Cell wall chitosaccharides are essential components and exposed patterns of the phytopathogenic oomycete Aphanomyces euteiches

Chitin is an essential component of fungal cell walls, where it forms a crystalline scaffold, and chitooligosaccharides derived from it are signaling molecules recognized by the hosts of pathogenic fungi. Oomycetes are cellulosic fungus-like microorganisms which most often lack chitin in their cell walls. Here we present the first study of the cell wall of the oomycete Aphanomyces euteiches, a major parasite of legume plants. Biochemical analyses demonstrated the presence of ca. 10% N-acetyl-D-glucosamine (GlcNAc) in the cell wall. Further characterization of the GlcNAc-containing material revealed that it corresponds to noncrystalline chitosaccharides associated with glucans, rather than to chitin per se. Two putative chitin synthase (CHS) genes were identified by data mining of an A. euteiches expressed sequence tag collection and Southern blot analysis, and full-length cDNA sequences of both genes were obtained. Phylogeny analysis indicated that oomycete CHS diversification occurred before the divergence of the major oomycete lineages. Remarkably, lectin labeling showed that the Aphanomyces euteiches chitosaccharides are exposed at the cell wall surface, and study of the effect of the CHS inhibitor nikkomycin Z demonstrated that they are involved in cell wall function. These data open new perspectives for the development of antioomycete drugs and further studies of the molecular mechanisms involved in the recognition of pathogenic oomycetes by the host plants.     

Infectivity and pathogenicity of the oomycete Aphanomyces invadans in Atlantic menhaden Brevoortia tyrannus

Atlantic menhaden Brevoortia tyrannus develop characteristic skin ulcers in response to infection by the oomycete Aphanomyces invadans. To investigate pathogenicity, we conducted a dose response study. Juvenile menhaden were inoculated subcutaneously with 0, 1, 5, 10, 100, and 500 secondary zoospores per fish and monitored for 37 d post-injection (p.i.). Survival rates declined with increasing zoospore dose, with significantly different survivorship curves for the different doses. Moribund and dead fish exhibited characteristic ulcerous lesions at the injection site starting at 13 d p.i. None of the sham-injected control fish (0 zoospore treatment) died. The LD50 (lethal dose killing 50% of exposed menhaden) for inoculated fish was estimated at 9.7 zoospores; however, some fish receiving an estimated single zoospore developed infections that resulted in death. Menhaden were also challenged by aqueous exposure and confirmed that A. invadans was highly pathogenic by this more environmentally realistic route. Fish that were acclimated to culture conditions for 30 d, and presumably free of skin damage, then aqueously exposed to 100 zoospores ml(-1), exhibited 14% lesion prevalence with 11% mortality. Net-handled fish that were similarly infected had a significantly higher lesion prevalence (64%) and mortality (64%). Control fish developed no lesions and did not die. Scanning electron microscopy of fish skin indicated that zoospores adhered to intact epidermis, germinated and penetrated the epithelium with a germ tube. Our results indicate that A. invadans is a primary pathogen of menhaden and is able to cause disease at very low zoospore concentrations.     

Development of a molecular method to detect and quantify Aphanomyces euteiches in soil

A real-time PCR assay using 136F/211R primers and 161T TaqMan probe for the detection and quantification of Aphanomyces euteiches in soil is presented. The specificity of primers was tested on 105 different A. euteiches isolates, mainly from France. A calibration curve was established with a plasmid pHS1 resulting from the target region cloned into the pCR4 Topo vector (Invitrogen). The target copy number was evaluated and was constant whatever the isolate. A DNA-based method was able to discriminate between different artificial infestation levels in soil with small SDs thus validating the relevance of the extraction and amplification method in soil samples. Furthermore, a good correlation was observed between inoculum quantity in soil estimated by qPCR and root rot severity in plant evaluated by bioassays. These steps are essential when considering the feasibility of using a DNA-based method as a fast and accurate way to evaluate inoculum quantity in soil.     

Association between border cell responses and localized root infection by pathogenic Aphanomyces euteiches

Background and Aims

The oomycete Aphanomyces euteiches causes up to 80 % crop loss in pea (Pisum sativum). Aphanomyces euteiches invades the root system leading to a complete arrest of root growth and ultimately to plant death. To date, disease control measures are limited to crop rotation and no resistant pea lines are available. The present study aims to get a deeper understanding of the early oomycete–plant interaction at the tissue and cellular levels.

Methods

Here, the process of root infection by A. euteiches on pea is investigated using flow cytometry and microscopic techniques. Dynamic changes in secondary metabolism are analysed with high-performance liquid chromatography with diode-array detection.

Key Results

Root infection is initiated in the elongation zone but not in the root cap and border cells. Border-cell production is significantly enhanced in response to root inoculation with changes in their size and morphology. The stimulatory effect of A. euteiches on border-cell production is dependent on the number of oospores inoculated. Interestingly, border cells respond to pathogen challenge by increasing the synthesis of the phytoalexin pisatin.

Conclusions

Distinctive responses to A. euteiches inoculation occur at the root tissue level. The findings suggest that root border cells in pea are involved in local defence of the root tip against A. euteiches. Root border cells constitute a convenient quantitative model to measure the molecular and cellular basis of plant–microbe interactions.     

Genotype-specific responses to the effects of commercial Trichoderma formulations in lentil (Lens culinaris ssp. culinaris) in the presence and absence of the oomycete pathogen Aphanomyces euteiches

Members of the endophytic fungal genus Trichoderma have been established as plant-beneficial microbes and are most successful commercial biologicals in the form of bio-fertilisers, biocontrol agents, and growth stimulators. We report the variable interactions among different lentil genotypes and Trichoderma strains in both the presence and absence of biotic stress (root-rot pathogen Aphanomyces euteiches). Two commercial Trichoderma formulations, namely RootShield^® (RS) and RootShield^® Plus (RSP) based on T. harzianum T22 and T. virens G41, respectively, were evaluated for control of Aphanomyces root rot and plant growth promotion in 23 wild and cultivated lentil genotypes. No significant disease control was recorded with either formulation in any lentil genotype. Significant genotype-specific plant growth promotion was observed in terms of root and shoot development and leaf parameters in a genotype-specific manner. Genotypes of Lens culinaris and Lens tomentosus, both in the primary lentil gene pool, demonstrated the maximum response. The overall effect of Trichoderma treatment was markedly higher under biotically stressed conditions in comparison to unstressed conditions. In many cases, negative responses were recorded, particularly in the absence of root-rot disease. L. tomentosus PI 572390 exhibited positive responses for most of the tested parameters. Our findings clearly indicate that, in the case of lentil, plant genotype plays a major role in interactions among the tested Trichoderma strains and the plant. Moreover, the influence of Trichoderma was greater and more favourable under conditions of biotic stress vs. the absence of stress.     

Nitrogen modulation of Medicago truncatula resistance to Aphanomyces euteiches depends on plant genotype

Nitrogen (N) availability can impact plant resistance to pathogens by the regulation of plant immunity. To better understand the links between N nutrition and plant defence, we analysed the impact of N availability on Medicago truncatula resistance to the root pathogen Aphanomyces euteiches. This oomycete is considered to be the most limiting factor for legume production. Ten plant genotypes were tested in vitro for their resistance to A. euteiches in either complete or nitrate‐deficient medium. N deficiency led to enhanced or reduced susceptibility depending on the plant genotype. Focusing on four genotypes displaying contrasting responses, we determined the impact of N deficiency on plant growth and shoot N concentration, and performed expression analyses on N‐ and defence‐related genes, as well as the quantification of soluble phenolics and different amino acids in roots. Our analyses suggest that N modulation of plant resistance is not linked to plant response to N deprivation or to mechanisms previously identified to be involved in plant resistance. Furthermore, our studies highlight a role of glutamine in mediating the susceptibility to A. euteiches in M. truncatula.     

Interactions between the vesicular-arbuscular mycorrhizal fungus Glomus fascicuhtum and Aphanomyces euteiches root rot of peas

Interactions between Glomus fasciculatum and Aphanomyces euteiches root rot of peas (Pisum sativum), were studied in pot experiments using irradiated soil. Infections with the pathogen were suppressed by VAM when plants were challenge inoculated after two weeks. No reduction of the pathogen was detected when the plants were inoculated with both fungi at the same time. The suppression of the pathogen, obtained by preinoculation with G. fasciculatum, was not reduced when the inoculum level of the pathogen was increased thirty times. The induced resistance to A. euteiches in VAM plants was partially a systemic effect. When root systems were split into two halves, one with mycorrhiza and one with A. euteiches, the oospore production was reduced in both root systems. The infection with the pathogen was only suppressed when both fungi were present in the same pot. The background for the induced resistance is discussed.     

A simple chemically,defined medium for the growth of Aphanomyces euteiches and some other oomycetes

A chemically-defined medium composed of glutathione, D-glucose, DL-asparagine, calcium and magnesium chlorides, and monobasic and dibasic potassium phosphates supported growth of several species of filamentous fungi which include seven isolates ofAphanomyces euteiches and single isolates ofA. leavis, A. stellatus, Achlya ambisexualis and several species ofPythium. Some growth occurred if a stoichiometric equivalent of the amino acids contained in glutathione were substituted for it. Dithiothreitol, a compound which keeps glutathione in the reduced form, inhibited growth ofA. euteiches at the concentrations tested. Replacement of the medium with a solution of known ionic composition caused the fungal colonies to produce and release zoospores and to produce oospores.     

Evolution of new metabolic pathways: prospectives of biotechnology

Many microorganisms possess a remarkable ability to construct new metabolic pathways by modifying and utilizing their existing DNA. Regulatory mutations permit the deregulation or constitutive synthesis of enzymes that possess gratuitous catalytic activity for new substrates. Often silent or cryptic genes, genes not known to be expressed or utilized by the parent strain, are mobilized to catalyse a critical reaction in establishing the new pathway.     

The chaperone Lhs1 contributes to the virulence of the fish-pathogenic oomycete Aphanomyces invadans

Oomycetes are fungal-like eukaryotes and many of them are pathogens that threaten natural ecosystems and cause huge financial losses for the aqua- and agriculture industry. Amongst them, Aphanomyces invadans causes Epizootic Ulcerative Syndrome (EUS) in fish which can be responsible for up to 100% mortality in aquaculture. As other eukaryotic pathogens, in order to establish and promote an infection, A. invadans secretes proteins, which are predicted to overcome host defence mechanisms and interfere with other processes inside the host. We investigated the role of Lhs1 which is part of an ER-resident complex that generally promotes the translocation of proteins from the cytoplasm into the ER for further processing and secretion. Interestingly, proteomic studies reveal that only a subset of virulence factors are affected by the silencing of AiLhs1 in A. invadans indicating various secretion pathways for different proteins. Importantly, changes in the secretome upon silencing of AiLhs1 significantly reduces the virulence of A. invadans in the infection model Galleriamellonella. Furthermore, we show that AiLhs1 is important for the production of zoospores and their cluster formation. This renders proteins required for protein ER translocation as interesting targets for the potential development of alternative disease control strategies in agri- and aquaculture.