Effect of phosphate and the arbuscular mycorrhizal fungus Glomus intraradices on disease severity of root rot of peas (Pisum sativum) caused by Aphanomyces euteiches

 The effects of inorganic phosphate levels and the presence of arbuscular mycorrhiza on disease severity of Aphanomyces euteiches in pea roots were studied. Disease severity on roots and epicotyl as well as the oospore number within infected root tissue were correlated with the phosphorus (P) level in the growth medium. The arbuscular mycorrhizal fungus Glomus intraradices increased P uptake and the P concentration in the plant but reduced disease development in peas. Polyacrylamide gel electrophoresis followed by densitometry of glucose-6-phosphate dehydrogenase specific to A.euteiches was used to measure the activity of the pathogen in roots. The enzyme activity increased with disease severity and disease incidence, except in plants supplemented with P at the highest level, where a peak in activity was seen 12 days after inoculation with the pathogen, followed by a decrease in activity. The epicotyl of mycorrhizal plants showed a reduction in disease severity although this part of the plants was not mycorrhizal. Thus, an induced systemic factor may be responsible for increased resistance in mycorrhizal plants. Accepted: 21 August 1998     

Green manures of oat,rape and sweet corn for reducing common root rot in pea (Pisum sativum) caused by Aphanomyces euteiches

Green manure crops of sweet corn, soybean, alfalfa, snap bean, rape, pea and of the two oat cultivars Dane and Troy were incorporated into the same soil and containers in which the crop had grown for five weeks. The soil was then evaluated for suppression of common root rot (Aphanomyces euteiches) of pea grown in infested pasteurized and non-pasteurized soils in the greenhouse. Pea biomass reduction and a plant bioassay for A. euteiches were used to measure the green manure suppression of disease. Green manures of sweet corn cv.Jubilee, oat cv.Troy, and rape cv.Humus significantly reduced pea biomass losses over the non-amended control soil treatments. Oat cv.Troyand sweet corn cv.Jubilee green manures significantly reduced inoculum density of A. euteiches over the corresponding fallow controls in inoculated pasteurized soil by 87% and 76%, respectively, and in inoculated non-pasteurized soil by 67% and 66%, respectively. Only the green manure of oat cv. Troy reduced inoculum density significantly below fallow.     

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.     

Aphanomyces root rot of cauliflower

Résumé L'influence du cycloheximide sur la composition chimique des parois de deux levures Rhodotorula après 24 heures et 4 jours de croissance dans deux milieux de culture différents a été étudiée.La composition en glucides neutres ne varie que faiblement en fonction de l'âge des cellules, la détermination du rapport des oses neutres n'indiquant qu'un léger enrichissement en mannose dans les parois des cellules âgées. Chez Rhodotorula glutinis, les teneurs en hexosamines et aminoacides augmentent nettement en fonction de l'âge des cellules, cette variation ne se produit pas chez Rhodotorula rubra. Les teneurs en chitine sont dépendantes de la nature du milieu de culture mais non de l'âge des cellules sauf chez Rhodotorula glutinis.L'influence du cycloheximide sur la composition chimique se traduit par une baisse globale en protéines et par une perturbation des concentrations d'aminoacides.Le rapport molaire des oses neutres indique une baisse systématique du galactose. Les valeurs des hexosamines sont également plus faibles; la diminution de la chitine est surtout très nette lorsque les cellules se développent dans un milieu normalement favorable à son élaboration, cependant il n'apparaît pas de relation directe entre les baisses des teneurs en hexosamines et en chitine.

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Summary The influence of cycloheximide on the chemical composition of the cell walls of two Rhodotorula yeasts, cultivated during 24 and 96 hours in two different media, has been studied.The composition, of the neutral sugars varies only weakly with the age of the cells, the determination of the molar ratio revealing only a little increase of mannose.Hexosamine and aminoacids contents in Rhodotorula glutinis cells distinctly increase in old cells; this variation does not appear in Rhodotorula rubra. The content of chitine depends on the culture conditions but not on the age of cells, except with Rhodotorula glutinis.The influence of cycloheximide on the chemical structure is responsable of a general decrease of protein and a perturbation of the concentration of the different aminoacids. The molar ratio of the neutral sugars indicates a decrease of galactose in the cell walls. The values of hexosamine are also lower; the decrease of chitine is especialy appearant when the cells are cultivated in a medium which normaly favors the synthesis of this polymer, but there is no direct relation between the decrease of hexosamin and chitin.

     

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.     

Quantitative trait locus responsible for resistance to Aphanomyces root rot (black root) caused by Aphanomyces cochlioides Drechs. in sugar beet

Aphanomyces root rot, caused by Aphanomyces cochlioides Drechs., is one of the most serious diseases of sugar beet (Beta vulgaris L.). Identification and characterization of resistance genes is a major task in sugar beet breeding. To ensure the effectiveness of marker-assisted screening for Aphanomyces root rot resistance, genetic analysis of mature plants’ phenotypic and molecular markers’ segregation was carried out. At a highly infested field site, some 187 F₂ and 66 F₃ individuals, derived from a cross between lines ‘NK-310mm-O’ (highly resistant) and ‘NK-184mm-O’ (susceptible), were tested, over two seasons, for their level of resistance to Aphanomyces root rot. This resistance was classified into six categories according to the extent and intensity of whole plant symptoms. Simultaneously, two selected RAPD and 159 ‘NK-310mm-O’-coupled AFLP were used in the construction of a linkage map of 695.7 cM. Each of nine resultant linkage groups was successfully anchored to one of nine sugar beet chromosomes by incorporating 16 STS markers. Combining data for phenotype and molecular marker segregation, a single QTL was identified on chromosome III. This QTL explained 20% of the variance in F₂ population (in the year 2002) and 65% in F₃ lines (2003), indicating that this QTL plays a major role in the Aphanomyces root rot resistance. This is the first report of the genetic mapping of resistance to Aphanomyces-caused diseases in sugar beet.     

The potential of antagonistic moroccan Streptomyces isolates for the biological control of damping‐off disease of pea (Pisum sativum L.) caused by Aphanomyces euteiches

Three hundred and fifty‐nine isolates of actinobacteria collected from different Moroccan soils were evaluated for their in vitro antimicrobial activity against the oomycete pathogen Aphanomyces euteiches, the causal agent of damping‐off of pea and other legumes. Eighty‐seven isolates (24%) had an inhibitory in vitro effect against A. euteiches. Fourteen bioactive isolates with the greatest inhibitory effect against A. euteiches and no inhibitory effect on plant beneficial rhizobia were tested for their ability to protect pea seeds and seedlings against the damping‐off disease using culture supernatants or spore suspensions as treatments. The two most protective isolates, OB21 and BA15, significantly reduced, compared to untreated control plants, damping‐off by 33% and 47%, respectively. The two bioactive isolates were classified as species of the genus Streptomyces based on 16S rDNA analysis and morphological and chemical characteristics.     

Root rot of onion caused byPythium irregulare andPythium coloratum

Pythium irregulare andPythium coloratum were isolated consistently from roots of onion plants exhibiting root rot and associated foliar symptoms in fields located in Organe County, NY.P. coloratum predominated following extremely wet weather in 1984 and 1986, whileP. irregulare was prevalent following moderately wet weather in 1985. Both species produced root rot symptoms similar to field symptoms when 12 week old onion plants (cv Downing Yellow Globe) were inoculated and incubated in a growth chamber at 14°C for 10 days. This is the first report ofP. irregulare andP. coloratum as causes of root rot of onion.     

Polyacrylamide Gel Electrophoresis (PAGE) and Densitometric Measurement of Enzyme Activity of the Pea Root Pathogen Aphanomyces euteiches in Pea Roots

A new method to measure enzyme activity of the fungal root pathogen Aphanomyces euteiches in pea roots is described. The specific enzymes of the fungus and the host were separated by polyacrylamide gel electrophoresis (PAGE) and the activity of fungal Glucose-6-phosphate dehydrogenase and Phosphoglucomutase were quantified by densitometry. Fungal activity could be correlated to the percentage infected root length and to the disease symptoms of the plants. The activity of A. euteiches was studied in a time course experiment with increasing levels of zoospore inoculum. The results indicated that an increase in inoculum level resulted in a faster disease development in the plants. The relation between fungal enzyme activity and infection level is discussed.     

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.     

Transcriptome of Aphanomyces euteiches: new oomycete putative pathogenicity factors and metabolic pathways

Aphanomyces euteiches is an oomycete pathogen that causes seedling blight and root rot of legumes, such as alfalfa and pea. The genus Aphanomyces is phylogenically distinct from well-studied oomycetes such as Phytophthora sp., and contains species pathogenic on plants and aquatic animals. To provide the first foray into gene diversity of A. euteiches, two cDNA libraries were constructed using mRNA extracted from mycelium grown in an artificial liquid medium or in contact to plant roots. A unigene set of 7,977 sequences was obtained from 18,864 high-quality expressed sequenced tags (ESTs) and characterized for potential functions. Comparisons with oomycete proteomes revealed major differences between the gene content of A. euteiches and those of Phytophthora species, leading to the identification of biosynthetic pathways absent in Phytophthora, of new putative pathogenicity genes and of expansion of gene families encoding extracellular proteins, notably different classes of proteases. Among the genes specific of A. euteiches are members of a new family of extracellular proteins putatively involved in adhesion, containing up to four protein domains similar to fungal cellulose binding domains. Comparison of A. euteiches sequences with proteomes of fully sequenced eukaryotic pathogens, including fungi, apicomplexa and trypanosomatids, allowed the identification of A. euteiches genes with close orthologs in these microorganisms but absent in other oomycetes sequenced so far, notably transporters and non-ribosomal peptide synthetases, and suggests the presence of a defense mechanism against oxidative stress which was initially characterized in the pathogenic trypanosomatids.     

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.     

Development of codominant simple sequence repeat, single nucleotide polymorphism and sequence characterized amplified region markers for the pea root rot pathogen, Aphanomyces euteiches

Three kinds of genetic markers including simple sequence repeats (SSRs), single nucleotide polymorphisms (SNPs) and sequence characterized amplified regions (SCARs) were developed from Aphanomyces euteiches. Of 69 loci tested, seven SSR, two SNP and two SCAR markers were codominantly polymorphic. These codominant markers and dominant markers described herein will facilitate population genetic and evolutionary studies of this important plant pathogen.     

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.     

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.