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The glucuronoyl esterases (GEs) that have been identified so far belong to family 15 of the carbohydrate esterases in the CAZy classification system and are presumed to target ester bonds between lignin alcohols and (4-O-methyl-)d-glucuronic acid residues of xylan. Few GEs have been cloned, expressed and characterised to date. Characterisation has been done on a variety of synthetic substrates; however, the number of commercially available substrates is very limited. We identified novel putative GEs from a wide taxonomic range of fungi and expressed the enzymes originating from Acremonium alcalophilum and Wolfiporia cocos as well as the previously described PcGE1 from Phanerochaete chrysosporium. All three fungal GEs were active on the commercially available compounds benzyl glucuronic acid (BnGlcA), allyl glucuronic acid (allylGlcA) and to a lower degree on methyl glucuronic acid (MeGlcA). The enzymes showed pH stability over a wide pH range and tolerated 6-h incubations of up to 50 °C. Kinetic parameters were determined for BnGlcA. This study shows the suitability of the commercially available model compounds BnGlcA, MeGlcA and allylGlcA in GE activity screening and characterisation experiments. We enriched the spectrum of characterised GEs with two new members of a relatively young enzyme family. Due to its biotechnological significance, this family deserves to be more extensively studied. The presented enzymes are promising candidates as auxiliary enzymes to improve saccharification of plant biomass. Electronic supplementary material The online version of this article (doi:10.1007/s00253-017-8266-9) contains supplementary material, which is available to authorized users.

Microorganisms play important roles in dairy products. They can be a source of spoilage, or they promote health or cause diseases. In dairy fermentations, microorganisms are of great importance due to their function as starter cultures and during the production process of dairy products. In order to better understand and improve the process, it is essential to identify the species that are involved and to monitor the development of microbial communities. There are several different approaches for the detection and analysis of the microbiota. The methods can be culture dependent and, for example, make use of morphological and physiological characters or DNA sequencing. Culture-independent methods include direct PCR and qPCR, but also electrophoresis-based profiling techniques as well as metagenomics. Descriptions of relevant methods are provided and their applications are discussed in this chapter.

International audience; 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.

Phylogenetic analysis of glycoside hydrolase family 2 including Aspergillus sequences and characterised beta-mannosidases from other organisms, clusters putative Aspergillus beta-mannosidases in two distinct clades (A and B). Aspergillus species have at least one paralog in each of the two clades. It appears that clade A members are extracellular and clade B members intracellular. Substrate specificity analysis of MndA of Aspergillus niger (clade A) and MndB of Aspergillus nidulans (clade B) show that MndB, in contrast to MndA, does not hydrolyse polymeric mannan and has probably evolved to hydrolyse small unbranched beta-mannosides like mannobiose. A 3D-model of MndB provides further insight.

The Glucuronoyl esterases (GE) have been proposed to target lignin-carbohydrate (LC) ester bonds between lignin moieties and glucuronic acid side groups of xylan, but to date, no direct observations of enzymatic cleavage on native LC ester bonds have been demonstrated. In the present investigation, LCC fractions from spruce and birch were treated with a recombinantly produced GE originating from Acremonium alcalophilum (AaGE1). A combination of size exclusion chromatography and (31) P NMR analyses of phosphitylated LCC samples, before and after AaGE1 treatment provided the first evidence for cleavage of the LC ester linkages existing in wood.

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.

A culture-independent survey of fungal diversity in four arable soils and one grassland in Lower Austria was conducted by RFLP and sequence analysis of clone libraries of the partial ITS/LSU-region. All soils were dominated by the ascomycetous orders Sordariales, Hypocreales and Helotiales, taxa that are known from traditional cultivation approaches to occur in agricultural soils. The most abundant genus in the investigated soils was Tetracladium, a hyphomycete which has been described as occurring predominantly in aquatic habitats, but was also found in agricultural soils. Additionally, soil clone group I (SCGI), a subphylum at the base of the Ascomycota with so far no cultivated members, was identified at high frequency in the grassland soil but was below detection limit in the four arable fields. In addition to this striking difference, general fungal community parameters like richness, diversity and evenness were similar between cropland and grassland soils. The presented data provide a fungal community inventory of agricultural soils and reveal the most prominent species.

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 ho- mogenized 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 composi- tions and soil properties, except gross N transformation rates, remained stable and reproducible during the test period in all soils. 15 N tracer studies showed that N uptake patterns of barley as well as plant growth were linear in the initial growth period. Overall, the presented microcosm system proved to be a powerful tool to elucidate N pathways in soil-plant-microbe systems. In future studies the microcosm system may greatly help generating new insights in the complex

Hygromycin B resistance was transferred to the sterile mycelia of Cadophora finlandia and Phialocephala fortinii by co-cultivation with Agrobacterium tumefaciens. Constitutively expressed green fluorescent protein (GFP) was also introduced using the same vector. Confocal laser scanning microscopy (CLSM) revealed strong fluorescence of transformants. Both traits were mitotically stable during one year of subculturing on non-selective growth medium. Southern blot analysis showed that the majority of the transformants contained single-copy integrations at random sites in the genome.

Species ofPyricularia(magnaporthe-like sexual morphs) are responsible for major diseases on grasses.Pyricularia oryzae(sexual morphMagnaporthe oryzae) is responsible for the major disease of rice called rice blast disease, and foliar diseases of wheat and millet, whilePyricularia grisea(sexual morphMagnaporthe grisea) is responsible for foliar diseases ofDigitaria.Magnaporthe salvinii, M. poaeandM. rhizophilaproduce asexual spores that differ from those ofPyricularia sensu strictothat has pyriform, 2-septate conidia produced on conidiophores with sympodial proliferation.Magnaporthe salviniiwas recently allocated toNakataea, whileM. poaeandM. rhizophilawere placed inMagnaporthiopsis. 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 ofPyricularia s. str.belong to a monophyletic clade that includes allP. oryzae/P. griseaisolates tested, defining thePyriculariaceae,which is sister to theOphioceraceae, representing two novel families. These clades are clearly distinct from species belonging to theGaeumannomyces pro parte/Magnaporthiopsis/Nakataeageneric complex that are monophyletic and define theMagnaporthaceae. A few magnaporthe- and pyricularia-like species are unrelated toMagnaporthaceaeandPyriculariaceae. Pyricularia oryzae/P. griseaisolates cluster into two related clades. Host plants such asEleusine, Oryza,SetariaorTriticumwere exclusively infected by isolates fromP. oryzae, while some host plant such asCenchrus, Echinochloa, Lolium, PennisetumorZingiberwere infected by differentPyriculariaspecies. 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 ofP. grisea/P. oryzaeis restricted toPyriculariaandNeopyricularia, 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 withinPyriculariaceaeis presented, and novelties are proposed based on morphological and phylogenetic data.

International audience; A gene (MoPRD1), related to xylose reductases, was identified in Magnaporthe oryzae. Recombinant MoPRD1 displays its highest specific reductase activity toward L-arabinose and D-xylose. K-m and V-max values using L-arabinose and D-xylose are similar. MoPRD1 was highly overexpressed 2-8 h after transfer of mycelium to D-xylose or L-arabinose, compared to D-glucose. Therefore, we conclude that MoPDR1 is a novel pentose reductase, which combines the activities and expression patterns of fungal L-arabinose and D-xylose reductases. Phylogenetic analysis shows that PRD1 defines a novel family of pentose reductases related to fungal D-xylose reductases, but distinct from fungal L-arabinose reductases. The presence of PRD1, L-arabinose and D-xylose reductases encoding genes in a given species is variable and likely related to their life style. (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Wenn in Innenraumen ein mikrobieller Befall vermutet wird oder wenn schon wahrnehmbare Hinweise auf diesen bestehen (beispielsweise makroskopisch sichtbarer Befall an Wanden oder schimmeltypische Geruche), sollte in jedem Fall eine Ortsbegehung stattfinden, um die Sachlage naher abzuklaren.

We sequenced and compared the genomes of the Dothideomycete fungal plant pathogens Cladosporium fulvum (Cfu) (syn. Passalora fulva) and Dothistroma septosporum (Dse) that are closely related phylogenetically, but have different lifestyles and hosts. Although both fungi grow extracellularly in close contact with host mesophyll cells, Cfu is a biotroph infecting tomato, while Dse is a hemibiotroph infecting pine. The genomes of these fungi have a similar set of genes (70% of gene content in both genomes are homologs), but differ significantly in size (Cfu >61.1-Mb; Dse 31.2-Mb), which is mainly due to the difference in repeat content (47.2% in Cfu versus 3.2% in Dse). Recent adaptation to different lifestyles and hosts is suggested by diverged sets of genes. Cfu contains an α-tomatinase gene that we predict might be required for detoxification of tomatine, while this gene is absent in Dse. Many genes encoding secreted proteins are unique to each species and the repeat-rich areas in Cfu are enriched for these species-specific genes. In contrast, conserved genes suggest common host ancestry. Homologs of Cfu effector genes, including Ecp2 and Avr4, are present in Dse and induce a Cf-Ecp2- and Cf-4-mediated hypersensitive response, respectively. Strikingly, genes involved in production of the toxin dothistromin, a likely virulence factor for Dse, are conserved in Cfu, but their expression differs markedly with essentially no expression by Cfu in planta. Likewise, Cfu has a carbohydrate-degrading enzyme catalog that is more similar to that of necrotrophs or hemibiotrophs and a larger pectinolytic gene arsenal than Dse, but many of these genes are not expressed in planta or are pseudogenized. Overall, comparison of their genomes suggests that these closely related plant pathogens had a common ancestral host but since adapted to different hosts and lifestyles by a combination of differentiated gene content, pseudogenization, and gene regulation., Author Summary We compared the genomes of two closely related pathogens with very different lifestyles and hosts: C. fulvum (Cfu), a biotroph of tomato, and D. septosporum (Dse), a hemibiotroph of pine. Some differences in gene content were identified that can be directly related to their different hosts, such as the presence of a gene involved in degradation of a tomato saponin only in Cfu. However, in general the two species share a surprisingly large proportion of genes. Dse has functional homologs of Cfu effector genes, while Cfu has genes for biosynthesis of dothistromin, a toxin probably associated with virulence in Dse. Cfu also has an unexpectedly large content of genes for biosynthesis of other secondary metabolites and degradation of plant cell walls compared to Dse, contrasting with its host preference and lifestyle. However, many of these genes were not expressed in planta or were pseudogenized. These results suggest that evolving species may retain genetic signatures of the host and lifestyle preferences of their ancestor and that evolution of new genes, gene regulation, and pseudogenization are important factors in adaptation.