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

RT-qPCR primers used to quantify expression of Colletotrichum lentis candidate effectors in an infection time-course on lentil cultivar Eston. (XLSX 10 kb

Reverse Genetics for Functional Genomics of Phytopathogenic Fungi and Oomycetes

Sequencing of over 40 fungal and oomycete genomes has been completed. The next major challenge in modern fungal/oomycete biology is now to translate this plethora of genome sequence information into biological functions. Reverse genetics has emerged as a seminal tool for functional genomics investigations. Techniques utilized for reverse genetics like targeted gene disruption/replacement, gene silencing, insertional mutagenesis, and targeting induced local lesions in genomes will contribute greatly to the understanding of gene function of fungal and oomycete pathogens. This paper provides an overview on high-throughput reverse genetics approaches to decode fungal/oomycete genomes

Rethinking pulse crop production in the era of Aphanomyces root rot

Non-Peer Reviewe

Characterization of Aphanomyces euteiches pathotypes infecting peas in Western Canada

Saskatchewan Pulse GrowersPeer ReviewedAphanomyces root rot, caused by the soil-borne 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 USA 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 x 103 zoospores mL-1 to the base of each plant. Root discoloration was scored 10 days post-inoculation using a 0-5 scale. Testing revealed that 38 of the isolates, including standard pathotype I isolate RB84 belonged to pathotype I, 4 isolates including standard pathotype III isolate Ae109 were pathotype III, and USA isolate Ae16-01 was a pathotype II isolate. An alfalfa isolate from Quebec was avirulent on all pea genotypes. These findings indicate that pathotype type I is predominant on the Canadian prairies

Peroxisomal Alanine: Glyoxylate Aminotransferase AGT1 Is Indispensable for Appressorium Function of the Rice Blast Pathogen, Magnaporthe oryzae

The role of β-oxidation and the glyoxylate cycle in fungal pathogenesis is well documented. However, an ambiguity still remains over their interaction in peroxisomes to facilitate fungal pathogenicity and virulence. In this report, we characterize a gene encoding an alanine, glyoxylate aminotransferase 1 (AGT1) in Magnaporthe oryzae, the causative agent of rice blast disease, and demonstrate that AGT1 is required for pathogenicity of M. oryzae. Targeted deletion of AGT1 resulted in the failure of penetration via appressoria; therefore, mutants lacking the gene were unable to induce blast symptoms on the hosts rice and barley. This penetration failure may be associated with a disruption in lipid mobilization during conidial germination as turgor generation in the appressorium requires mobilization of lipid reserves from the conidium. Analysis of enhanced green fluorescent protein expression using the transcriptional and translational fusion with the AGT1 promoter and open reading frame, respectively, revealed that AGT1 expressed constitutively in all in vitro grown cell types and during in planta colonization, and localized in peroxisomes. Peroxisomal localization was further confirmed by colocalization with red fluorescent protein fused with the peroxisomal targeting signal 1. Surprisingly, conidia produced by the Δagt1 mutant were unable to form appressoria on artificial inductive surfaces, even after prolonged incubation. When supplemented with nicotinamide adenine dinucleotide (NAD+)+pyruvate, appressorium formation was restored on an artificial inductive surface. Taken together, our data indicate that AGT1-dependent pyruvate formation by transferring an amino group of alanine to glyoxylate, an intermediate of the glyoxylate cycle is required for lipid mobilization and utilization. This pyruvate can be converted to non-fermentable carbon sources, which may require reoxidation of NADH generated by the β-oxidation of fatty acids to NAD+ in peroxisomes. Therefore, it may provide a means to maintain redox homeostasis in appressoria

Comparison of the epidemiology of ascochyta blights on grain legumes

International audienceAsochyta blights of grain legumes are caused by fungal pathogens in the genus Ascochyta. Different species infect the different legume species, and in pea three species including Phoma medicaginis var. pinodella have been implicated in ascochyta blight. The impact of the diseases varies between crops, countries, seasons and cropping systems, and yield loss data collected under well-defined conditions is scarce. However, ascochyta blights are considered major diseases in many areas where legumes are grown. Symptoms appear on all aerial parts of the plant, and lesions are similar for most of the species, except for M. pinodes and P. medicaginis var. pinodella. Infected seed, stubble and/or air-borne ascospores are major sources of primary inoculum. Their importance varies between species and also between regions. All Ascochyta spp. produce rain-splashed conidia during the cropping season which are responsible for the spread of the disease within the crop canopy. Only in pea are ascospores involved in secondary disease spread. Limited data suggests that Ascochyta spp. may be hemibiotrophs; however, toxins characteristic for necrotrophs have been isolated from some of the species. Modelling of ascochyta blights is still in the developmental stage and implementation of such models for disease forecasting is the exceptio

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

Cataloging proteins putatively secreted during the biotrophy-necrotrophy transition of the anthracnose pathogen Colletotrichum truncatum

Hemibiotrophic phytopathogenic fungi cause devastating diseases in agronomically important crops. These fungal pathogens exploit a stealth bi-phasic infection strategy to colonize host plants. Their morphological and nutritional transition from biotrophy (characterized by voluminous intracellular primary hyphae) to necrotrophy (characterized by thin secondary hyphae) known as the biotrophy-necrotrophy switch (hemibiotrophy) is critical in symptom and disease development. To establish successful hemibiotrophic parasitism, pathogens likely secrete suites of proteins at the switch that constitute the biotrophy-necrotrophy switch secretome. To catalog such proteins, a directional cDNA library was constructed from mRNA isolated from infected Lens culinaris leaflet tissues displaying the switch of Colletotrichum truncatum, and 5,000 expressed sequence tags (ESTs) were generated. Four potential groups [hydrolytic enzymes, cell envelope-associated proteins (CEAPs), candidate effectors and proteins with diverse functions] were identified from pathogen-derived ESTs. Expression profiling of transcripts encoding CEAPs and candidate effectors in an infection time-course revealed that the majority of these transcripts were expressed or induced during the necrotrophic phase and repressed during the biotrophic phase of in planta colonization, indicating the massive accumulation of proteins at the switch. Taken together, our data suggest that the hemibiotrophic mode of fungal proliferation entails complex interactions of a pathogen with its host wherein the pathogen requires live host cells prior to switching to the necrotrophic phase. The microbial proteins employed during pathogenesis are likely to have defined roles at specific stages of pathogenesis