László Hornok

Mating type sequences in asexually reproducing Fusarium species.

ABSTRACT To assess the potential for mating in several Fusarium species with no known sexual stage, we developed degenerate and semidegenerate oligonucleotide primers to identify conserved mating type (MAT) sequences in these fungi. The putative α and high-mobility-group (HMG) box sequences from Fusarium avenaceum, F. culmorum, F. poae, and F. semitectum were compared to similar sequences that were described previously for other members of the genus. The DNA sequences of the regions flanking the amplified MAT regions were obtained by inverse PCR. These data were used to develop diagnostic primers suitable for the clear amplification of conserved mating type sequences from any member of the genus Fusarium. By using these diagnostic primers, we identified mating types of 122 strains belonging to 22 species of Fusarium. The α box and the HMG box from the mating type genes are transcribed in F. avenaceum, F. culmorum, F. poae, and F. semitectum. The novelty of the PCR-based mating type identification system that we developed is that this method can be used on a wide range of Fusarium species, which have proven or expected teleomorphs in different ascomycetous genera, including Calonectria, Gibberella, and Nectria.

Relationship Between the Fungal Complex Causing Fusarium Head Blight of Wheat and Environmental Conditions

ABSTRACT Over 4 years, the environmental conditions and the causal agents of Fusarium head blight (FHB) disease of wheat were determined in field sites in four European countries: Hungary, Ireland, Italy, and the United Kingdom. Polymerase chain reaction-based methods were used to detect each species causing FHB and quantify its DNA (as a measurement of fungal abundance) in the samples. Canonical correspondence analysis (CCA) was used to determine the relationship of the incidence and abundance of each species with weather variables. CCA indicated that little variability in the species prevalence data was explained by the weather variables. In contrast, a greater proportion of variability in abundance data was accounted for by the weather variables. Most samples contained two or more species and statistical analysis suggested that these species tended to coexist at field sites. CCA also indicated that there were differences in the relationships of the prevalence and abundance of the six FHB species with environmental variables. Fusarium poae was associated with relatively drier and warmer conditions, whereas F. graminearum was associated with warmer/humid conditions. F. avenaceum and F. culmorum were both associated with niches of cooler/wet/humid conditions. Two Microdochium species were associated with regions of relatively cool/moderate temperatures and frequent rainfalls of short duration. The results also suggested that environmental conditions differentially affect the infection and colonization processes, and the comparative abundance of the six species.

N-starvation stress induced FUM gene expression and fumonisin production is mediated via the HOG-type MAPK pathway in Fusarium proliferatum

During cultivation of a wild type strain of Fusarium proliferatum on ammonium dihydrogen phosphate containing defined medium, expression levels of FUM1 and FUM8, members of the fumonisin biosynthesis gene cluster significantly increased when ammonium ion concentration of the culture medium decreased below 10 mM, indicating that N-depletion triggers the fumonisin biosynthesis genes. Deletion of Fphog1, a HOG-type MAP kinase gene resulted in further increases in FUM1 and FUM8 expression under nitrogen starvation (absence of any N-source) conditions. Fumonisin B1 (FB1) production paralleled with increased FUM gene expression: significant amounts of FB1 were measured in culture filtrates of the DeltaFphog1 deleted mutant after five days culturing, whereas only traces of FB1 could be detected in filtrates of the wild type and the restored strain (R1) complemented with the wild-type Fphog1-24 gene. N-starvation strongly retarded the growth of the DeltaFphog1 mutant in comparison to wild type. The up-regulation of fumonisin biosynthesis genes in the DeltaFphog1 mutant could be explained by the increased sensitivity of these strains to N-starvation stress that appears in the absence of an intact HOG-type MAPK pathway.

Expression of cmg1, an Exo-β-1,3-Glucanase Gene from Coniothyrium minitans, Increases during Sclerotial Parasitism

ABSTRACT During sclerotial infection of Sclerotinia sclerotiorumthe mycoparasite Coniothyrium minitans penetrates through the host cell wall, which contains β-1,3-glucan as its major component. A PCR-based strategy was used to clone a β-1,3-glucanase-encoding gene, designated cmg1, from a cDNA library of the fungus. The nucleotide and deduced amino acid sequences of this gene showed high levels of similarity to the sequences of other fungal exo-β-1,3-glucanase genes. The calculated molecular mass of the deduced protein (without the predicted 24-amino-acid N-terminal secretion signal peptide) was 83,346 Da, and the estimated pI was 4.73. Saccharomyces cerevisiaeINVSc1 expressing the cmg1 gene secreted a ∼100-kDa β-1,3-glucanase enzyme (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) into the culture medium. N-terminal sequence analysis of the purified recombinant enzyme revealed that the secreted enzyme starts at Ala-32, seven amino acids downstream from the predicted signal peptidase cleavage site. The purified recombinant glucanase inhibited in vitro mycelial growth ofS. sclerotiorum by 35 and 85% at concentrations of 300 and 600 μg ml−1, respectively. A single copy of thecmg1 gene is present in the genome of C. minitans. Northern analyses indicated increases in the transcript levels of cmg1 due to both carbon starvation and the presence of ground sclerotia of S. sclerotiorum; only slight repression was observed in the presence of 2% glucose. Expression of cmg1 increased during parasitic interaction with S. sclerotiorum.

Within-field variability of Fusarium head blight pathogens and their associated mycotoxins

Within-field variability in the Fusarium head blight (FHB) and its associated mycotoxins was studied in four European countries. At each of 14 sites, each FHB pathogen and associated mycotoxins were quantified in 16 quadrat samples at harvest. Overall, the incidence of quadrat samples with detectable and quantifiable pathogen DNA was significantly lower in the grain than in the corresponding chaff. Deoxynivalenol (DON) was the most frequently detected toxin in the samples and its accumulation was most strongly associated with the presence of Fusarium graminearum. Nivalenol (NIV) accumulation was significantly associated only with the presence of F. culmorum. Zearalenone (ZON) accumulation was strongly associated with the presence of all three pathogens (F. graminearum, F. culmorum and F. poae). The levels of both DON and ZON concentrations were positively related to the amount of F. graminearum DNA in the grain or in the chaff. The presence/absence of FHB pathogens within a single quadrat appeared to be independent of each other. The presence of a particular FHB pathogen and the amount of its DNA, as well as the associated mycotoxin(s), varied greatly among samples at each site. This study demonstrated the large extent of within-field variability of FHB and its associated mycotoxins, and the importance of representative sampling in FHB studies.

Cloning and Heterologous Expression of a β-d-Mannosidase (EC 3.2.1.25)-Encoding Gene from Thermobifida fusca TM51

ABSTRACT Thermobifida fusca TM51, a thermophilic actinomycete isolated from composted horse manure, was found to produce a number of lignocellulose-degrading hydrolases, including endoglucanases, exoglucanases, endoxylanases, β-xylosidases, endomannanases, and β-mannosidases, when grown on cellulose or hemicellulose as carbon sources. β-Mannosidases (EC 3.2.1.25), although contributing to the hydrolysis of hemicellulose fractions, such as galacto-mannans, constitute a lesser-known group of the lytic enzyme systems due to their low representation in the proteins secreted by hemicellulolytic microorganisms. An expression library of T. fusca, prepared in Streptomyces lividans TK24, was screened for β-mannosidase activity to clone genes coding for mannosidases. One positive clone was identified, and a β-mannosidase-encoding gene (manB) was isolated. Sequence analysis of the deduced amino acid sequence of the putative ManB protein revealed substantial similarity to known mannosidases in family 2 of the glycosyl hydrolase enzymes. The calculated molecular mass of the predicted protein was 94 kDa, with an estimated pI of 4.87. S. lividans was used as heterologous expression host for the putative β-mannosidase gene of T. fusca. The purified gene product obtained from the culture filtrate of S. lividans was then subjected to more-detailed biochemical analysis. Temperature and pH optima of the recombinant enzyme were 53°C and 7.17, respectively. Substrate specificity tests revealed that the enzyme exerts only β-d-mannosidase activity. Its kinetic parameters, determined on para-nitrophenyl β-d-mannopyranoside (pNP-βM) substrate were as follows: Km = 180 μM and Vmax = 5.96 μmol min−1 mg−1; the inhibition constant for mannose was Ki = 5.5 mM. Glucono-lacton had no effect on the enzyme activity. A moderate trans-glycosidase activity was also observed when the enzyme was incubated in the presence of pNP-αM and pNP-βM; under these conditions mannosyl groups were transferred by the enzyme from pNP-βM to pNP-αM resulting in the synthesis of small amounts (1 to 2%) of disaccharides.

Electrophoretic karyotypes and gene mapping in eight species of the Fusarium sections Arthrosporiella and Sporotrichiella

Pulsed-field gel electrophoresis was used to identify karyotypes for eight species of the Fusarium sections Arthrosporiella and Sporotrichiella. The total number of chromosome-sized DNA molecules varied from six to nine, depending on the species. The sizes of chromosomes ranged from 0.4 to approximately 6.5 Mb which gave estimates of genome size of between 27.0 and 29.9 Mb. When fractionated chromosomes of the eight species were probed with Tox5, a gene coding for the key-enzyme of trichothecene biosynthesis, strong hybridization signals developed in F. poae and F. sporotrichioides, suggesting that of the eight species examined only these two have the genetic potentiality to produce trichothecene mycotoxins. By using heterologous probes from Aspergillus different rRNA loci have also been mapped on Fusarium chromosomes.

Fphog1, a HOG‐type MAP kinase gene, is involved in multistress response in Fusarium proliferatum

ΔFphog1 mutants of Fusarium proliferatum obtained by targeted gene disruption of Fphog1, an orthologue of the Saccharomyces cerevisiae hog1 MAPK gene showed increased sensitivity towards different abiotic stressors including UV‐irradiation, heat, salt, osmotic and hydrogen peroxide treatments. Incubation of the ΔFphog1 mutants under hyperosmotic conditions was accompanied with prolonged growth arrest, inhibition of conidial germination, morphological abnormalities and time‐dependent increase of the cell death rate. The wild type Fphog1 gene, under the control of its own promoter, was able to rescue the multistress sensitivity of the mutant strain. Real time qPCR data demonstrated that under salt and sorbitol stress conditions the Fphog1 gene is not subject of transcriptional regulation. Levels of reactive oxygen species (ROS), mitochondrial membrane permeability transition, nuclear disintegration and DNA fragmentation, indicators of programmed cell death (PCD) all showed significant increases under osmotic stress conditions in the ΔFphog1 mutant in comparison to the wild type strain. These results suggest that an important function of Fphog1 is attenuating apoptotic phenotypes under salt and sorbitol stressors. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Advances in molecular diagnosis of toxigenic Fusarium species: A review

The development of advanced molecular diagnosis for the critical toxigenic Fusarium species is considered in this review. The specific topics discussed are (1) isolation of mating type genes of Gibberella complex, (2) molecular detection of Fusarium–producing fumonisins, (3) molecular detection of Fusarium–producing trichothecenes and enniatins. Particular attention is given to the development of PCR assays for genes involved in the toxin biosynthesis that would permit the early detection of Fusarium species–producing toxins and potentially even reveal which particular toxin may be present within a food or feed product. Most of these data have been obtained within the ‘De–Tox Fungi’ project supported by the European Commission (QLK1–CT–1998–01380).

Thermobifida cellulolytica sp. nov., a novel lignocellulose-decomposing actinomycete

Four actinomycete strains, isolated from the overheated region of manure compost, were assigned to the genus Thermobifida on the basis of morphological, physiological and biochemical characteristics. All strains produced single, ovoid, heat-sensitive spores on dichotomically branched aerial hyphae. On the basis of chemotaxonomic traits, these isolates showed strong affinity towards members of the genus Thermobifida. Cell-wall analysis revealed the presence of meso-diaminopimelic acid, but no other characteristic amino acids or sugars in the murein (cell wall type III). According to polar lipid analysis, all strains showed PL II-type phospholipid composition; phosphatidylethanolamine and glycolipid were detected together with some unidentified phospholipids. The isoprenoid quinone composition of the new isolates differed slightly from that of the other two Thermobifida species described thus far. The partial 16S rDNA sequence similarity of the four strains reached 99.8-100%, whereas a nearly complete 16S rDNA sequence of TB100T, the representative strain of this collection, showed only 97.4 and 97.8% similarity to the corresponding rDNA sequences of the type strains of Thermobifida fusca and Thermobifida alba, respectively. These four isolates constituted a homogeneous group with levels of DNA-DNA homology ranging from 94.6 to 99.1%. The DNA-DNA relative homology values of strain TB100T to Thermobifida fusca ATCC 27730T and Thermobifida alba DSM 43795T were 48.1 and 57%, respectively. On the basis of phenotypic, chemotaxonomic and genotypic data, the strains are assigned to a new species within the genus Thermobifida under the name Thermobifida cellulolytica sp. nov. The type strain is TB100T (= DSM 44535T = NCAIM B01997T).