Cryptic subspecies and beauvericin production by Fusarium subglutinans from Europe.

Fusarium subglutinans is a maize ear rot pathogen and producer of beauvericin and other mycotoxins. This species has recently been split into two major phylogenetic within-species groups based on RFLP DNA sequence polymorphisms identified in the histone H3 and beta-tubulin sequences. A Pan European collection of the fungus originating mostly from maize was subjected to phylogenetic analysis by RFLP grouping and to chemical analysis for beauvericin production. Of the 62 isolates belonging to Group 1, 48 (77%) produced from 10 to 532 microg/g of beauvericin, whereas none of the 39 Group 2 isolates synthesized detectable amounts of the mycotoxin. The association between RFLP group and beauvericin production is consistent with the existence of two reproductively isolated subgroups within F. subglutinans and indicates that the toxicological risk of isolates of F. subglutinans depends on the group with which they are affiliated.

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.

Assignment of a PCR-amplified chitinase sequence cloned from Trichoderma hamatum to resolved chromosomes of potential biocontrol species of Trichoderma.

A 1424 bp DNA sequence containing the genetic determinants of the chitinase enzyme was identified in Trichoderma humatum by PCR amplification. High levels of similarity were observed between this sequence, named Th-ch (T. hamatum chitinase), and the 42 kDa chitinase genes known from T. harzianum. Chromosome-sized DNAs of five potential biocontrol species of Trichoderma were separated by pulsed-field gel electrophoresis. The total number of chromosomes was six in all the species, with sizes ranging from 3.7 to 7.7 Mb; estimated genome sizes were between 30.5 and 35.8 Mb. When fractionated chromosomes of the five species were probed with radiolabelled Th-ch, strong hybridization signals developed in all cases, but the physical position of these signals varied among species indicating a polymorphic chromosomal location of the highly conserved 42 kDa chitinase gene within the genus Trichoderma.

Homologous transformation of Trichoderma hamatum with an endochitinase encoding gene, resulting in increased levels of chitinase activity.

A 42-kDa endochitinase encoding gene, Tham-ch, was cloned by screening the genomic library of Trichoderma hamatum strain Tam-61 with a PCR-amplified chitinase sequence from the same fungus. Tham-ch with its own regulatory sequences was reintroduced into the host strain. The integration of the transforming construct was stable only in one copy. Homologous integration occurred in nine transformants, while non-homologous integration was detected in one transformant. All but one transformant expressed higher levels of chitinase activity in comparison to the wild-type recipient strain; the maximum level of increase was 5-fold. Duplicating the copy number of the highly conserved approximately 42-kDa endochitinase encoding gene appears to be one potential means by which the biocontrol capability of the Trichoderma species might be improved.

High-frequency occurrence of virus-like particles with double-stranded RNA genome in Fusarium poae.

Fifty-five geographically different strains of Fusarium poae were assayed for the presence of extrachromosomal nucleic acid elements. All strains were found to harbour double-stranded RNA (dsRNA) elements and encapsidated virus-like particles (VLP). There were great individual differences in dsRNA patterns of the various strains, but numbers and sizes characteristic for a given isolate remained unchanged after repeated subculturing of the fungi. Morphological alterations or signs of degeneration were not observed in dsRNA-containing isolates. This is the first report on the ubiquitous occurrence of dsRNAs in a hyphomycete fungus species.

Surface structures of new and lesser known species of thermobifida as revealed by scanning electron microscopy.

Surface structures of representatives of the genus Thermobifida were examined by scanning electron microscopy. Spores formed at the tips of multibranched sporophores initially resembled short sausages; then, upon maturation, they gradually built up their typical ovoid shape. Characteristic differences were observed between T. cellulolytica strain TB108 and T. fusca strains TM51. The spores of TB108 were larger (0.8x 1.3 microm) than those of TM51 (0.6 x 1.1 microm) in consequence of the more thickened outer squamous layer. When Thermobifida strains were grown on cellulose as sole carbon source, the mycelium was found to coil around the cellulose crystals and multiple protuberances emerged, resulting in a scabrous appearance to the mycelial surface. The presence of these cellulosome-like structures yielded a 24.5% surface enlargement of the scabrous mycelium as compared with the smooth one. The cellulosome emergence pattern paralleled the proportional increase in free endoglucanase activity measured during the culturing of these actinomycetes in the presence of cellulose.

[Treatment of anal fistulas]. / Az analis fistula kezelése.

The authors describe the traditional operative technique for correction of anal fistulae and analyse the outcome of surgical treatment. During a 5-years period between 1994 and 1998, 286 patients underwent surgery for anal fistula in the department--more than one--third of this population presented with recurrent disease. During the operation, the extrasphincteric segment of the anal fistula is excised and the margin of the sinus is marsupialized. Introducing a rubber band through the sinus tract eliminates lesions that penetrate the sphincter. As the tied band shears through the encircled sphincter muscle, the rate of transsection is controlled individually, by adjusting the tightness of the rubber band as necessary. The inner opening of the fistula is often difficult to identify and consequently, excision may be incomplete. This is a serious pitfall that commonly leads to recurrence. According to the authors' experience, flushing the fistula tract with hydrogen peroxide is the most effective methods for pinpointing the inner meatus. Using this technique, postoperative recurrence was detected in 30 patients (10%). Moderate impairment of anal continence had been observed in 57 patients (20%); however, this never progressed to permanent incontinence.

[Unusually large stromal tumor of the rectum causing obstruction]. / Obstrukciót okozó szokatlan nagyságú stromális rectum tumor.

A male, 74 years old patient with perineal, sacral pain and with defecation disorders attended the outpatient clinic of HIETE. The origine of the complains was a retrorectal, fist like, rectum narrowing tumor. The tumor was covered by normal mucosa from rectal side. Preoperative examinations--endoscopy, CT, MRI transrectal US--detected a tumor with size 7 x 6 x 5 cm, growing from the muscular wall of the rectum, with no connection with the surrounding tissues. Deep biopsy revealed malignant mesenchymal tumor. After preoperative irradiation abdominoperineal rectum amputation was performed. The recovery was uneventful. The definitive hystological examination proved a gastrointestinal stromal tumor (GIST). This type of tumor rarely occurs in the large intestine or in the rectum, that why the publishing can be interesting.

[Possibilities in the preservation and restoration of anal continence]. / Az analis continentia megórzésének és helyreállításának lehetóségei.

The preservation of anal continence and the improvement of the patients' quality of life in general are primary objectives of colorectal surgery. Earlier the loss of the entire rectum, colon required a definitive stoma. This review describes surgical procedures designed to preserve anal continence. This paper also describes operative techniques designed to improve impaired sphincter function. Total extirpation of the mesorectum reduces local recurrence of rectal tumours. At the same time, this operation requires formation of the anastomosis low, at the level of the levator muscle. Low colorectal or coloanal anastomoses are associated with higher incidence of suture leakage and poor functional outcome. The distance between anastomosis and anal verge was less than 7 cm in 249 sphincter-sparing rectal resections performed during the examined 6-year period in our institute. Different techniques to perform anastomoses were applied, the prevalence of suture leakage and the functional results are analysed. Restorative proctocolectomy has dramatically improved the treatment of familial polyposis and ulcerative colitis with rectal involvement. Although proctocolectomy is necessary to cure the disease, acceptable faecal continence can be achieved by creating ileoanal anastomosis with ileal reservoir. We discuss our results after 43 operations. Weakness of the sphincter apparatus is the most common cause of continence problems. Occasionally, the sphincter is no longer suitable for reconstruction because of extensive damage or denervation. In such cases, the levator muscles or--if neither these are of acceptable quality--the gluteus maximus muscle can be used to repair the external sphincter. Anterior levator plasty involves tightening the levator plate by suturing its arches together between the rectum and the vagina. This procedure enhances the resistance of the sphincter barrier primarily by increasing functional sphincter length. The functional outcome of this procedure was acceptable in two-thirds of the 52 operations. Post anal repair was performed only in 3 patients. This method comprises reinforcing the levator plate through an access between the external and the internal sphincters. When the levator plate is unsuitable, bilateral gluteus plasty can be performed to increase the strength of sphincter muscles. As the gluteus is a striated muscle it can improve only the of the external sphincter function. Therefore this procedure can restore acceptable continence to hard stool only. This is demonstrated by our clinical experience obtained in 10 patients.

Expression patterns of cel5A-cel5B, two endoglucanase encoding genes of Thermobifida fusca.

Expression patterns of cel5A and cel5B, two endoglucanase encoding genes of Thermobifida fusca were compared by quantitative real-time PCR. With Avicel as carbon source the transcript level of cel5A continuously increased until the 10th hour of incubation and then a sharp decrease was observed, whereas cel5B presented a slow constitutive expression on this substrate. When the microcrystalline cellulose powder MN300 was used as the inducing carbon source, the expression patterns of the two genes were similar. A low initial level of expression was followed by a rapid increase at the 5th hour of incubation; a transient repression was then observed at the 10th hour but after this sampling time, the expression levels started to increase again. The relative expression levels of cel5A were always higher than those of cel5B. Differences in transcription patterns of these two genes can be explained with the imperfect structure of the CelR binding regulatory region of cel5B.

Mating type loci inFusarium: structure and function.

Sex in fungi is regulated by highly dissimilar mating type loci named idiomorphs. The genus Fusarium harbours both sexual as well as esexual species and each appears to contain one or the other idiomorph. The structure of these loci is highly conserved, suggesting a cryptic sexual cycle in these socalled asexual species. Alternatively, idiomorphs could regulate additional hitherto unrecognized biological processes. Such processes could be elucidated by expression profiling using mutants disrupted in their mating type loci.

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).

Chromosomes, karyotype analysis, chromosome rearrangements in fungi.

In this review the organization of fungal chromosomes and the methods used for karyotype analysis are briefly summarized. The role of chromosome rearrangement, supernumerary chromosomes and repeated DNA sequences in the genetic change of fungi is evaluated.

Comparison of Fusarium acuminatum and Fusarium culmorum isolates by means of tandem-crossed immunoelectrophoresis.

F. acuminatum and F. culmorum strains were compared by means of tandem-crossed immunoelectrophoresis in order to estimate the possibilities of serological classification in Fusarium sections "Gibbosum" and "Discolor". On the basis of qualitative similarity the two species could be distinguished well. By the use of anti-F. acuminatum serum a similarity of SSM = 0.52 was found between F. acuminatum and F. culmorum, but the SSM coefficient reached a value of 0.67 when the anti-F. culmorum serum was tested. This asymmetric nature of the qualitative similarity is discussed. In the majority of cases the quantitative differences of the common antigens did not allow differentiation between the species.

Isoelectric focusing isozyme profiles and taxonomic distances among Fusarium species of the sections Arthrosporiella and Sporotrichiella.

Isozymes from 18 isolates representing seven species of the Fusarium sections Arthrosporiella and Sporotrichiella were compared by isoelectric focusing in polyacrylamide gels. Of the six enzyme systems tested esterase and malate dehydrogenase showed the largest variation. A numerical analysis of the pI values determined for acid phosphatase, esterase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, phosphoglucose isomerase and phosphoglucomutase resulted in a dendrogam demonstrating the taxonomical relationships of the seven species. Fusarium avenaceum and Fusarium pallidoroseum were the two most closely related species. The high degree of isoenzyme dissimilarity among Fusarium chlamydosporum, Fusarium poae, Fusarium tricinctum, the fungi that produce pyriform or citriform microconidia, suggests that they are distinct species and their reduction to a variety level is not reasonable. The taxonomical distinctness of Fusarium camptoceras, a lesser known and rarely occurring fungus was also proven.

A case of Fournier's gangrene.

Authors report their case of Fournier's gangrene on the basis of their own observations and relying on data of the literature. Complete healing can be expected of immediate radical surgery and of aggressive intensive and antibiotic treatments.

Primary structure and transcription patterns of RPL36, a ribosomal protein-encoding gene of the mycoparasitic fungus, Trichoderma hamatum.

We report the isolation and expression profiles of a single-copy gene from the mycoparasitic fungus Trichoderma hamatum encoding a 60 S cytoplasmic ribosomal protein. The gene, named RPL36, was cloned through its nutrient-mediated expression, using mRNA differential screening. Its predicted ORF, interrupted by two introns, encoded a 105-aa polypeptide. The deduced rpL36 protein showed high overall homologies with other L36-type ribosomal proteins isolated from yeast, rat and human. Analysis of the promoter region of RPL36 revealed the presence of two ribosomal protein gene (RPG) boxes and a T-rich region known to be involved in the regulation of most ribosomal protein genes. Expression of RPL36 was tightly regulated by carbon and nitrogen availability. The mRNA levels of this gene decreased upon exposure of the mycelium to different stresses, whereas the addition of cycloheximide resulted in a super-induction. Levels of RPL36 transcripts also increased during mycoparasitic interaction between T. hamatum and Botrytis cinerea.

Expression of cmg1, an exo-beta-1,3-glucanase gene from Coniothyrium minitans, increases during sclerotial parasitism.

During sclerotial infection of Sclerotinia sclerotiorum the mycoparasite Coniothyrium minitans penetrates through the host cell wall, which contains beta-1,3-glucan as its major component. A PCR-based strategy was used to clone a beta-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-beta-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 cerevisiae INVSc1 expressing the cmg1 gene secreted a approximately 100-kDa beta-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 of S. sclerotiorum by 35 and 85% at concentrations of 300 and 600 microg x ml(-1), respectively. A single copy of the cmg1 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.

Mini-chromosomes in Fusarium sporotrichioides are mosaics of dispersed repeats and unique sequences.

Variations in trichothecene patterns of 26 Fusarium sporotrichioides isolates from different plant and geographic origins showed no correlation with electrophoretic karyotype polymorphisms. When intact chromosomes were examined, interisolate karyotype differences were observed only in the mini-chromosome range. Further polymorphisms were revealed in Notl-digested samples. By summing the Notl fragments the average genome size of F. sporotrichioides was estimated to be 20.4 Mb. Mini-chromosomes shared common sequences with the larger ones; however, clones (RMS-1 and RMS-2) specific to these structures have also been found. These clones contained no coding region and no promising similarities were observed when they were compared to sequences held at GenBank. Mini-chromosomes in F. sporotrichioides constitute a mosaic composed of dispersed repeats and unique sequences. This mosaic structure was maintained in all noninterbreeding, genetically isolated strains examined.

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.