to more durable resistance to ascochyta blight.

Assessment of the Effect of Seed Infection with Ascochyta pisi on Pea in Western Canada.

The role of seed infection with Ascochyta pisi using naturally infected seeds with an incidence from 0.5 to 14.5% was studied in field pea experiments in western Canada at locations with historically low inoculum pressure. A significant effect of A. pisi seed infection on the emergence of seedlings was observed in one experiment and when all data were pooled, but emergence was only reduced minimally, and symptoms of A. pisi on the aerial parts of the seedlings were rarely observed. The level of seed infection at planting had no impact on A. pisi disease severity on mature plants, on seed yield and size, or on the incidence of A. pisi infection of harvested seeds although A. pisi was the dominant species recovered from seeds. Results suggest that the disease did not progress significantly from seeds to seedlings, hence did not contribute to infection of aerial parts of the plants, and therefore infected seeds cannot be regarded as a source of inoculum in the epidemiology of this pathogen under western Canadian growing conditions. Assessing seed components of seeds with varying levels of A. pisi infection and seed staining revealed that the pathogen was present in all components of the seed, regardless of the severity of seed staining. This indicates that infected seeds may be an important way for the pathogen to survive in nature.

Genotype-Dependent Interaction of Lentil Lines with Ascochyta lentis.

Ascochyta blight of lentil is a prevalent disease in many lentil producing regions and can cause major yield and grain quality losses. The most environmentally acceptable and economically profitable method of control is to develop varieties with high levels of durable resistance. Genetic studies to date suggest that ascochyta blight resistance genes (R-gene) in lentil lines CDC Robin, ILL 7537, 964a-46, and ILL 1704 are non-allelic. To understand how different R-genes manifest resistance in these genotypes and an accession of Lens ervoides, L-01-827A, with high level of resistance to ascochyta blight, cellular and molecular defense responses were compared after inoculation with the causal pathogen Ascochyta lentis. Pathogenicity testing of the resistant lines to A. lentis inoculation revealed significantly lower disease severity on CDC Robin and ILL 7537 compared to ILL 1704 and 964a-46, and no symptoms of disease were observed on L-01-827A. Histological examinations indicated that cell death triggered by the pathogen might be disrupted as a mechanism of resistance in CDC Robin. In contrast, limiting colonization of epidermal cells by A. lentis is a suggested mechanism of resistance in 964a-46. A time-series comparison of the expressions of hallmark genes in salicylic acid (SA) and jasmonic acid (JA) signal transduction pathways between CDC Robin and 964a-46 was conducted. These partially resistant genotypes differed in the timing and the magnitude of SA and JA signaling pathway activation. The SA signaling pathway was only triggered in 964a-46, whereas the JA pathway was triggered in both partially resistant genotypes CDC Robin and 964a-46. The expression of JA-associated genes was lower in 964a-46 than CDC Robin. These observations corroborate the existence of diverse ascochyta blight resistance mechanisms in lentil genotypes carrying different R-genes.

QTL mapping reveals genetic determinants of fungal disease resistance in the wild lentil species Lens ervoides.

Lens ervoides, a wild relative of lentil is an important source of allelic diversity for enhancing the genetic resistance of the cultivated species against economically important fungal diseases, such as anthracnose and Stemphylium blight caused by Colletotrichum lentis and Stemphylium botryosum, respectively. To unravel the genetic control underlying resistance to these fungal diseases, a recombinant inbred line (RIL) population (n = 94, F9) originating from a cross between two L. ervoides accessions, L01-827A and IG 72815, was genotyped on the Illumina HiSeq 2500 platform. A total of 289.07 million 100 bp paired-end reads were generated, giving an average 7.53-fold genomic coverage to the RILs and identifying 2,180 high-quality SNPs that assembled in 543 unique haplotypes. Seven linkage groups were resolved among haplotypes, equal to the haploid chromosome number in L. ervoides. The genetic map spanned a cumulative distance of 740.94 cM. Composite interval mapping revealed five QTLs with a significant association with resistance to C. lentis race 0, six QTLs for C. lentis race 1 resistance, and three QTLs for S. botryosum resistance. Taken together, the data obtained in the study reveal that the expression of resistance to fungal diseases in L. ervoides is a result of rearrangement of resistant alleles contributed by both parental accessions.