Signatures of adaptation to obligate biotrophy in the Hyaloperonospora arabidopsidis genome

Many oomycete and fungal plant pathogens are obligate biotrophs, which extract nutrients only from living plant tissue and cannot grow apart from their hosts. Although these pathogens cause substantial crop losses, little is known about the molecular basis or evolution of obligate biotrophy. Here, we report the genome sequence of the oomycete Hyaloperonospora arabidopsidis (Hpa), an obligate biotroph and natural pathogen of Arabidopsis thaliana. In comparison with genomes of related, hemibiotrophic Phytophthora species, the Hpa genome exhibits dramatic reductions in genes encoding (i) RXLR effectors and other secreted pathogenicity proteins, (ii) enzymes for assimilation of inorganic nitrogen and sulfur, and (iii) proteins associated with zoospore formation and motility. These attributes comprise a genomic signature of evolution toward obligate biotrophy.     

Control of seed-borne pathogens on legumes by microbial and other alternative seed treatments

Greenhouse trials were carried out in order to test the efficacy of different seed treatments as alternatives to chemicals against Colletotrichum lindemuthianum cause of anthracnose on bean and Ascochyta spp. cause of Ascochyta blights on pea, respectively. Resistance inducers, commercially formulated microorganisms, non-formulated selected strains of different microorganisms (fungi, bacteria and yeasts) and plant extracts were applied as dry or liquid seed treatments on naturally infested seeds. Seedling emergence and disease incidence and/or severity were recorded. Almost all seed treatments turned out to be ineffective in controlling the Ascochyta infections, which is in line with the literature stating that these pathogens are difficult to control. The only alternative treatments that gave some control of Ascochyta spp. were thyme oil and a strain of Clonostachys rosea. The resistance inducers tested successfully controlled infections of bean by C. lindemuthianum. Among the formulated microorganisms, Bacillus subtilis-based formulations provided the best protection from anthracnose. Some strains of Pseudomonas putida, a disease-suppressive, saprophytic strain of Fusarium oxysporum and the mustard powder-based product Tillecur also proved to be effective against bean anthracnose. However, among the resistance inducers as well as among the other groups, certain agents caused a significant reduction of plant emergence. Different alternative seed treatments can therefore be used for the control of C. lindemuthianum on bean, while on pea only thyme oil and a strain of Clonostachys rosea showed some effectiveness against Ascochyta spp.     

Evaluation of non-chemical seed treatment methods for the control of Alternaria dauci and A. radicina on carrot seeds

The current study was initiated to evaluate the efficacy of physical methods (hot water, aerated steam, electron treatment) and agents of natural origin (resistance inducers, plant derived products, micro-organisms) as seed treatments of carrots for control of Alternaria dauci and A. radicina. Control of both Alternaria species by seed treatment with the resistance inducers was generally poor. Results were also not satisfactory with most of the formulated commercial micro-organism preparations. Based on the average of five field trials, one of these, BA 2552 (Pseudomonas chlororaphis), provided a low but significant increase in plant stand. Among the experimental micro-organisms, the best results were obtained with Pseudomonas sp. strain MF 416 and Clonostachys rosea strain IK726. A similar level of efficacy was provided by seed treatment with an emulsion (1%) of thyme oil in water. Good and consistent control was generally achieved with the physical methods aerated steam, hot water and electron treatment. Aerated steam treatment was, apart from the thiram-containing chemical standard, the best single treatment, and its performance may at least partially be due to extensive pre-testing, resulting in dosages optimally adapted to the respective seed lot. In some of the experiments the effect of the hot water treatment, which was tested at a fixed, not specifically adapted dosage, was significantly improved when combined with a Pseudomonas sp. MF 416 or C. rosea IK726 treatment. The results are discussed in relation to the outcome of experiments in which the same seed treatment methods and agents were tested in other seed-borne vegetable pathosystems.     

An octahedral coordination complex of iron(VI)

The hexavalent state, considered to be the highest oxidation level accessible for iron, has previously been found only in the tetrahedral ferrate dianion, FeO4(2-). We report the photochemical synthesis of another Fe(VI) compound, an octahedrally coordinated dication bearing a terminal nitrido ligand. M?ssbauer and x-ray absorption spectra, supported by density functional theory, are consistent with the octahedral structure having an FeN triple bond of 1.57 angstroms and a singlet d2(xy) ground electronic configuration. The compound is stable at 77 kelvin and yields a high-spin Fe(III) species upon warming.     

Analysis of diversity in populations of plant pathogens: the barley powdery mildew pathogen across Europe

Understanding population genetics and evolution within species requires recognition of variation within and between populations and the ability to distinguish between the potential causes of an observed distribution of variation. For this aim several established indices of diversity, and a novel one, were applied to population samples of the barley powdery mildew pathogen,Erysiphe graminis f. sp.hordei. Random spore samples were obtained from the air along transects through regions of interest across large parts of Europe in 1990. Significant geographical differences in diversity of virulence genotypes occurred among regional sub-samples. Diversity was highest in the samples from eastern Germany, Denmark and Austria, whereas the lowest values were found in the samples from Italy, southern France and parts of western Germany. Diversity in the pathogen population was generally related to the degree of diversification of host resistance in time and space, although there was considerable variation in ranking among different measures of diversity. Sensitivity to sample size proved to be the major problem with the use of several established indices of diversity. Working with very large sample sizes we used multiple random subsamples of various smaller sizes to determine how the mean index values changed with changing sample size. The Shannon index proved to be considerably affected by sample size, in contrast to the Simpson index that was therefore used as a global measure of diversity. Limits of confidence were estimated for the Simpson index using the bootstrap method of numerical resampling. The two aspects contributing to global diversity, richness and evenness, were considered separately to allow meaningful interpretation of the Simpson index. Random sub-sampling was used to reduce the influence of sample size differences for these measures. Dissimilarity, a novel measure of diversity for use in plant pathology, indicates the average number of major genes for host resistance against which pathotypes in a sample respond differently. It is thus able to account for the genetic relationship among pathotypes, which is not considered by any other index. The approaches developed in this study help to compare major forces driving evolution of large-scale populations of the barley mildew pathogen.     

Development and evaluation of an enzyme-linked immunosorbent assay (ELISA) for the detection of loose smut of barley (Ustilago nuda)

Polyclonal antibodies were raised against mycelium from the logarithmic growth phase of a shake culture of Ustilago nuda, and a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) with biotinylated detection antibodies was developed. The detection limit of the assay was 15 ng total protein ml^–1 for the homologous antigen and 50 ng ml^–1 for a spore extract, respectively. Other species of Ustilago reacted with the antibodies. Cross-reactivity was highest with U. tritici. No signal was obtained with the tested isolates of Tilletia, Rhizoctonia, Pythium and Fusarium. With naturally infected barley seeds, the results of the ELISAs were always in good agreement with those obtained with the routinely used seed embryo test. However, when seeds grown from artificially inoculated florets were used, the ELISA indicated significantly higher infestation levels than the embryo test. Results of assays with halved seeds from the same lot showed that high amounts of mycelium were present in the non-embryo half. This and especially the relatively long duration of the assay suggested that the ELISA (as conducted here) may not be suitable as a routine method for analysing seed infection with U. nuda. With samples from barley seedlings grown from infected seeds the results of the immunoassay again corresponded very well with the infection level determined by staining of the seed embryo, irrespective of the mode of floret inoculation (natural or artificial). Potential fields of application of the ELISA include the early prediction of the efficacy of protection agents, e.g. in screenings for seed treatments, the elucidation of the biology of the fungus and characterisation of resistance mechanisms.     

Effect of varied soil nitrogen supply on growth and nutrient uptake of young Norway spruce plants grown in a shaded environment

To understand the effect of increased soil N supply on tree growth and nutrient uptake, three-year-old Norway spruce seedlings were grown in pots on low-nutrient mineral forest soil supplemented with N in mineral or organic form. Outdoor shaded growth conditions were used, to test the hypothesis that shaded plants are particularly susceptible to high soil N supply. Plants were harvested eleven months after planting. Shoot growth was not affected by the N supply, but N concentrations in needles and roots were increased in plants supplied with mineral N (150 or 300 mg N [kg soil]^—1). Root growth was drastically reduced and root/shoot ratios were decreased in plants with higher N uptake. A high supply of mineral N to soil also decreased the concentrations of other essential elements (P, K) in the needles and thus had effects on plant growth which may impair the stress resistance of trees. Organic N in the form of keratin (150 mg N [kg soil]^—1) did not influence plant growth significantly. The adverse effects of high mineral N supply were particularly pronounced under shaded conditions in comparison to results from other experiments using higher light intensity and temperature conditions.     

Recent evolution of multiple resistance of Blumeria (Erysiphe) graminis f.sp. tritici to selected DMI and morpholine fungicides in France

Fungicides inhibiting sterol biosynthesis are frequently used for powdery mildew control and can be subdivided into sterol demethylation inhibitors (DMIs) and morpholines with different modes of action. Whereas fungicide resistance to DMIs (Rdmi) and morpholines (Rmor) has been continuously monitored, there are no data available on the combination of Rdmi and Rmor, which led us to ask whether multiple resistance to triadimenol (Rtria), representing DMIs and to fenpropimorph (Rfen), representing the morpholines, evolved in France from 1993 to 1996. The method used allowed testing of both chemicals simultaneously, with the same inoculum. In 1993, the resistance factor of the mean (RFM) of the French wheat mildew population was 9·59 for Rtria and 5·11 for Rfen. Resistance increased, leading to RFMs of close to 14 for Rtria and 8 for Rfen at the end of the study. From the analysis of single colony isolates (SCI) that are genetically uniform, the presence of multiple resistance and its increase were evident and in line with the results of bulk isolates. Covariance of resistance to both chemicals was close to one. In contrast to the increase of Rfen, the use of morpholines decreased. These effects are supposed to result from multiple selection due to the use of mixtures of DMIs and morpholines that have been favoured in recent years. Fungicide sensitivity is, in general, not normally but lognormally distributed in a population. A new way to evaluate and describe lognormal data is presented. It is easy and convenient to use and provides solutions for current problems in the literature with lognormal distributions. Multiple resistance, its evolution and persistance are discussed in relation to fungicide use and to implications for anti-resistance strategies. © 1998 Society of Chemical Industry     

Host matrix has major impact on the morphology of <Emphasis Type="Italic">Pseudoperonospora cubensis</Emphasis>

Oomycetes contain some of the economically most important pathogens of flowering plants. Most have a rather narrow host range, often being restricted to single host species. In downy mildews and other obligate biotrophic plant parasites, like powdery mildews and rusts, delimitating species on grounds of morphological characteristics is often hardly possible and thus often based on only subtle differences. This has led to the widespread application of a broad species concept for these organisms. Consequently, despite the fact that morphological differences were reported for Pseudoperonospora cubensis from different host species, the corresponding new pathogen species were not accepted as being independent, and the host range of Pseudoperonospora cubensis is reported to encompass more than 50 host species in the Cucurbitaceae in temperate to tropical climates. However, recent studies have reported narrow host ranges for other downy mildew genera and advocated a narrow species concept. Here, we report successful colonisation of five different tribes of the Cucurbitaceae by a strain of Pseudoperonospora cubensis and demonstrate that the host matrix has a major impact on the morphology of the pathogen. On the basis of five morphological criteria significant differences could be found for all hosts. These differences were more pronounced in phylogenetically unrelated than in related hosts. Our results provide evidence for a broad host range of Pseudoperonospora cubensis and demonstrate that species delimitation based on morphological characters is not feasible in Pseudoperonospora on Cucurbitaceae. Also in other biotrophic plant pathogens, the situation could be similar, thus necessitating thorough morphological, molecular phylogenetic and cross inoculation experiments for species recognition.     

Bremia lactucae populations on cultivated lettuce originate from prickly lettuce and are interconnected with the wild pathosystem

European Journal of Plant Pathology - Maize rough dwarf disease (MRDD) is one of the most important viral diseases around the world. Rice black-streaked dwarf virus (RBSDV) and maize rough dwarf...