Ferenc Kevei

Immunochemical detection of ochratoxin A in black Aspergillus strains

One hundred and fifty-seven strains belonging to Aspergillus section Nigri were tested for ochratoxin A production using three different methods: a relatively new immunochemical method based on an enzyme-linked immunosorbent assay (ELISA), thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The monoclonal antibody-based ELISA technique was successfully used to screen for low levels of ochratoxin A in the black Aspergilli without concentrating the culture filtrates. The results were confirmed by TLC and HPLC analysis and chemical derivatization. These latter methods required concentrated filtrates. Ochratoxin A was detected in the culture filtrates of five of the 12 A. carbonarius strains, none of the 45 A.japonicus strains and three of the 100 isolates in the A. niger aggregate (A. foetidus, A. awamori and A. niger).

Restriction fragment length polymorphisms in the mitochondrial DNAs of the Aspergillus niger aggregate

Restriction fragment length polymorphisms (RFLPs) were detected in the mitochondrial and nuclear DNAs of isolates belonging to the Aspergillus niger aggregate. These strains can be classified into two well-defined groups according to the electrophoretic patterns of their mtDNAs obtained with some restriction enzymes, such as EcoR I, Pvu II, Hae III and Bgl II. Strains belonging to both groups show some further minor variation. Estimates of the molecular weight of these mitochondrial DNAs were between 30 and 32·5 kb. The patterns produced by the nuclear ribosomal DNA repeats in digests of total DNA made it possible to classify the black Aspergillus strains into two groups, each further subdivided into two. The two main groups established from the mitochondrial and ribosomal DNA patterns coincide with each other. A rapid and easy method was also worked out to classify large numbers of field isolates of black aspergilli into one of the groups described using mtDNA patterns obtained by the double-digestion of total DNA samples with Hae III and Bgl II.

Molecular Diversity of Agriculturally Important Aspergillus Species

Although Aspergillus species are not usually considered as serious plant pathogens, Aspergilli are frequently encountered in plant products. The most important consequence of their presence is mycotoxin contamination. The main mycotoxins produced by Aspergilli are the aflatoxins, ochratoxin A and patulin, which are produced by a variety of Aspergillus species in different plant commodities. Phylogenetic analysis of sequences of the ribosomal RNA gene cluster is useful for clarifying taxonomic relationships among toxigenic Aspergilli causing pre- and postharvest contamination of agricultural products. Molecular data has enabled us to clarify the taxonomy of black Aspergilli, A. flavus and its relatives, and sections Circumdati and Clavati, which include ochratoxin and patulin-producing species. Phylogenetically unrelated species were found to produce the same mycotoxins, indicating that mycotoxin-producing abilities of the isolates have been lost (or gained) several times during the evolution of the genus. The data also indicate that biosynthetic gene-based probes are necessary for molecular detection of these mycotoxin-producing organisms. The organisation of the biosynthetic genes of patulin and ochratoxins is unknown, although experiments are in progress in several laboratories to clarify the genetic background of biosynthesis of these mycotoxins. Identification of biosynthetic genes responsible for mycotoxin production is essential for clarifying the evolution of mycotoxin biosynthesis in Aspergilli, and to develop specific gene probes for the detection of mycotoxin-producing Aspergilli in agricultural products.

Isolation and characterization of a new keratinolytic Bacillus licheniformis strain

A keratin-degrading bacterium strain (K-508) was isolated from partially degraded feathers and characterized. This isolate exhibited a high chicken feather-degrading activity when cultured in feather-containing broth with a growth optimum of pH 7.0 and 47 °C. On the basis of its phenotypic characteristics (quickly moving, Gram-positive rods), the results of metabolic tests and rDNA sequence analysis, it was identified as Bacillus licheniformis. Its fermentation broth showed activity on N-Bz-l-Phe-l-Val-l-Arg-p-nitroanilide, N-Suc-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide, N-CBZ-Gly-Gly-l-Leu-p-nitroanilide and N-CBZ-l-Ala-l-Ala-l-Leu-p-nitroanilide as chromogenic protease substrates at near neutral pH. Both trypsin-like and chymotrypsin-like proteases were constitutively secreted by this strain.

Interspecific Hybridization between Aspergillus nidulans and Aspergillus rugulosus by Fusion of Somatic Protoplasts

Summary: Interspecific heterokaryons have been produced between auxotrophic mutants of Aspergillus nidulans and Aspergillus rugulosus by polyethylene glycol induced fusion of somatic protoplasts. The heterokaryons grew very slowly producing colonies of irregular shape and, on complete medium, the parental strains were readily segregated. However, during long-term cultivation on minimal medium many of the heterokaryons gave rise to vigorously growing sectors characterized by the secretion of a brown pigment and by their stability when subcultured on complete medium. Protoplasts isolated from the heterokaryons gave rise to the same new colony type when regenerated on minimal medium, and to the new colony type plus the parental types on complete medium. The new colony type was assumed to be an interspecific ‘hybrid’ and showed normal vegetative morphology, regular colony shape and size but produced few conidia. Conidia formed early in the development of the ‘hybrid’ gave rise to ‘hybrid’ colonies. Various sizes of conidia were observed; the largest were uninucleate and the smallest were enucleate. Comparisons of conidial size, numbers of nuclei in cells and DNA content per nucleus for ‘hybrid’ and parental strains indicated that the ‘hybrid’ was diploid. Conidia from older cultures of the ‘hybrid’ were heterogeneous with ‘hybrid’, parental and recombinant colony types developing on germination. Cultivation of the ‘hybrid’ in the presence of benomyl gave segregation of parental and recombinant sectors.

Factors affecting the spread of double-stranded RNA viruses in Aspergillus nidulans

Viruses are common in asexual Aspergilli but not in sexual Aspergilli. We found no viruses in 112 isolates of the sexual Aspergillus nidulans. We have investigated factors that could play a role in preventing the spread of mycoviruses through populations of A. nidulans. Experiments were performed with A. nidulans strains infected with viruses originating from A. niger. Horizontal virus transmission was restricted but not prevented by somatic incompatibility. Viruses were transmitted vertically via conidiospores but not via ascospores. Competition experiments revealed no effect of virus infection on host fitness. Outcrossing was found to limit the spread of viruses significantly more than selfing. It is concluded that the exclusion of viruses from sexual Aspergilli could be due to the formation of new somatic incompatibility groups by sexual recombination.

Isolation and characterization of protease overproducing mutants of Trichoderma harzianum

Several Trichoderma strains have been reported to be effective in controlling plant diseases, and the action of fungal hydrolytic enzymes is considered as the main mechanism involved in the antagonistic process. Strain Trichoderma harzianum T334 is a potential biocontrol agent against plant pathogenic fungi with the ability to produce low levels of proteases constitutively. To improve its fungal antagonistic capacity, mutagenetic program was undertaken for the construction of protease overproducing derivates. The mutant strains were obtained by means of UV-irradiation and were selected for p-fluorophenyl-alanine resistance or altered colony morphology. It was revealed by means of specific chromogenic protease substrates that both trypsin-like and chymotrypsin-like protease secretion was elevated in most of the mutant strains. The profiles of isoenzymes were different between the mutants and the wild-type strain, when examined by gel filtration chromatography. Certain mutants proved to be better antagonists against plant pathogens in in vitro antagonism experiments. This study suggests the possibility of using mutants with improved constitutive extracellular protease secretion against plant pathogenic fungi.

Molecular polymorphism and phenotypic variation in Aspergillus carbonarius

Thirteen collection strains and field isolates of Aspergillus carbonarius were examined by using various genotypic and phenotypic approaches. Restriction fragment length polymorphism analysis of the ribosomal RNA gene cluster and the mitochondrial DNA of the strains revealed only slight variations, except for one field isolate (IN7), which exhibited completely different ribosomal RNA gene cluster and mitochondrial DNA patterns. The mitochondrial DNAs of these strains were found to be much larger (45 to 57 kb) than those found earlier in the A. niger aggregate. Strain-specific characters could be detected by the random amplified polymorphic DNA technique. Isoenzyme analysis and examination of carbon source utilisation patterns of the strains also revealed some intraspecific variability, though much smaller than that observed by using DNA-based techniques. The dendrograms constructed based on genotypic and phenotypic data suggest that strain IN7 might represent a new subspecies of A. carbonarius.

Molecular and phenotypic characterization of Aspergillus japonicus and Aspergillus aculeatus strains with special regard to their mitochondrial DNA polymorphisms

Forty Aspergillus japonicus and A. aculeatus strains, most of them wild-type isolates, were examined using various molecular and phenotypic techniques. The rDNAs proved to be invariable (even strains of the species A. aculeatus exhibited the same restriction profile), while the strains could be classified into seven different mtDNA RFLP groups. Hybridisation data suggest that six of these mtDNA types have certain common restriction sites, while mtDNA type 7, which was exhibited by some A. aculeatus strains, probably has quite different mtDNA organisation and their size was smallest among the strains studied. The RAPD technique and isoenzyme analysis revealed some variabilities within these RFLP groups and strain specific features could also be recognised. Carbon source assimilation spectra were found to be very distinctive for strains of A. japonicus, A. aculeatus and A. niger, providing a useful tool for pre-characterising new wild-type isolates of black Aspergilli. Only a limited correlation was observed between the dendrograms based on genotypic and phenotypic characters.

Further studies on protoplast fusion and interspecific hybridization within the Aspergillus nidulans group

Hybridization of eight species of the Aspergillus nidulans group was attempted using auxotrophic mutants and protoplast fusion methods. Viable fusion products were obtained from eight crosses. Allodiploid hybrids were recovered from crosses involving A. nidulans with A. rugulosus, A. quadrilineatus, A. nidulans var. echinulatus and A. violaceus, although some mutants only gave heterokaryons. Crosses involving these latter species also gave heterokaryons. Crosses between A. nidulans and A. unguis, A. stellatus and A. heterothallicus were unsuccessful. Fusions involving three parents gave heterokaryons made up of only two of them.