Of the two potato glycoalkaloids, α-chaconine and α-solanine, α-chaconine was the more inhibitory on spore germination in Alternaria brassicicola and Phoma medicaginis, and on growth in liquid culture of these species and also Ascobolus crenulatus and Rhizoctonia solani. At 50 μM or less, solanine caused little (<20%) or no inhibition but, in combination with comparable concentrations of chaconine (which were sometimes also below the activity threshold), synergistic inhibitory effects (up to 100% inhibition) were apparent. A scanning electron microscopic examination of agar-grown cultures of P. medicaginis revealed that both glycoalkaloids at 100 μM inhibited hyphal extension, but that chaconine particularly increased branch formation (three-fold) as well as hyphal thickness and ‘beading’. Glycoalkaloid-containing liquid medium from R. solani cultures did not contain glycoalkaloid hydrolysis products. There was a 17% reduction in the solanine content of the medium, but the chaconine level was not significantly different from that in uninoculated controls. Neither glycoalkaloids nor their hydrolysis products were detectable in the washed mycelium of such cultures.

The genetic structure of three newly established Cryphonectria parasitica populations (Choëx, Weggis, Murg) was analysed and compared to an older, post-epidemic population (Claro). Vegetative compatibility (vc) type and DNA fingerprint analysis revealed an almost clonal C. parasitica population in Choëx, Weggis and Murg. These stands are all situated in northern Switzerland, outside the main range of European chestnut. Only one vc type and one dominant DNA fingerprint was found in Choëx and Murg. In Weggis, two vc types and two dominating DNA fingerprints were found. The European Cryphonectria hypovirus (CHV1) was not detected in these three populations. In contrast, the population in Claro, situated within the main range of European chestnut in southern Switzerland, had much higher vc type and DNA fingerprint diversity. DNA fingerprints were correlated to vc types in Claro and in Weggis. Mating type determination revealed one strongly dominating mating type in each of the three northern populations, but not in Claro. From these results we conclude that C. parasitica disseminated almost exclusively by means of asexual reproduction in Choëx, Weggis and Murg, whereas in Claro sexual reproduction also played an important role. Additionally, founder effects and restricted gene flow were dominating factors in shaping the genetic structure of the three northern populations.

Samples of standing senescent culms of Panicum maximum, Pennisetum purpureum, Phragmites australis, Miscanthus floridulus, Saccharum arundinaceum and Thysanolaena maxima (Gramineae), and Schoenoplectus litoralis (Cyperaceae) were collected in Hong Kong between 1997 to 1999. A total of 205 fungal taxa were identified on these samples, including 61 ascomycetes, and 144 mitosporic taxa. Common fungal genera included Diaporthe, Leptosphaeria, Massarina, Ophiobolus and Ophioceras (ascomycetes), and Monodictys, Phaeoisaria, Periconia, Phoma, Phomopsis, Rhinocladiella, Septoria and Sporidesmium (mitosporic taxa). Different grass species were host to different fungal communities and diversities of taxa. Diversity indices for fungi on the hosts varied from 3·3 to 8·7, the highest index being from Pennisetum purpureum, and were overall higher from species offering more durable, strongly sclerenchymatic substrates. No single saprobic fungus collected in this study is thought to be specific to any one grass, however, certain fungi tended to reoccur on single grass species, but not on adjacent grasses. A ‘core fungal group’ was commonly associated with the decaying grasses and this ‘core’ was thought to be important in nutrient cycling in the grasses. A comparison of the fungi occurring on grasses with those on other monocotyledonous hosts is made. The numbers of fungi known to occur on Juncus roemerianus and Phragmites australis are briefly summarised.

Net blotch caused by Pyrenophora teres is a serious disease of barley in many cereal production areas world-wide, including the Western Cape province of South Africa. The pathogen occurs as two forms, P. teres f. teres, which produces net-blotch symptoms, and P. teres f. maculata which produces leaf spots. Pyrenophora japonica and P. hordei, which have also been reported in South Africa, also produce spots on susceptible barley cultivars. Using RAPD markers, spot-forming isolates from the South African population were found to be relatively uniform. Single ascospores were obtained from pseudothecia after in vitro mating had occurred between a verified P. teres net-blotch isolate from Denmark and a representative Pyrenophora leaf spot isolate from South Africa. Using amplified fragment length polymorphism (AFLP) and RAPD markers, recombination was demonstrated in the progeny which had DNA banding patterns different from the two parental isolates. Pathogenicity trials also confirmed that recombination had taken place during mating. Inoculations were conducted on differential cultivars susceptible to the net-blotch or leaf spot forms. The two parents induced typical net-blotch or leaf spot symptoms whereas the progeny mostly induced a jagged spot symptom on each cultivar. Fungicide sensitivity tests using the ergosterol biosynthesis inhibitors triademinol, bromuconazole and triticonazole showed that, due to recombination, some progeny could have increased resistance to these fungicides. Due to mating and subsequent recombination between a net blotch isolate of P. teres and a representative leaf spot isolate, it was concluded that the latter was P. teres f. maculata. These results contrast with the earlier belief that Pyrenophora leaf spot isolates in the Western Cape are P. japonica and P. hordei.

In the present study we investigated the abundance and spatial distribution of dark septate root endophytes (DSE) in a 3×3 m plot in a spruce stand (Picea abies). A total of 144 DSE isolates were obtained by means of a hierarchical sampling design. Most roots were colonised, as DSE were isolated from 81.7% of root segments. ISSR–PCR fingerprinting was used to identify 21 unique ISSR types. Dominant types were isolated from adjacent points that covered an area of up to 6.8 m2 of the study plot, and ISSR types were intermingled extensively. Frequency of isolation of the different ISSR types was uneven with two dominant types that accounted for 38% and 28% of all DSE isolates, respectively. Seven DSE strains representing six different ISSR types were identified as Phialocephala fortinii based on the morphology of fertile conidiophores and/or ITS 1 and 2 sequence comparisons.

PCR-based methods to detect Verticillium chlamydosporium on infected plant roots were developed. Arbitrary ERIC primers and those based on rRNA genes, to identify fungi grown in pure culture, were unsuitable for DNA extracted from nematode-infested roots, because of interference by plant and nematode DNA. A novel method utilizing specific primers designed from an amplified and cloned fragment of the V. chlamydosporium β-tubulin gene was developed. Although it could not discriminate between different isolates of V. chlamydosporium, one primer set could identify the fungus on tomato roots infested with root-knot nematodes. The V. chlamydosporium β-tubulin sequence data showed close homology to sequences from plant endophytic Acremonium and Epichloë species and the saprotrophic Trichoderma viride.

Verticillium fungicola, a severe mycopathogen of the cultivated mushroom Agaricus bisporus, was successfully transformed using both PEG-mediated and Agrobacterium-mediated techniques. PEG-mediated co-transformation was successful with hygromycin B resistance (hph), uidA (β-glucuronidase GUS), and green fluorescent protein (GFP) genes. Agrobacterium-mediated transformation was successful with the hph gene. Transformation frequencies of up to 102 transformants per μg DNA and 4068 transformants per 105 conidia were obtained for PEG-mediated and Agrobacterium-mediated transformation respectively. Expression of integrated genes in co-transformants was stable after 18 months of successive sub-culturing on non-selective medium, and following storage at −80 °C in glycerol. Molecular analysis of PEG-mediated transformants showed integration of the transforming genes into the target genome. Molecular analysis of Agrobacterium-mediated transformants showed integration of transforming DNA as single copies within the target genome. Co-transformants exhibited symptoms of disease in inoculation experiments and were at least as virulent as the wild-type fungus. GFP and GUS expression were observed in-vivo with the GFP-tagged strain showing great potential as a tool in epidemiological and host-pathogen interaction studies. The development of transformation systems for V. fungicola will allow in-depth molecular studies of the interaction of this organism with A. bisporus.

About 15000 species of ascomycete fungi lack a known sexual state. For fungi with asexual states in the anamorph genera Embellisia, Ulocladium, and Alternaria, six species have known sexual states but more than 50 species do not. In sexual filamentous ascomycetes, opposite mating type information at the MAT1 locus regulates mating and the opposite mating type genes each have a clonal, non-recombining phylogenetic history. We used PCR to amplify and sequence fragments of the opposite mating type genes from three supposedly asexual species, A. brassicae, A. brassicicola and A. tenuissima. Each haploid fungal isolate had just one mating type, but both mating types were present in all the three species. We sequenced the ribosomal ITS regions for isolates of opposite mating type, for the three asexual species and four known related sexual species. In a phylogenetic analysis including other ITS sequences from GenBank®, the three asexual species were not closely related to any of the known sexual species. Isolates of opposite mating type but the same species had identical ITS sequences. During any period of asexual evolutionary history, lineages of each mating type would have had a separate evolutionary history and any ITS substitutions shared between isolates of opposite mating type would have had to accumulate by convergence. Allowing for varying substitution rates and assuming a Poisson distribution of substitutions, the probability that isolates of opposite mating type shared an ITS substitution through convergence was low. This suggests that isolates of opposite mating type of A. brassicae, A. brassicicola and A. tenuissima were exchanging substitutions through sexual or parasexual reproduction while the ITS was evolving. If sexuality was lost, it was lost after the period of evolutionary history represented by the shared substitutions.

Leaf-cutting ants finely fragment the leaf material that they bring to the nest and place faecal droplets on this substrate before incorporating it in the upper part of the fungus-garden. The faecal droplets contain enzymes of which some have been shown to be of fungal origin. Here we explicitly address the enzymatic activity of faecal droplets in the leaf-cutting ants Acromyrmex echinatior and Atta colombica. We used isoelectric focusing and specific staining to show that faecal droplets of both species contain carboxymethylcellulases, laccases, proteases, and pectinases (both pectin esterases and pectin lysases) and we demonstrate that these enzymes originate from the symbiotic fungus and not from the ants themselves. The level of activity of fungal pectin lyase in faecal droplets indicates that fungal enzymes may be protected and possibly concentrated during their passage through the ant gut. This would imply that enzymes that are transferred by ants from mature parts of the fungus gardens may play an important role for the colonization of new substrate by new mycelium of the same fungal clone.

In a continuation of studies started by de Ruiter et al. (1993), all known species of the Mortierella isabellina-group (Micromucor/Umbelopsis clade of O'Donnell et al. 2001) and a few other Mucorales and species of Mortierella were investigated by RFLP (including ITS1, 5.8S, ITS 2 and the 5′ end of the large subunit rDNA gene) and ITS1 sequence analyses. This monophyletic group is unrelated to Mortierella and is only distantly related to the core group of the Mucoraceae. M. longicollis falls outside the Umbelopsis clade. Molecular data resolved two subclades within the M. isabellina-group; however, they are not correlated with any differences in sporangial wall and shape, spore pigmentation and shape, or sporangiophore branching. Therefore we subsume all taxa in one genus, Umbelopsis. The new family Umbelopsidaceae and the new combinations U. isabellina, U. ramanniana, and U. autotrophica are proposed.

Macrophomina phaseolina, the causal agent of charcoal root or collar rot, is an important plant pathogen especially in soybean and cotton. Single primers of simple sequence repeats (SSR) or microsatellite markers have been used for the characterization of genetic variability of different populations of M. phaseolina obtained from soybean and cotton grown in India and the USA. Genetic similarity between isolates was calculated, and cluster analysis was used to generate a dendrogram showing relationships between isolates collected from the two hosts. Forty isolates could be clustered into three major groups corresponding to their hosts and geographical region. The wide distribution of microsatellites in M. phaseolina genome was assessed by agarose gel electrophoresis of the PCR products generated by direct amplification of inter SSR regions DNA. This is the first report of the use of microsatellite markers to characterize the charcoal root rot pathogen. The SSR fingerprints (0.25–3.5 kb) generated using DNA from different populations of M. phaseolina of two hosts indicated that these repeats are interspersed within the genome of this pathogen. The variability found within closely related isolates of M. phaseolina indicated that such microsatellites are useful in population studies and represents a step towards identification of potential isolate diagnostic markers specific to soybean and cotton.

Genetic variation within Gremmeniella abietina var. abietina was studied using the Random Amplified Microsatellite-technique (RAMS). Ninety-three isolates were investigated, originating mostly from Finland, but also from Canada, U.S.A., Japan, Norway, Italy, Iceland and Sweden. Four banding pattern types were observed corresponding to the present division of this species in Asian, North American and two types of European races. Furthermore, an additional banding pattern was also observed. Intraracial variation was observed within all races, the North American one being the most polymorphic. Isolates of the large tree type (LTT) Gremmeniella from North America, Italy and Iceland contained RAMS alleles not observed in Finland, Sweden or Norway. Therefore, the isolates of LTT Gremmeniella should not be transported even within the area of its natural occurrence.

Two types of genetic markers, vegetative compatibility and RAPD, were evaluated for their ability to assess genetic variation among 38 isolates of the entomopathogenic fungus, Paecilomyces fumosoroseus. Both markers revealed significant polymorphisms. Nitrate non-utilizing mutants were generated from chlorate-containing media and used as forcing markers to observe heterokaryon formation and determine vegetative compatibility. Nineteen separate vegetative compatibility groups (VCGs) were identified, only seven having at least two members and 12 represented by single isolates. No correlation was observed between VCG and either insect host or geographic origin. Nine RAPD primers produced 365 unique fragments and could distinguish three different phenetic groups. RAPD profiles conclusively showed that strains within some VCGs were genetically unrelated, indicating that these strains were not clonal in origin. The large number of VCGs suggests that formation of heterokaryons and possible genetic exchange are uncommon. DNA fingerprinting with RAPD-PCR provides high resolution of genotype distribution and could be useful in tracking the fate of biocontrol agents released into the environment.

The effect of hydroxyl radical scavengers dimethyl sulphoxide, p-nitrosodimethylaniline, ethanol, benzoate, salicylate and thiourea was studied on sclerotial differentiation and growth of Sclerotinia sclerotiorum and Rhizoctonia solani. There is a correlation between both scavenger concentration and scavenger-hydroxyl radical reaction rate with delay and inhibition of differentiation at growth-noninhibiting scavenger concentrations. Growth-inhibiting scavenger concentrations further increased delay and inhibition of differentiation, and eventually stopped fungal growth, acting as antifungal antioxidant alternatives to traditional fungicides. p-nitrosodimethylaniline (with the highest hydroxyl radical reaction rate) was the most effective inhibitor of sclerotial differentiation and growth. The results strongly support the theory that oxygen free radicals induce differentiation in sclerotium-producing phytopathogenic fungi.

This paper synthesizes the work done on decolourization of bleach plant effluent and synthetic dyes using fungi from marine habitats. Three fungi obtained from marine habitats removed up to 60–95% of Kraft paper mill bleach plant effluent colour. One of the cultures could bring about decolourization of bleach plant effluent at an alkaline pH of 8.2. All the three fungi produced the lignin-modifying enzymes laccase and manganese-dependent peroxidase (MNP) and one of the cultures, Flavodon flavus (isolate 312), also produced lignin peroxidase (LIP) when grown in low nitrogen medium. Production of these enzymes increased in the presence of paper mill effluent in all the three isolates. Isolate Flavodon flavus (312) decolourized synthetic dyes like Azure B, Brilliant green, Congo red, Crystal violet, Poly-R, Poly-B and Remazol Brilliant Blue R in low nitrogen medium prepared with distilled as well as with artificial sea water. Decolourization of dyes was also observed in a rich nutrient medium like malt extract broth. About 60–90% of decolourization was achieved in these media within 3–10 days. Visible spectra of most of the dyes incubated with this culture indicated degradation at the end of the experiment. Manganese-dependent peroxidase enzyme was always produced in cultures of 312 containing dyes and in some cases, a direct correlation existed between % decolourization and MNP activity, suggesting its involvement in this process. This culture with its capacity to degrade paper mill effluent and synthetic dyes appears to be a potential candidate for bioremediation of coloured industrial effluents.

Fusarium equiseti and F. pallidoroseum are frequently reported as secondary colonizers of plant tissues. In this study they were isolated from the embryos of weathered cottonseed. Most isolates tested produced equisetin, an antibiotic, when grown on potato dextrose agar, rice, surface-sterilized cottonseed, or autoclaved cottonseed. This is the first report of equisetin from F. pallidoroseum. Equisetin was extracted from cultures of F. equiseti and F. pallidoroseum with acetone and dichloromethane, and partially purified by TLC. Two epimers of equisetin, designated as EQ and epi-EQ, were separated by HPLC. EQ or epi-EQ at 2·5–10 μg ml−1 suppressed germination or inhibited growth of various monocotyledonous and dicotyledonous seed, when the seed were incubated at 30°C under aqueous shake conditions. The two epimers also inhibited the growth of young seedlings and caused necrotic lesions on the roots, cotyledons, and coleoptiles of tested plant seedlings. The results suggest that equisetin may be a pathogenic factor of F. equiseti and F. pallidoroseum on seed and seedling health of cotton and other plants.

Rust fungi cause economically important diseases of cereals, and their ability to rapidly evolve new virulent races has hindered attempts to control them by genetic resistance. PCR-based molecular tools may assist in understanding the genetic structure of pathogen populations. The high multiplex DNA fingerprinting techniques, amplified fragment length polymorphisms (AFLP), selectively amplified microsatellites (SAM) and sequence-specific amplification polymorphisms (S-SAP) were assessed for their potential in investigations of the genetic relationships among isolates of the wheat rust pathogens, Puccinia graminis f. sp. tritici (Pgt), Puccinia triticina (Pt), and P. striiformis f. sp. tritici (Pst), the oat stem rust pathogen P. graminis f. sp. avenae (Pga), and a putative new P. striiformis special form tentatively designated Barley grass yellow rust (Bgyr). Marker information content, as indicated by the number of species-specific fragments, polymorphic fragments among pathotypes, percentage of polymorphic loci, and the marker index, was highest for the SAM assay, followed by the AFLP and S-SAP assays. UPGMA analysis revealed that all marker types efficiently discriminated the five different taxa and Mantel tests revealed significant correlations between the marker types. Within pathogen groups, the marker types differed in the amount of variation detected among isolates; however, the major differences were consistent and polymorphism was generally low. This was reflected by the AMOVA analysis that significantly partitioned 90% of the genetic variation between taxa. Of the three marker types, SAMS were the most informative, and have the potential for the development of locus-specific microsatellites.

Phenotypic and genotypic variability of 18 endophytic Guignardia strains from different host plants (Anacardiaceae: Anacardium giganteum, Myracrodruon urundeuva, Spondias mombin; Apocynaceae: Aspidosperma polyneuron; Ericaceae: Rhododendron sp.; Fabaceae: Bowdichia nitida; Leguminosae: Cassia occidentalis; Rutaceae: Citrus aurantium) growing in different sites in Brazil was assessed by means of morphometric measurements and inter-single-sequence-repeat-anchored polymerase chain reaction (ISSR-PCR) amplifications of the DNA. Morphology of conidia and ascospores and growth rates of the Brazilian isolates corresponded well with those of G. mangiferae. Multiple correspondence analysis (MCA) of the ISSR-PCR data yielded three groups of strains, which did, however, not correspond either to the host or to the geographic origin. The same individual tree hosted genotypically different strains indicating multiple infections. Phylogeny based on ITS1-5.8S-ITS2 sequence analyses confirmed conspecificity of the Brazilian isolates with G. mangiferae (syn. G. endophyllicola).

We investigated the influence of changes in external phosphorus (P) concentration on the proportion of phosphatase-active structures of the arbuscular mycorrhizal fungus Gigaspora margarita associated with Allium cepa. The P treatment was started when mycorrhizal colonisation had been established, and plant systems were harvested three times after the start of the P treatment. Higher shoot dry weights and P contents were observed in the high-P treated plants at the last harvest. We did not find any change in the proportion of phosphatase-active extraradical mycelium following P treatment. However, the proportion of alkaline phosphatase-active mycelium was positively correlated for extraradical and intraradical mycelium. Also, the proportion of alkaline phosphatase-active arbuscules seemed to increase with the shoot fresh weight, whereas the proportion of acid phosphatase-active arbuscules decreased with higher shoot P concentration and dry weight. We have shown experimentally that the intraradical mycelium of G. margarita, but not the extraradical mycelium, responds metabolically to plant P concentration, and possibly also to external P availability.