An effective colonization of the host plant tissue by the necrotrophic fungus Sclerotinia sclerotiorum requires the secretion of the non-host specific toxin oxalic acid (OA), which is known to suppress the generation of reactive oxygen intermediates (ROI). A full-length cDNA coding for an oxalate decarboxylase (TOXDC), which converts OA into CO 2 and formate, was isolated from the basidiomycete Trametes versicolor. It was overexpressed in tobacco plants to study the role of ROI and OA in the interaction between tobacco and S. sclerotiorum. The transgenic plants contained less OA and showed a delayed colonization of S. sclerotiorum; furthermore a strong ROI accumulation and nearly no catalase activity compared to the wild type (WT) plants could be detected. In addition, inoculation experiments with transgenic catalase-deficient plants (CAT1AS) and in vitro studies showed that S. sclerotiorum copes with strong ROI stress. Our results indicate that OA supports the infection process caused by S. sclerotiorum and the fungus itself is able to tolerate high ROI concentrations.

The potential of surface active proteins to affect gushing upon their formation in situ during fermentation was investigated. This was achieved by cloning the genes of two hydrophobins of F. culmorum and of a wheat lipid transfer protein (LTP1500) in Saccharomyces cerevisiae, with expression of these genes under control of the constitutive TPI (triose phosphate isomerase) promoter. The transgenic yeast clones were used for fermentation of wort. The resulting beers were bottled and examined for the occurrence of gushing. Gushing was induced by the class II hydrophobin FcHyd5p of the fungus F. culmorum, found in naturally occurring cases of gushing worldwide.

Aims: The objective of this study was to evaluate the capability of Fusarium culmorum to produce non-hydrophobin surface-active proteins in vitro, to isolate and characterize such proteins from liquid cultures, to analyse their effect on overfoaming (gushing) of beer and to elucidate their prevalence in pure cultures and infected malt. Methods and Results: A 20 kDa protein was isolated from liquid cultures of F. culmorum BBA 62182 upon enrichment by foaming. BLAST search with Nterminal and internal sequences of the protein revealed high homology with a hypothetical protein predicted within the F. graminearum PH1 genome sequence. Oligonucleotide primers designed to bind 30 nt upstream and downstream of the predicted gene were used to amplify a 695 nt PCR fragment from genomic DNA of F. culmorum BBA 62182. Cloning and sequencing of the product revealed a 635 nt open reading frame which had 98% homology to the predicted F. graminearium PH1 gene code. Removal of a 59 nt intron and translation resulted in a 191 amino acid protein of 20AE754 kDa with a calculated pI of 9AE1. Amino acids obtained by Edman sequencing of fragments within the 20 kDa protein were 100% homologous with the sequence deduced from the DNA sequence. According to its properties, the new protein was termed alkaline foam protein A (AfpA). Sequence comparison revealed some homologies with proteins in Emericella nidulans, which are involved in phialide development and response to antifungal agents. Homologies with other hypothetical fungal proteins suggest a new group of proteins, for which we suggest the name fungispumins. Addition of AfpA to beer showed that overfoaming (gushing) is not induced in stable beer but can significantly enhance this effect in beer showing moderate gushing.

Deoxynivalenol (DON) transformation products from selected time course experiments were analyzed by thin-layer chromatography. With the strainAlternaria alternata f. sp.lycopersici AS27-3, one major metabolite of DON in ethyl acetate was observed. This unidentified metabolite was more polar than DON and has a Rf value of 0.71. Derivatization indicated that this metabolite was probably an unidentified trichothecene. Screening of 29 other microbial isolates (bacteria, yeast, filamentous fungi) for DON transformation did not result in any active organism.

Double-stranded RNAs (dsRNAs) have been found in two isolates of the plant pathogenic fungus Fusarium graminearum which produce trichothecene mycotoxins. The isolates 8.2 and 19.2 had dsRNAs in the size of about 2.0 kb and 6.0 kb, respectively, which were associated with capsid proteins and persisted within the cytoplasm of the infected host cells as encapsidated virus-like particles (VLPs). The dsRNAs contained in the VLP pellets were the same size as the dsRNA isolated in total nucleic acid preparations. In the VLP pellets the isolate 19.2 had a second dsRNA with the size of about 1.6 kb. After mycovirus purification one icosahedral particle of about 28 nm in diameter from the isolate 8.2 and two icosahedral particles of about 28 nm and 38 to 40 nm in diameter from the isolate 19.2 could be identified with electron microscopy. SDS-PAGE analysis of the VLPs from the isolate 8.2 revealed one major protein component of approximately 65 kDa, while the isolate 19.2 had two major protein bands at about 94 kDa and 105 kDa. Both isolates were studied for potential trichothecene production. Tox5 PCR showed a 658 bp fragment in each isolate. In addition, both strains were able to produce the trichothecenes deoxynivalenol (DON), the derivatives acetyl-DON (3-A-DON, 15-A-DON) and nivalenol (NIV) in vitro.