This work aimed to evaluate the genetic diversity of Botrytis cinerea in Chile and to determine whether the two genetically different groups transposa and vacuma, described in France, are present in the country. Isolates collected in Chile from grapes, tomato, kiwifruit and blueberry were analysed using molecular markers. We developed a PCR test to identify the two groups of B. cinerea, transposa (with the transposable elements Boty and Flipper) and vacuma (with neither). As described in France, both kind of isolates were found to be present and sympatric in Chile. Isolates containing the transposable element Boty alone (boty isolates) were also detected. The frequencies of transposa, boty and vacuma isolates were significantly different on kiwifruit compared to grapes, tomato and blueberry. RAPD analysis revealed a high degree of genetic diversity, with no widespread clonal lineages. Dendrograms, analysis of molecular variance and Fst values revealed the existence of genetic differentiation in our sample between isolates from the different host plants. PCR-RFLP markers also showed that isolates sampled from grapes and tomato were genetically differentiated. Additional sampling is required to confirm these findings.

Seventy-one strains of fungi belonging to 53 species of Zygomycota and mitosporic fungi were isolated from soils containing different levels of 2,4,6-trinitrotoluene (TNT). Three moderately contaminated samples (500 ppm) and an uncontaminated control contained a rich diversity of species whereas few fungi were isolated from a heavily contaminated sample (1000 ppm). Most isolates were capable of biotransforming TNT into aminodinitrotoluenes and azoxy-dimers, although they differed widely in their tolerance of TNT. Spore germination was generally more strongly inhibited by TNT than hyphal growth. Among the Zygomycota, Absidia and Cunninghamella were highly TNT-tolerant and attacked the substrate even when this had precipitated at 1000 ppm, whereas 12 of the 14 Mortierella isolates were intolerant of TNT above 50 ppm. Among the mitosporic fungi, Acremonium, Cylindrocarpon, Gliocladium and Trichoderma but not Penicillium species were highly tolerant of TNT and had a high capacity to biotransform this compound. Large variations in these properties were determined between species and even strains belonging to the same species, and between different isolates from the same soil sample. Populations of soil microfungi therefore show a high natural variation of TNT tolerance and biotransformation ability, irrespective of any previous long-term exposure to this xenobiotic.

The ergocalciferol (vitamin D2) content of individual fruit bodies of the edible mushroom Cantharellus cibarius (chanterelle) was measured with HPLC 2–6 years after drying. The concentrations of ergocalciferol in different fruit bodies varied between 0·12 and 6·30 μg g DW−1, with a mean of 1·43 μg g DW−1. This result indicates that dried and stored chanterelles contain significant amounts of ergocalciferol. The huge range in concentration challenges the validity of food tables based on means from pooled samples. The range may reflect differences in exposure to sun light, as fruit bodies within the same cluster showed little variation. Our study indicates that mycelial cultivation for ergocalciferol production is an interesting prospect if strains and incubation conditions are selected carefully. No significant difference in ergocalciferol concentration between pigmented and albino forms was found.

The electrophoretic karyotype (EK) of 24 strains of Botryotinia fuckeliana was resolved to determine the extent of chromosomal polymorphism among them. Based on EK variation, nine different profiles with a number of bands ranging from 5 to 8 were found. Those profiles were not related to the origins of the strains, hosts, or year of isolation. Inheritance of chromosomal bands was studied by analyzing the EK of the progeny from crosses between sexually compatible B. fuckeliana strains that had different karyotype. The EKs of 44 monoascospore strains showed 12 new EK profiles, suggesting that chromosomal rearrangements generated after meiotic recombination provides EK variability in this fungus. Also, we report here that the degree of EK variability indicates that sexual reproduction under natural conditions might be quite important in the life cycle of B. fuckeliana. Hybridization analysis showed that, in most strains, the rDNA is located in both a chromosomal band of high molecular weight and different size depending on the strain and a band of low molecular weight, revealing evidence for chromosome rearrangements in this plant pathogen.

The dynamics of competitive colonisation of necrotic cyclamen tissue by the plant pathogenic fungus Botrytis cinerea and the saprophytic fungal antagonist Ulocladium atrum were studied immuno-histologically, while sporulation was studied macroscopically. The effect of different time intervals between inoculation of both fungi on resource capture by each species was explored. Colonisation and sporulation were used as indicators for competitive resource capture and the effectiveness of biological control of B. cinerea, using U. atrum. Mycelial biomass and sporulation showed logistical time courses in both species, in monocultures as well as in mixed cultures. Final colonisation and sporulation levels were lowered by competition, indicating competitive resource capture. Analysis of the extent to which sporulation of either fungus could be reduced by co-inoculation with the other fungus at different times, showed that B. cinerea can be completely excluded by ‘early’ pre-inoculation with U. atrum, but not vice versa, indicating that U. atrum can exploit resources in the leaf that are not accessible to B. cinerea. A model of competitive substrate colonisation and resource capture was developed on the basis of the experimental results. Model results confirm that competition for resources provides a sufficient biological explanation for the dynamic interactions between the fungi. The model provides a tool to optimise dose and timing of U. atrum applications providing effective biological control of B. cinerea.

The production of laccase, an enzyme of industrial interest, was screened among species of the genus Pycnoporus, in particular P. sanguineus. Strains were isolated from various tropical Chinese environments and phylogenetically compared to ones deposited in international collections. Molecular clustering, based on ribosomal ITS1-5.8S-ITS2 genomic sequence analysis, showed that the Chinese strains of P. sanguineus formed an homogeneous phylogenetic group distinguished by its laccase-overproducing character. The dikaryotic strain P. sanguineus G05 was selected for its ability to produce up to 40 000 U l−1 laccase in the presence of 2,5-xylidine, Tween 80 and maize bran. Since fruit bodies of P. sanguineus could be formed in the laboratory, monokaryotic laccase-hyperproducing strains were isolated using classic genetical methods. Among these isolates, strain G05.10 synthesized up to 71 000 U l−1 laccase, with a productivity of 5069 U l−1 d−1. The laccase was purified and identified as a 70 kDa protein with an acidic pI, and was very stable at high temperatures.

Polygalacturonase (PG) secretion was analysed from five independent isolates of Botrytis cinerea grown in minimal liquid media enriched with pectin as the only carbon source. To acquire information about the biochemical characteristics of the PGs secreted in vitro, we combined enzyme-activity assays with Western blot analysis and isoelectro-focusing (IEF). All isolates secreted at least four PGs isozymes with a similar electrophoretic patterns, easily distinguishable by western blots. IEF enabled the isoelectric point of the PGs for each isolate to be determined. Wide variability in isoelectric point was observed between the PGs of each isolate, ranging from above pH 9.0 to below pH 5.0. For one of the isolates, each PG secreted in vitro was further purified by chromatofocusing and analysed for endo/eso-polygalacturonase activity by thin layer chromatography. This combination of analytical techniques and enzyme assays provided a view of the pool of PGs produced by B. cinerea and defined the biochemical features of each isozyme.

The Claviceps purpurea population colonising British Spartina salt marsh stands is characterised by unusually long cylindrical conidia (average 9·5–12 μm) and sclerotia floating on the water surface. RAPD, AFLP and rDNA comparison defined these isolates as the third genetically distinct homogenous population (G3) of C. purpurea. The same morphological and genetical markers were found also in S. alterniflora isolates from Spartina from the USA. All G3 isolates belonged to a chemotype producing ergocristine and ergocryptine. In phylogenetic trees based on rDNA and AFLP, a G1 population from fields and meadows appeared as the sister clade to the one formed by G3 (Spartina) and G2 (wet and shady habitats), both with floating sclerotia and elongated conidia. British stands of S. anglica were probably colonised by isolates introduced from America, rather than by isolates from species of neighbouring biotopes.

Monilinia fructigena isolates from Japan were compared with isolates from Europe. General colony characteristics, stroma formation, growth rate and conidial dimensions were determined for six isolates each from both groups, as well as sporulation intensity on potato dextrose agar (PDA) and germ tube features. Potential differences in pathogenicity were tested on the pear cultivars ‘Conference’ and ‘Doyenné du Comice’, and on the apple cultivar ‘Cox's Orange Pippin’. A marked difference in stroma formation occurred, the area of stromatal plates ranged from 4·1 to 5·2 cm2 in the Japanese group, and from 0 to 0·9 cm2 in the European. The mean growth rate was significantly higher for Japanese isolates (t-test, P = 0·01). Length and width of conidia were significantly greater in European isolates (t-test, P = 0·01). Conidia measured on average 19×11·5 μm in European isolates, and 16×10 μm in Japanese ones when grown on cherry agar. On fruits, the difference in conidium size was even more pronounced. Sporulation intensity on PDA and germ tube features did not differ between both groups. No differences were found in latency period, lesion growth rate or sporulation intensity on apple and pear fruits between both groups. Together with previously published differences in the ITS region of ribosomal DNA, our results show that the Japanese isolates belong to a distinct species, Monilia polystroma sp. nov. A description of the anamorph is given, as well as a table summarising key features for all four brown rot associated Monilia species.

Ganoderma boninense was determined to be heterothallic and tetrapolar with multiple alleles at both mating type loci. Mycelial interactions amongst siblings were assessed to determine if closely related isolates of G. boninense exhibited vegetative incompatibility. Interactions amongst monokaryotic and dikaryotic mycelia generally grouped into four categories ranging from strong antagonism to the absence of antagonism. Pairing of dikaryotic mycelia always resulted in antagonism and the degree of antagonism generally correlated with the relatedness of the isolates. Genetically different dikaryons therefore constitute discrete individuals. This is the first report of such interactions in G. boninense and these findings provide the basis for further studies on natural populations of this species.

Economic mass production is a crucial stage in the development of biocontrol agents. This paper compares different solid substrates with and without preceding liquid culture for efficacy and economics in the scaled-up production of the mycoparasitic biocontrol agent Clonostachys rosea. In a basic liquid medium (BLM) containing (in g l−1): molasses (80), neopeptone (10) and yeast extract (2), Clonostachys rosea produced 107 conidia ml−1 after 4 d in agitated culture. Spore viability was 99% and did not differ from that of conidia derived from potato dextrose agar plates. Guata, an inert polyester fibre, coated with BLM and cassava starch (30 g l−1), was the best solid matrix yielding 109 conidia g−1 after 20 d. On a volume basis after extraction and on a production unit basis, Guata was in a similar range to Biodac®, a granular cellulose complex, which had also been enriched with BLM, producing up to 107 conidia ml−1 or over 1010 conidia per 23×28 cm polypropylene bag, the production unit in which all solid matrices were incubated. Rice was the lowest-yielding solid matrix regardless of quality. Chlamydospores were not observed in liquid or on solid media. All conidia produced on solid matrices had a viability >90%. Conidia produced on Biodac had a higher viability (95.6%) than spores produced on Guata (93.1%) after 24 h on potato-dextrose plates. However, production on Biodac was more variable and negatively affected by prefermentation in liquid culture, whereas production on Guata and rice was unaffected by the origin of the starting inoculum. Guata of medium thickness (1.27 cm) was superior to thicker (2.54 cm) or thinner (0.64 cm) sheets with respect to both yield per gram and per production unit. Although prefermentation neither improved yield nor reduced cost, it increased throughput by a factor five and may be advantageous where fermentor and/or incubator space is limited. Biodac and Guata were the most economical solid matrices; inoculum to treat one hectare (3×1012 conidia) cost US $ 27, whereas rice-based production cost US $ 613 under Costa Rican conditions. Liquid culture alone would be fast but too costly: US $ 1078 ha−1. Experiments to further reduce costs in the Guata system are suggested.

A saprophytic, lignin-decomposing basidiomycete fungal isolate (BB1), identified as a member of the russuloid lineage closest to the genus Peniophora, plays a role in soil aggregation and stabilization. In liquid media this fungus secretes large amounts of extracellular materials or mucilage that act as soil binding agents. We investigated the nature of these materials using periodate treatment and lectin cytochemistry, and studied whether or not these materials are involved in soil aggregation. Water stability of artificial fungal amended soil aggregates was destroyed by periodate treatment suggesting that polysaccharides produced by the basidiomycete were involved in soil aggregation. Binding patterns of fluorescently labeled lectins were also investigated to determine specific carbohydrate moieties present in the fungal mucilage. Fluorescently conjugated lectins (Ulex europaeus Agglutinin I and Lotus tetragonolobus lectin) bound to extracellular mucilage indicating that this basidiomycete mucilage contains fucosyl sugar residues. We also demonstrated that water stability of artificial soil aggregates prepared with fungal mycelia pretreated with L(−)fucose lectins were significantly reduced. This study indicates that fungal-derived material containing fucosyl residues plays a role in soil adhesion.

Eight species of ectomycorrhizal (ECM) fungi in the genera Amanita, Gymnoboletus, Lactarius, and Russula were isolated from subtropical plant communities in eastern Australia. Two species were isolated from each of rainforest, Nothofagus forest, Eucalyptus forest, and Eucalyptus dominated wallum (heath) forest. These communities differ strongly in their soluble soil nitrogen (N) composition. The ability of the fungi to use inorganic (nitrate, ammonium) and organic (amide, peptide, protein) nitrogen sources was determined. As the fungi did not grow in liquid culture, a ‘floating culture’ technique was devised that allows hyphal growth on a screen floating on liquid medium. With some exceptions, fungal biomass production in floating culture closely reflected fungal growth on solid media assessed by total colony glucosamine content. Most isolates grown in floating culture had similar glucosamine concentrations on all N sources, with isolate specific concentrations ranging from 6 to 12 μg glucosamine g−1DW. However, Russula spp. had up to 1·7-fold higher glucosamine concentrations when growing with glutamine or ammonium compared to nitrate, glutathione or protein. Floating cultures supplied with 0·5, 1·5, 4·5, or 10 mM N mostly produced greatest biomass with 4·5 mM N. In vitro nitrate reductase activity (NRA) ranged from very low (0·03 μmol NO2 g−1 fw h−1) in Russula sp. (wallum) to high (2·16 μmol NO2 g−1 fw h−1) in Gymnoboletus sp. (rainforest) and mirrored the fungi's ability to use nitrate as a N source. All Russula spp. (wallum, Nothofagus and Eucalyptus forests), Lactarius sp. (rainforest) and Amanita sp. (wallum) utilized ammonium and glutamine but had little ability to use other N sources. In contrast, Amanita species (Nothofagus and Eucalyptus forests) grew on all N sources but produced most biomass with ammonium and glutamine. Only Gymnoboletus sp. (rainforest) showed similar growth with nitrate and ammonium as N sources. Fungal N source use was not associated with taxonomic groups, but is discussed in the context of soil N sources in the different habitats.

The potential as biocontrol agents of the apple scab pathogen, Venturia inaequalis and the effect of environmental factors on growth, pycnidial production and spore germination of four isolates of Microsphaeropsis were examined on culture media and leaf discs. V. inaequalis ascospore production was reduced by 52, 77, 89 and 95% on leaf discs treated with the isolates IMI 294735, P176A, DAOM 198536 and P130A, respectively. For all isolates, optimum temperature for mycelial growth was 25 °C. More pycnidia were produced on culture media than on leaf discs. On culture media, optimum temperature was from 15 to 25 °, while on leaf discs it varied among isolates. Pycnidial production was inhibited by darkness and a minimum of 8 h light d−1 was required. Spores of isolates P130A and DAOM 198536 germinated at temperatures above 15 ° and at pH 4–5, as compared to 10 ° and pH 4–8 for isolates P176A and IMI 294735. The best candidates were P130A and DAOM 198536 and optimum conditions for growth, pycnidial production and spore germination were temperatures between 20 and 25 °, pH approx. 4 and light.

The genetic structure of Pyrenophora teres, the causal agent of net blotch of barley, was examined in two fields 30 km apart in the south-western Cape of South Africa. The two fields respectively represented a net- and spot-type population, the two types being distinguished on the basis of symptom expression on differentially susceptible cultivars. The number of isolates sampled from each field was 36 for the net-type population and 29 for the spot-type population. Samples were collected from infected barley leaves from two separate quadrants in each field, the two quadrants positioned in corners of the fields, diagonal to one another. Of the 40 10-mer random oligonucleotide primers screened, five produced scorable, reproducible DNA bands suitable for the determination of population structure. A total of 65 loci were produced of which 54 were polymorphic. Genetic analysis of bands produced by one of the primers has revealed single locus segregation in a mating between a net- and spot-type isolate, indicating that RAPD bands can be interpreted as alleles at genetic loci. Total gene diversities determined for all loci resulted in mean indices of 0.063 and 0.082 being obtained respectively for the net- and spot-type populations. Genetic diversity among the two populations was divided into within- (variation between sampling quadrants) and among population components using Nei's GST. A coefficient of genetic differentiation (GS) of 0.0149 was obtained between quadrants within populations while a coefficient (GT) of 0.63 was obtained between the two populations. Genotypic variation revealed 13 distinct multilocus genotypes (haplotypes) in the net-type population while there were 12 in the spot-type population. UPGMA cluster analysis of the two populations together with six progeny from a mating between a net- and spot-type isolate resulted in three main clusters being produced, one for each population and one for the progeny. One isolate collected from the net-type population that did not cluster with the other net-type isolates clustered directly next to the cluster containing the sexual progeny. This isolate also contained a unique spot-type DNA band. This suggested that sexual recombination may be occurring between net- and spot-type isolates under field conditions.

Recently, a powdery mildew anamorph infecting snowberry (Symphoricarpos albus) has been reported as a new plant pathogen in several European countries. We report here its occurrence in the UK, Germany and, for the first time, Switzerland. Based on morphological and scanning electron microscope (SEM) patterns, this novel pathogen appeared similar to the anamorph of a common North American powdery mildew, Erysiphe symphoricarpi (syn. Microsphaera symphoricarpi). A phylogenetic analysis of the internal transcribed spacer (ITS) sequences of the ribosomal DNA (rDNA) of an English and a North American snowberry powdery mildew fungus showed that they are conspecific. Ascomata of the pathogen were found only in one European collection, that being in Germany in 2002. The similarity in morphology of the ascomata also confirmed the co-identity of the European and American snowberry powdery mildews.

The root systems of Carex flacca and C. pilulifera, growing in the Burren (western Ireland), were shown to be colonized by Cortinarius (Dermocybe) cinnamomeus. This basidiomycete formed ectomycorrhiza-like structures, which possessed a distinct fungal mantle (85–100 μm thick), hyphal infection in epidermal cells, rhizomorphs and extramatrical hyphae, but lacked a Hartig net. These ectomycorrhiza-like structures were formed on first-order lateral roots but were distinct, morphologically and anatomically, from dauciform roots. Structures typical of arbuscular mycorrhizas were never observed. The colonization of root systems of C. flacca by C. cinnamomeus was confirmed by PCR/RFLPs and DNA sequencing of the ITS region of rDNA.

The thermophilic fungus Thermomyces lanuginosus was cultivated in shake-flasks for up to 120 h with low-molecular-weight dextran as carbon source. Maximal biomass was attained after 48 h of growth whereas extracellular α-amylase activity was highest in the stationary phase with maximum activity after 96 h of cultivation. A similar pattern was found for total extracellular protein. Pulse-labeling of proteins showed that α-amylase and other unidentified proteins were de novo synthesised in the stationary phase. Using specific primers with sequences from α-amylase from T. lanuginosus, RT-PCR analysis showed that α-amylase transcription did not start until late in the growth phase and reached a maximum more than 24 h after maximal biomass was obtained.

Six species of the genus Polyporus s. str. from which monosporic cultures were obtained, were studied to investigate their sexual compatibility. All species tested have an heterothallic multifactorial tetrapolar system. Results of intraspecific matings indicated a certain reproductive isolation of the Argentine and Brazilian collections of P. tenuiculus. Interspecific matings confirmed the separation of the species P. ciliatus and P. tucumanensis, as well as P. dictyopus and P. melanopus. Di-mon matings allowed the separation of P. tenuiculus from P. philippinensis.

We compared the degradative enzymology of fusaria in a small clade of closely related species as determined by phylogenetic analysis of nuclear 28S rDNA sequences. The clade includes entomogenous species and at least one recently discovered lichenicolous species. The lichenicolous Fusarium sp. and three entomogenous species were tested for the ability to degrade lichen tissue in both the presence and the absence of antibiotic secondary compounds. Only the undescribed lichenicolous Fusarium sp. showed degradative ability irrespective of lichen secondary chemistry and this was correlated with its ability to hydrolyse enzymatically the antibiotic depside, lecanoric acid. Similarly, the lichenicolous Fusarium sp. exhibited the greatest capacity for degrading lichen cell walls and the highest levels of cell wall-degrading polysaccharidases. These results correlate the ability to exploit lichen substrates with the production of specific enzyme activities able to overcome the chemical and physical barriers that characterize lichen hosts.