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BackgroundThe toxigenic fungal plant pathogen Fusarium graminearum compromises wheat production worldwide. Azole fungicides play a prominent role in controlling this pathogen. Sequencing of its genome stimulated the development of high-throughput technologies to study mechanisms of coping with fungicide stress and adaptation to fungicides at a previously unprecedented precision. DNA-microarrays have been used to analyze genome-wide gene expression patterns and uncovered complex transcriptional responses. A recently developed one-color multiplex array format allowed flexible, effective, and parallel examinations of eight RNA samples.ResultsWe took advantage of the 8 × 15 k Agilent format to design, evaluate, and apply a novel microarray covering the whole F. graminearum genome to analyze transcriptional responses to azole fungicide treatment. Comparative statistical analysis of expression profiles uncovered 1058 genes that were significantly differentially expressed after azole-treatment. Quantitative RT-PCR analysis for 31 selected genes indicated high conformity to results from the microarray hybridization. Among the 596 genes with significantly increased transcript levels, analyses using GeneOntology and FunCat annotations detected the ergosterol-biosynthesis pathway genes as the category most significantly responding, confirming the mode-of-action of azole fungicides. Cyp51A, which is one of the three F. graminearum paralogs of Cyp51 encoding the target of azoles, was the most consistently differentially expressed gene of the entire study. A molecular phylogeny analyzing the relationships of the three CYP51 proteins in the context of 38 fungal genomes belonging to the Pezizomycotina indicated that CYP51C (FGSG_11024) groups with a new clade of CYP51 proteins. The transcriptional profiles for genes encoding ABC transporters and transcription factors suggested several involved in mechanisms alleviating the impact of the fungicide. Comparative analyses with published microarray experiments obtained from two different nutritional stress conditions identified subsets of genes responding to different types of stress. Some of the genes that responded only to tebuconazole treatment appeared to be unique to the F. graminearum genome.ConclusionsThe novel F. graminearum 8 × 15 k microarray is a reliable and efficient high-throughput tool for genome-wide expression profiling experiments in fungicide research, and beyond, as shown by our data obtained for azole responses. The array data contribute to understanding mechanisms of fungicide resistance and allow identifying fungicide targets.

Fungal endophytes mainly belong to the phylum Ascomycota and colonize plants without producing symptoms. We report on the isolation of seed-borne fungal endophytes from Phragmites australis (common reed) that were ascribed to the genus Stagonospora. Nested polymerase chain reaction (PCR) assays revealed that a Stagonospora sp. regularly colonized reed as shown for a period of three years. In spring, it was only detected in roots, whereas in autumn, it could frequently be found in all organs, including seeds. Microcosm experiments revealed that seeds harbored viable propagules of the fungus that colonized the developing germling, indicating vertical transmission. Endophytic growth was confirmed by immunofluorescence microscopy, reisolation of the fungus after surface sterilization, and PCR. Aseptic microcosms were established for studying fungal contributions towards host vitality. Several Stagonospora isolates enhanced reed biomass. Seed-borne endophytic Stagonospora spp. thus can provide improved vigor to common reed, which could be most important when seed-derived germlings establish new reed stands.Additional keywords: ITS phylogeny, Phaeosphaeria.Fungi living in close association with plants can have effects on their host that range from detrimental to beneficial, depending on the partners involved and additional biotic and abiotic factors. Mycorrhiza is a well-documented type of a symbiotic interaction that is found at roots of the majority of higher plants. It is well known that mycorrhizal fungi are important for retrieving nutrients from soil and allocating them to their hosts (Hodge et al. 2001;Landeweert et al. 2001;Sharma et al. 1997). Endophytes represent another type of a putative symbiotic interaction between fungi and plants. Endophytes live within plant tissue without producing overt symptoms. Endophytic fungi have been recovered from all vegetative organs and from a broad range of plants growing from tropical to alpine habitats, indicating that these associations are widespread in nature (Carroll 1988;Petrini et al. 1992). Endophytes represent a wide taxonomical range of fungi, mostly within the phylum Ascomycota. Caroll (1988) distinguished two types of endophytic mutualism. Constitutive endophytic mutualism is typified by a systemic infection of grasses in genera Festuca, Lolium, and a few others in the family Poaceae with fungi of the anamorph genus Neotyphodium and the corresponding teleomorph genus Epichloë (family Clavicipitaceae, phylum Ascomycota) (Schardl 2001). These fungi proliferate in infected plants and are often vertically transmitted by seeds to the next generation of the host. It has been shown that these fungi improve the vitality of their hosts by increasing resistance against grazing, insects, drought, and microbial parasites (Clay 1992;Leuchtmann 1992;Schardl 2001). Furthermore, they improve biomass production and nutrient status (Arachevaleta et al. 1989;Groppe et al. 1999;Latch et al. 1985;Malinowski et al. 2000). On the other hand, Caroll (1988) characterized inducible en...

A molecular approach was applied to investigate the colonisation of arbuscular mycorrhizal fungi (AMF) on the wetland grass Phragmites australis. A PCR assay targeting the traditional families of the Glomeromycota yielded products that were used to construct libraries of 18S rDNA. Five hundred and forty six clones were typed by restriction analysis and 76 representatives were sequenced. The majority corresponded to a wide range of taxa within Glomus group A, a few belonged to the "Diversisporaceae" and none to the genera Scutellospora or Acaulospora. Among these sequences, some were very similar to those reported earlier, e.g. Glomus mosseae and G. fasciculatum, other pointed to various new taxa. Although this wetland habitat harboured just one single plant species, phylogenetic analysis exhibited 21 AMF phylotypes, which is in the same range as reported for other natural ecosystems composed of more diverse host communities. Diversity indices supported the perception that the AMF mycoflora associated with this natural grass "monoculture" is not depauperate as it had been described for grasses of crop monocultures. Soil conditions determined the mycorrhizal state of the host, since AMF were not detected at the lakeward front of the reed belt, which is permanently waterlogged.

The endophyte Piriformospora indica colonizes roots of a range of host plants and increases biomass production and resistance to fungal pathogens and, thus has been considered a biocontrol fungus. However, the field performance of this fungus has not yet been tested in temperate climates. Therefore, we evaluated the performance of this fungus in different substrata under greenhouse and practical field conditions. Roots of winter wheat were colonized efficiently, and biomass was particularly increased on poor substrata. In greenhouse experiments, symptom severity of a typical leaf (Blumeria graminis f. sp. tritici), stem base (Pseudocercosporella herpotrichoides), and root (Fusarium culmorum) pathogen was reduced significantly. However, in field experiments, symptoms caused by the leaf pathogen did not differ in Piriformospora indica-colonized compared with control plants. In the field, Pseudocercosporella herpotrichoides disease severity was significantly reduced in plants colonized by the endophyte. Increased numbers of sheath layers and hydrogen peroxide concentrations after B. graminis attack were detected in Piriformospora indica-colonized plants, suggesting that root colonization causes induction of systemic resistance or priming of the host plant. Although the endophyte is not well suited for growth at Central European temperature conditions, it remains to be shown whether P. indica is more suitable for tropical or subtropical farming.