Community profiling and gene expression of fungal assimilatory nitrate reductases in agricultural soil

Although fungi contribute significantly to the microbial biomass in terrestrial ecosystems, little is known about their contribution to biogeochemical nitrogen cycles. Agricultural soils usually contain comparably high amounts of inorganic nitrogen, mainly in the form of nitrate. Many studies focused on bacterial and archaeal turnover of nitrate by nitrification, denitrification and assimilation, whereas the fungal role remained largely neglected. To enable research on the fungal contribution to the biogeochemical nitrogen cycle tools for monitoring the presence and expression of fungal assimilatory nitrate reductase genes were developed. To the ∼100 currently available fungal full-length gene sequences, another 109 partial sequences were added by amplification from individual culture isolates, representing all major orders occurring in agricultural soils. The extended database led to the discovery of new horizontal gene transfer events within the fungal kingdom. The newly developed PCR primers were used to study gene pools and gene expression of fungal nitrate reductases in agricultural soils. The availability of the extended database allowed affiliation of many sequences to known species, genera or families. Energy supply by a carbon source seems to be the major regulator of nitrate reductase gene expression for fungi in agricultural soils, which is in good agreement with the high energy demand of complete reduction of nitrate to ammonium

Regulation of plant polysaccharide utilisation in Magnaporthe oryzae and other ascomycetous fungi

Magnaporthe oryzae is a fungal plant pathogen of many grasses including rice. Since arabinoxylan is one of the major components of the plant cell wall of grasses, M. oryzae is likely to degrade this polysaccharide for supporting its growth in infected leaves. D-xylose is released from arabinoxylan by fungal xylanolytic enzymes and catabolised through the pentose pathway. This PhD thesis describes the characterisation of the pathway for pentose utilisation in M. oryzae and the discovery of a novel pentose reductase. In fungi, the expression of genes involved in xylan degradation and D-xylose utilisation is under control of the transcriptional activator XlnR. A detailed characterisation of the homolog of XlnR in M. oryzae, Xlr1, was performed in the course of this thesis. Investigation of an XLR1 disruption strain demonstrated that XLR1 encodes the transcriptional activator of the pentose catabolic pathway in M. oryzae. However, the xylanolytic system of this fungus was induced even in the absence of Xlr1. A detailed transcriptome analysis demonstrated that a number of genes involved in xylan degradation were strongly dependent on Xlr1 while a second set of xylanolytic genes appeared to be under control of another yet unknown regulatory system. XlnR homologs are commonly found in filamentous ascomycetes and often assumed to have the same function in different fungi. A comparison of five filamentous fungi showed significant differences in the proteome secreted by these fungi in response to xylan as well as regarding the set of genes that is controlled by XlnR in the individual species. This comparison emphasizes the functional diversity of a fine-tuned (hemi-) cellulolytic regulatory system in filamentous fungi, which might be related to the adaptation of fungi to their specific biotopes. This PhD thesis deepens our knowledge about regulation of plant polysaccharide degradation and utilisation in filamentous fungi with a special focus on the rice blast fungus M. oryzae. The family, to which M. oryzae belongs to (Magnaporthaceae), also includes other important plant pathogens (e.g. rice stem rot fungus, summer patch). The taxonomic relationships of Magnaporthe and Pyricularia species were investigated which led to the identification of novel species, which are also described in this PhD thesis

Xlr1 is involved in the transcriptional control of the pentose catabolic pathway, but not hemi-cellulolytic enzymes in Magnaporthe oryzae

Magnaporthe oryzae is a fungal plant pathogen of many grasses including rice. Since arabinoxylan is one of the major components of the plant cell wall of grasses, M. oryzae is likely to degrade this polysaccharide for supporting its growth in infected leaves. D-Xylose is released from arabinoxylan by fungal depolymerising enzymes and catabolized through the pentose pathway. The expression of genes involved in these pathways is under control of the transcriptional activator XlnR/Xlr1, conserved among filamentous ascomycetes. In this study, we identified M. oryzae genes involved in the pentose catabolic pathway (PCP) and their function during infection, including the XlnR homolog, XLR1, through the phenotypic analysis of targeted null mutants. Growth of the Delta xlr1 strain was reduced on D-xylose and xylan, but unaffected on L-arabinose and arabinan. A strong reduction of PCP gene expression was observed in the Delta xlr1 strain on D-xylose and L-arabinose. However, there was no significant difference in xylanolytic and cellulolytic enzyme activities between the Delta xlr1 mutant and the reference strain. These data demonstrate that XLR1 encodes the transcriptional activator of the PCP in M. oryzae, but does not appear to play a role in the regulation of the (hemi-) cellulolytic system in this fungus. This indicates only partial similarity in function between Xlr1 and A. niger XlnR. The deletion mutant of D-xylulose kinase encoding gene (XKI1) is clearly unable to grow on either D-xylose or L-arabinose and showed reduced growth on xylitol, L-arabitol and xylan. Delta xki1 displayed an interesting molecular phenotype as it over-expressed other PCP genes as well as genes encoding (hemi-) cellulolytic enzymes. However, neither Delta xlr1 nor Delta xki1 showed significant differences in their pathogeny on rice and barley compared to the wild type, suggesting that p-xylose catabolism is not required for fungal growth in infected leaves. (C) 2013 Elsevier Inc. All rights reserved

Resolving the polyphyletic nature of Pyricularia (Pyriculariaceae)

BGPI : équipe 5International audienceSpecies of Pyricularia (magnaporthe-like sexual morphs) are responsible for major diseases on grasses. Pyricularia oryzae (sexual morph Magnaporthe oryzae) is responsible for the major disease of rice called rice blast disease, and foliar diseases of wheat and millet, while Pyricularia grisea (sexual morph Magnaporthe grisea) is responsible for foliar diseases of Digitaria. Magnaporthe salvinii, M. poae and M. rhizophila produce asexual spores that differ from those of Pyricularia sensu stricto that has pyriform, 2-septate conidia produced on conidiophores with sympodial proliferation. Magnaporthe salvinii was recently allocated to Nakataea, while M. poae and M. rhizophila were placed in Magnaporthiopsis. To clarify the taxonomic relationships among species that are magnaporthe- or pyricularia-like in morphology, we analysed phylogenetic relationships among isolates representing a wide range of host plants by using partial DNA sequences of multiple genes such as LSU, ITS, RPB1, actin and calmodulin. Species of Pyricularia s. str. belong to a monophyletic clade that includes all P. oryzae/P. grisea isolates tested, defining the Pyriculariaceae, which is sister to the Ophioceraceae, representing two novel families. These clades are clearly distinct from species belonging to the Gaeumannomyces pro parte/Magnaporthiopsis/Nakataea generic complex that are monophyletic and define the Magnaporthaceae. A few magnaporthe- and pyricularia-like species are unrelated to Magnaporthaceae and Pyriculariaceae. Pyricularia oryzae/P. grisea isolates cluster into two related clades. Host plants such as Eleusine, Oryza, Setaria or Triticum were exclusively infected by isolates from P. oryzae, while some host plant such as Cenchrus, Echinochloa, Lolium, Pennisetum or Zingiber were infected by different Pyricularia species. This demonstrates that host range cannot be used as taxonomic criterion without extensive pathotyping. Our results also show that the typical pyriform, 2-septate conidium morphology of P. grisea/P. oryzae is restricted to Pyricularia and Neopyricularia, while most other genera have obclavate to more ellipsoid 2-septate conidia. Some related genera (Deightoniella, Macgarvieomyces) have evolved 1-septate conidia. Therefore, conidium morphology cannot be used as taxonomic criterion at generic level without phylogenetic data. We also identified 10 novel genera, and seven novel species. A re-evaluation of generic and species concepts within Pyriculariaceae is presented, and novelties are proposed based on morphological and phylogenetic data

A cost-effective high-throughput microcosm system for studying nitrogen dynamics at the plant-microbe-soil interface. Plant Soil 317

Abstract In the present study a new microcosm system was evaluated for its suitability to investigate nitrogen dynamics between soils, plants and microbes. Five different agricultural soils were homogenized and transferred in the test tubes, and kept under controlled conditions in a climate chamber for 4weeks. Soils differed clearly in nitrogen pools and microbial population structures but less in their activities. Bacterial and fungal community compositions and soil properties, except gross N transformation rates, remained stable and reproducible during the test period in all soils