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Peronospora salviae‐officinalis, the causal agent of downy mildew on common sage, is an obligate biotrophic pathogen. It grows in the intercellular spaces of the leaf tissue of sage and forms intracellular haustoria to interface with host cells. Although P. salviae‐officinalis was described as a species of its own 10 years ago, the infection process remains obscure. To address this, a histological study of various infection events, from the adhesion of conidia on the leaf surface to de novo sporulation is presented here. As histological studies of oomycetes are challenging due to the lack of chitin in their cell wall, we also present an improved method for staining downy mildews for confocal laser scanning microscopy as well as evaluating the potential of autofluorescence of fixed nonstained samples. For staining, a 1:1 mixture of aniline blue and trypan blue was found most suitable and was used for staining of oomycete and plant structures, allowing discrimination between them as well as the visualization of plant immune responses. The method was also used to examine samples of Peronospora lamii on Lamium purpureum and Peronospora belbahrii on Ocimum basilicum, demonstrating the potential of the presented histological method for studying the infection processes of downy mildews in general.

Cell-cell fusion in fungi is required for colony formation, nutrient transfer and signal transduction. Disruption of genes required for hyphal fusion in Epichloe festucae, a mutualistic symbiont of Lolium grasses, severely disrupts the host interaction phenotype. They examined whether symB and symC, the E. festucae homologs of Podospora anserina self-signaling genes IDC2 and IDC3, are required for E. festucae hyphal fusion and host symbiosis. Deletion mutants of these genes were defective in hyphal cell fusion, formed intra-hyphal hyphae, and had enhanced conidiation. SymB-GFP and SymC-mRFP1 localize to plasma membrane, septa and points of hyphal cell fusion. Plants infected with ΔsymB and ΔsymC strains were severely stunted. Hyphae of the mutants colonized vascular bundles, were more abundant than wild type in the intercellular spaces and formed intra-hyphal hyphae. Although these phenotypes are identical to those previously observed for cell wall integrity MAP kinase mutants no difference was observed in the basal level of MpkA phosphorylation or its cellular localization in the mutant backgrounds. Both genes contain binding sites for the transcription factor ProA. Collectively these results show that SymB and SymC are key components of a conserved signaling network for E. festucae to maintain a mutualistic symbiotic interaction within L. perenne.

Summary Epichloë festucae forms a mutualistic symbiotic association with Lolium perenne. This biotrophic fungus systemically colonizes the intercellular spaces of aerial tissues to form an endophytic hyphal network. E. festucae also grows as an epiphyte, but the mechanism for leaf surface colonization is not known. Here we identify an appressorium‐like structure, which we call an expressorium that allows endophytic hyphae to penetrate the cuticle from the inside of the leaf to establish an epiphytic hyphal net on the surface of the leaf.We used a combination of scanning electron, transmission electron and confocal laser scanning microscopy to characterize this novel fungal structure and determine the composition of the hyphal cell wall using aniline blue and wheat germ agglutinin labelled with Alexafluor‐488.Expressoria differentiate immediately below the cuticle in the leaf blade and leaf sheath intercalary cell division zones where the hyphae grow by tip growth. Differentiation of this structure requires components of both the NoxA and NoxB NADPH oxidase complexes. Major remodelling of the hyphal cell wall occurs following exit from the leaf.These results establish that the symbiotic association of E. festucae with L. perenne involves an interconnected hyphal network of both endophytic and epiphytic hyphae., See also the Commentary on this article by Genre & Lanfranco, 211: 5–7.

Polymer-modified asphalts (PMAs) were prepared using Bachaquero asphalt and styrene–butadiene–styrene (SBS) type copolymers. Their rheological behavior was compared to that of unmodified asphalt and of a compatible commercial PMA. Materials were submitted to frequency sweeps between 10−1 and 102 rad/s from 0 to 50°C. Storage stability tests were performed for 72 h at 160°C. Ring and ball softening points from the top and the bottom of the blends were compared and were used along with fluorescence microscopy to evaluate stability. Samples prepared with styrene–ethylene–butylene–styrene (SEBS) showed improved compatibility and stability as compared with SBS-modified asphalts, probably because of a higher stability to thermal degradation from the absence of double bonds. An additional improvement in stability and compatibility was observed for SEBS functionalized with maleic anhydride (SEBS-g-MAH)–modified blends. Better compatibility, however, did not improve rheological behavior at low temperatures. The systems studied are so complex from a chemical point of view that the rheological criteria normally used to predict compatibility of polyblends did not give enough information regarding the compatibility of the modified asphalts. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 1772–1782, 2003