The antifungal activity of flavone and flavanone was tested in three different concentrations, 0·5, 2 and 8 × 10−4 M, against 34 fungi. In general, flavone was the substance with the higher activity, and could at 2 × 10−4 M completely inhibit several fungi. No linear relationships between the concentration and the antifungal activity could be observed for some fungi. Chaetomium globosum was the only fungus which was totally inhibited by flavanone at 8 × 10−4 M. Several fungi did, however, show a higher inhibition rate at 2 × 10−4 M.

The extracellular subtilisin-like protease activity of several fungal pathogens of nematodes, insects and plants was recorded after batch cultivation. Nematophagous and entomophagous isolates of Verticillium, Paecilomyces, Beauveria and Metarhizium produced Suc-(Ala)2-Pro-Phe-pNA hydrolysing enzymes that differed in serological properties based on Western blot analysis with polyclonal antibodies. The antibodies used were raised against the main subtilisin-like proteases from one species from each of the four genera. From each strain of V. chlamydosporium tested, between one and four isoforms with different pI values were separated by isoelectric focusing and visualized by enzymoblots. The occurrence of multiple isoforms with unique N-terminal sequences in single strains might suggest the presence of subtilisin gene families. Using as a probe a fragment of the Pr1 gene from M. anisopliae, RFLPs were observed that confirmed qualitative differences between subtilisin-like genes within and between species. In contrast to these pathogens of invertebrates, plant-pathogenic species of Verticillium did not produce subtilisin-like proteases, nor were homologous genes detected. Such activity, however, and the corresponding genes, were detected in species weakly pathogenic to plants, or saprotrophs.

A technique based on the polymerase chain reaction (PCR) for the specific detection of Phytophthora medicaginis was developed using nucleotide sequence information of the ribosomal DNA (rDNA) regions. The complete IGS 2 region between the 5 S gene of one rDNA repeat and the small subunit of the adjacent repeat was sequenced for P. medicaginis and related species. The entire nucleotide sequence length of the IGS 2 of P. medicaginis was 3566 bp. A pair of oligonucleotide primers (PPED04 and PPED05), which allowed amplification of a specific fragment (364 bp) within the IGS 2 of P. medicaginis using the PCR, was designed. Specific amplification of this fragment from P. medicaginis was highly sensitive, detecting template DNA as low as 4 ng and in a host-pathogen DNA ratio of 1000000: 1. Specific PCR amplification using PPED04 and PPED05 was successful in detecting P. medicaginis in lucerne stems infected under glasshouse conditions and field infected lucerne roots. The procedures developed in this work have application to improved identification and detection of a wide range of Phytophthora spp. in plants and soil.

The susceptibility of mustard beetle, Phaedon cochleariae, larvae to the insect pathogenic fungus Metarhizium anisopliae, was influenced by the topography of the host plant, the dosage and the larval instar. At high doses (>109 conidia ml−1) more insects were likely to acquire conidia. Most of the inoculum adhered to the mid-ventral region of the abdomen. Inoculum applied to the leaf became diluted during leaf expansion and this decreased beetle mortality. Leaf expansion was slower at 10°C than at alternating temperatures of 23/10° or when constant at 23°. Slightly more larvae acquired conidia when fed on oilseed rape than Chinese cabbage or turnip. Most conidia adhered to the legs and the mid-ventral region of the abdomen. Mortality was usually less on oilseed rape than Chinese cabbage and turnip. These observations suggest that fungistatic compounds of oilseed rape were interfering with the infection process.

Four group I introns, designated Bb1, Bb2, Bb3 and Bb4, were identified in the entomopathogenic hyphomycete Beauveria bassiana. Sequence analyses of these introns verified that they were invariably inserted at specific target sequences after conserved positions Ec2563, Ec2449, Ec2066 and Ec1921 of the large nuclear subunit (LSU) rDNA 3′-end. Secondary structure modelling confirmed that Bb1 and Bb3 belonged to subgroup IE while Bb2 and Bb4 belonged to subgroup IC1. Intron presence, distribution and size-variation were studied in a population of 125 B. bassiana strains using site-specific primers. Nucleotide sequences and secondary structures were compared and showed considerable variations usually at P1, P6 and P9 helices, but concomitantly, high homology between members of the same site-specific group. Intron distribution studies revealed that few (7.2%) strains were intron-less, most contained one (28%), two (48%) or three (16%) introns, while only one strain contained all four introns. Bb4-like introns (Ec1921) were the most abundant (86.4%), whereas the other three introns were evenly represented (ca 30%) in the B. bassiana population. Analysis of intron genotype distribution indicated a tenuous dependence upon geographic origin or insect host species. Phylogenetic analysis of all B. bassiana LSU introns and their close relatives from other entomopathogenic fungi showed a strong correlation between specific insertion sites and intron subgroups, fully supported by corresponding clades, suggesting common ancestry of the site specific LSU introns.

21 strains with all typical morphological characteristics of eight Verticillium species (Phyllachorales) were studied in this work, together with representatives from four Hypocreales species (11 strains), that were previously classified as members of the genus. The PCR products from two nuclear genes, i.e. the ITS1-5.8S-ITS2 region and RNA polymerase II largest subunit gene (rpb1), together with four mitochondrial genes, i.e. the small ribosomal rRNA subunit (rns), the two NADH dehydrogenase subunit genes (nad1 and nad3), and the cytochrome oxidase subunit III gene (cox3) were sequenced and analyzed. Similarly, antibodies raised against one strain of each of the species examined (V. nubilum and V. theobromae excluded) were used against the proteins of all other strains. The number and relative area of precipitates formed after crossed electrophoreses served to estimate the degree of immunochemical relatedness. Combined molecular and immunochemical data clarified the phylogenetic relationships of all true Verticillium species and provided a convincing insight into the evolutionary relation of the sect. Nigrescentia with members of the sect. Verticillium and sect. Prostrata that until recently were included in Verticillium.

A molecular phylogenetic analysis of rDNA was performed for seven Caloplaca, seven Xanthoria, one Fulgensia and five outgroup species. Phylogenetic hypotheses are constructed based on nuclear small and large subunit rDNA, separately and in combination. Three strongly supported major monophyletic groups were revealed within the Teloschistaceae. One group represents the Xanthoria fallax-group. The second group includes three subgroups: (1) X. parietina and X. elegans; (2) basal placodioid Caloplaca species followed by speciations leading to X. polycarpa and X. candelaria; and (3) a mixture of placodioid and endolithic Caloplaca species. The third main monophyletic group represents a heterogeneous assemblage of Caloplaca and Fulgensia species with a drastically different metabolite content. We report here that the two genera Caloplaca and Xanthoria, as well as the subgenus Gasparrinia, are all polyphyletic. The taxonomic significance of thallus morphology in Teloschistaceae and the current delimitation of the genus Xanthoria is discussed in light of these results.

Amplification of the internal transcribed spacer (ITS) of ribosomal RNA genes using PCR and subsequent RFLP analysis offers promise for improved species-level identification of ectomycorrhizal fungi. Intrageneric homogeneity and intraspecific heterogeneity of these characters are, however, frequent. A diverse group of ectomycorrhizal fungi with worldwide distribution was characterized by ITS-RFLP analysis. DNA of 64 identified and five unknown ectomycorrhizal fungi was extracted and their ITS regions were selectively amplified with ITS1-F and ITS4 ribosomal RNA gene primers. Each PCR product was digested with seven restriction enzymes. The resulting restriction fragments were compared by distance matrix analysis. Restriction enzyme analysis of amplified fragments was sufficient for distinguishing all but two closely related species. Intraspecific variation was observed in Cenococcum geophilum and Pisolithus arhizus, providing further evidence that these taxa represent species complexes.

Using the mating type genes (MAT-1 and MAT-2) from Cochliobolus heterostrophus (a pathogen of maize) as probes, MAT genes from C. carbonum (also a maize pathogen) and C. victoriae (an oat pathogen) were cloned. Sequence analyses and functional tests showed that C. carbonum MAT-1 and C. victoriae MAT-2 are structurally similar to their C. heterostrophus homologues, and all three genes function comparably when expressed in C. heterostrophus. Gel blot analyses of DNAs from C. heterostrophus or C. carbonum, probed with C. heterostrophus MAT-specific fragments, revealed that field isolates from diverse geographical locations contained either MAT-1 or MAT-2, but never both. The entire currently existing collection of C. victoriae isolates, however, contained only MAT-2, and all members of the collection were found to be either female sterile or completely infertile. Thus, crosses between isolates of C. victoriae were not possible, although crosses between C. victoriae and C. carbonum (but not C. heterostrophus) were readily made, confirming previous reports that these two species are interfertile. These observations, combined with careful scrutiny of the literature, have led to the hypothesis that MAT-1 female fertile isolates of C. victoriae have never existed. All available evidence is consistent with the notion that the entire C. victoriae field population was derived from a single progenitor strain, which arose as the result of horizontal transfer of genes for pathogenicity to oats into a strain of C. carbonum that was MAT-2 and female sterile.

The basidiomycete genus Armillaria causes root rot and death to woody plants in boreal, temperate and tropical regions of the world. Armillaria root rot has been described from various parts of Africa on many different hosts. However, very little is known regarding the evolutionary relationships among Armillaria species in Africa. The aim of this study was to determine the phylogenetic relationships between isolates originating from different regions in Africa using nDNA sequences from two non-coding gene regions. The ITS and the IGS-1 regions of the ribosomal DNA operon were sequenced and analysed using different phylogenetic tree searching methods. Phylogenetic trees grouped the African taxa in two strongly supported clades. One of these represented A. fuscipes and the other an undescribed but distinct species.

A total of 500 strains of the thermophilic fungus Thermomyces lanuginosus were screened for their ability to produce proteases. A selected strain, T. lanuginosus P134, showed high enzymatic activity for proteases. The maximal yield of proteases was obtained with 4% casein, 4% glucose and 4% yeast extract in submerged culture. The protease system was shown to contain at least two protease isoenzymes by SDS–PAGE with gels containing gelatin as protease substrate. The effects of inhibitors imply that the two enzymes appear to be serine proteases. The crude proteases of T. lanuginosus P134 displayed unusual properties. The proteases were shown to be one of the most thermostable proteases so far isolated in fungi. The proteases were 100% stable at 50 °C. The half-life at 60 and 70° was approximately 160 min and 60 min, respectively. Temperature optimum of activity was 70°. The crude proteases were stable over a broad pH range (4–11). Optimum pH of activity was at 5·0 and 9·0.

The endophytic behaviour of Sclerotinia pseudotuberosa, cause of black rot of fruits of Castanea sativa, was investigated in asymptomatic tissues of the host, including nuts, buds and bark. A nested polymerase chain reaction (PCR) assay was developed for the specific detection of S. pseudotuberosa. Two specific primers, RAC1 and RAC2 were designed within the ITS1-5.8S-ITS2 region of rDNA by comparison of sequences of 24 isolates of S. pseudotuberosa and related species. Specificity of primers was tested against isolates of related and unrelated genera and the total DNA of C. sativa. The assay proved to be highly sensitive and capable of detecting picograms of S. pseudotuberosa DNA. Our study showed that S. pseudotuberosa was present asymptomatically in all the chestnut tissues tested. It is discussed that latency of S. pseudotuberosa in chestnut tissues may represent a adaptive strategy of the pathogen for rapid and massive host colonization in favourable environmental conditions.

Phylogenetic analyses of the ITS1-5.8S-ITS2 region of the ribosomal RNA gene cluster were carried out with two short-cycled (aecial/telial) European rusts on Asteraceae, Puccinia distincta causing the current pan-European epidemic on Bellis perennis, and P. lagenophorae causing a similar disease on Senecio spp., as well as the macrocyclic P. obscura which alternates between B. perennis (pycnial and aecial host) and Luzula spp. (main host). All three species formed a well-resolved cluster when compared with the ITS sequences of a range of other rust fungi, using both parsimony and distance methods. The sequences of P. distincta and P. lagenophorae differed from each other at three positions whereas P. obscura differed from P. distincta at 37 points. Together with consistent morphological and epidemiological differences across Europe, these data support the recognition of P. distincta as a separate species from P. lagenophorae. Both may be derived from P. obscura, although the precise evolutionary history remains obscure.

Aspects of the ecology of oospores of Phytophthora infestans were studied in the highlands of central Mexico. From an investigation of a random sample of strains, it was found that isolates differed in their average capability to form oospores when engaged in compatible pairings. Most crosses produced large numbers of oospores but a few yielded none and some yielded only a few oospores. The results reveal that oospore production and fecundity is dependent on both isolates and the combining ability of a specific combination of parental strains. On average, 14% of the oospores produced were viable as determined by the plasmolysis method. Viability ranged from a low 1% in one cross to a high of 29% in another cross. Oospores were found in 10–20% of naturally infected Solanum demissum leaves from two different collections, and leaflets with two lesions per leaflet produced more oospores than did leaflets with 3–5 lesions per leaflet. There was no consistent trend for preferential mating between isolates from the same location or host.

Recombination between two strains of Metarhizium anisopliae, subcultured on artificial medium and passed through an insect host was investigated. When grown on agar culture, or passed through a host, strains did not show detectable genetic changes. When passed through an insect in the presence of the other strain, genotypic changes in the isolates, as detected by esterase electrophoresis, restriction digestion of total genomic DNA and RAPD-PCR, were apparent. The instability of molecular markers suggests recombination between strains.

A piece of Baltic amber (Tertiary, Eocene) contains an inclusion of a springtail (Collembola) which is overgrown by an Aspergillus species. The fossil fungus is described as A. collembolorum sp. nov. The excellent mode of preservation of the numerous conidiophores is remarkable and can be explained by sporulation in liquid resin. This is the second report of a fossil Aspergillus, the first being from Dominican amber.

The phylogeny of Nephroma was studied by nucleotide sequences of the mitochondrial ribosomal small subunit (mtSSU rDNA) and the internal transcribed spacers of the nuclear ribosomal repeat (ITS), together with chemical characters. The biological material included both bipartite and tripartite species and all Nephroma species native to northern Europe. Phylogenetic analyses demonstrated that all Nephroma species form a monophyletic group and that Peltigera constitutes the sister group to Nephroma. The two gene regions revealed qualitatively similar relationships within Nephroma and chemical characters had a minor impact on tree topologies. The results demonstrated that tripartite Nephroma species do not form a monophyletic group within the genus, this being in agreement with previous findings from bi- and tripartite Peltigera species. The results also indicated that N. resupinatum does not form a monophyletic group with all other bipartite Nephroma species but form a sister group to the studied Nephroma taxa. Furthermore, N. helveticums. lat. is highly variable and seems to represent aggregates of closely related taxa. Also N. laevigatum and N. resupinatum are genetically variable. All European Nephroma species can be rapidly and accurately identified on the basis of their fungal ITS sequences. This will prove useful in ecological and environmental studies.

Host-parasite interactions of Agaricus bisporus and Verticillium fungicola were studied in paired-cultures. Different growth reactions were found according to the culture medium used, but no apparent alterations could be detected by light and electron microscopy in A. bisporus vegetative mycelia. In vitro, different hydrolytic enzymes are produced by V. fungicola in cultures with A. bisporus vegetative mycelial cell walls as the only carbon source. In vitro, this enzyme preparation is able to partially digest isolated A. bisporus vegetative mycelial cell walls. These results suggest that although apparent attachment of the mycoparasite to the host cell surface seems to occur, there is no in vivo A. bisporus vegetative mycelial cell wall degradation.

In this investigation, we have determined the life span of the fruit bodies of Agaricus bisporus grown in a protected cultivation environment as 36 d. We describe the morphological changes occurring in ageing mushrooms and find that signs of senescence become visible around day 18; they steadily increase in amplitude towards the end of the fruit bodies' life. Cytologically, karyolysis and focal cytoplasmolysis were the first signs followed by indications of an increased permeability of the cytoplasmic membranes and by structural changes of the cell wall. These changes result in extracellular aggregations of the lytic cell remains encapsulating or bridging the hyphal cells. Cells of the stipe tissues were transformed to empty cylinders or had irregularly collapsed. Most basidia and subhymenial cells remained alive even on day 36. When the mushrooms were cultivated according to our usual growth procedures, about 50% of the fruit bodies were infected by Trichoderma harzianum and/or Pseudomonas tolaasii on day 18; all fruit bodies died on day 24 due to diffuse bacterial and mycotic infections manifesting themselves by tissue necrosis and discoloration of the caps and stipes. When none of the fruit bodies was harvested at the time of the first flush they soon formed a canopy of pilei and their growth ceased. On day 14, all these fruit bodies showed widespread mixed infections followed by a diffuse liquefaction necrosis of all tissues including the hymenium. Post-harvest fruit bodies kept at ambient temperature or at 2 °C at low r.h., or at 20° at high r.h. revealed a diffuse cell wall destruction which was followed by cytoplasmic degeneration in due course. Fruit bodies kept refrigerated had the least gross and cell structural changes during our observation period of 7 d. We conclude that the morphological changes occurring in post-harvest and in senescent fruit bodies of Agaricus bisporus are different.

DNA sequence analysis of the internal transcribed spacer region 1 (ITS1) and random amplified polymorphic DNA (RAPD) markers were used to survey genetic variability in relation to agronomic and regional factors among 60 isolates of Thanatephorus cucumeris (anamorph Rhizoctonia solani) collected from lesions on potato stems or sclerotia of potato tubers. Based on comparative sequence analysis it was shown that all isolates belonged to anastomosis group 3 subgroup Potato Type (AG-3 PT). ITS1 sequence polymorphisms were found within 45 of the 60 isolates showing that different types of the ITS-region are present in individual isolates. Cloning and sequence analysis of the ITS1 region from three selected isolates with sequence polymorphism showed that two different ITS1-types were present in each isolate. RAPD analysis identified 51 RAPD-phenotypes among the 60 investigated isolates indicating a high level of diversity within the subgroup AG-3 PT. Putative clonal isolates with identical RAPD- and ITS1-types were identified within fields, and in one case the same phenotype was found in two different fields separated by several hundred kilometers. Population subdivision analysis based on phenotypic as well as genotypic diversities showed differentiation among populations from different fields when isolates were sampled from tubers, indicating restricted gene flow among soil populations. Low differentiation was seen among field populations sampled from stems, indicating that gene flow is taking place. The population structure was not influenced by the previous crop in the rotation nor by the two cultivars ‘Sava’ and ‘Bintje’.