The aim of this study was to clarify the reproductive mode in the wood-rotting fungus Phellinus nigrolimitatus, using molecular markers and pairing experiments of cultural isolates. A 511 bp segment of the translation elongation factor 1a (EF1a) gene and the internal and intergenic transcribed spacer sequences (ITS and IGS) of the nuclear ribosomal DNA (nrDNA), were amplified by PCR from somatic cultures derived directly from basidiomes and single spore cultures of the fungus. Sequence analyses of the partial EF1a sequence (511 bp) and ITS (732 bp) of single spore cultures gave five and fourteen variable sites, respectively. Five somatic isolates yielded double ITS sequences, which suggested the existence of different ITS types within the same isolate. Restriction digestion of the partial EF1a and the complete ITS and IGS (3·4±0·2 kbp) sequences with HinfI and HaeIII gave restriction fragment length polymorphisms (RFLPs) among the isolates. The sum of fragments exceeded the lengths of the initial PCR product in several somatic isolates, which indicated the existence of EF1a and nrDNA heterotypes within isolates. Segregation of ITS and IGS RFLPs in single spore isolates suggested heterokaryosis in somatic isolates of P. nigrolimitatus. The polymorphic EF1a and ITS restriction sites were used as codominant markers in a test for Hardy–Weinberg expectations. The molecular data suggests a predominant outcrossing reproductive mode in P. nigrolimitatus. Results from cultural pairing experiments with single spore isolates corresponded poorly with the molecular data, suggesting that laboratory pairing experiments do not necessarily provide reliable information of the mode of reproduction in natural populations of fungi.

Inter-simple sequence repeat (ISSR) markers were used to investigate genetic diversity among 39 isolates of Beauveria spp. (36 B. bassiana, one B. brongniartii, one B. amorpha, and one B. velata) isolated from different insect hosts and geographical origins. Eighteen among 33 primers that contained different simple sequence repeats (SSR) were chosen for their reproducibility and high polymorphism. Considerable intraspecific and interspecific variability among the isolates of Beauveria spp. was revealed. One hundred sixty-eight highly reproducible fragments were amplified in all 39 isolates with an average of 9.3 markers per primer; among these, 161 (95.8%) were polymorphic. For 36 B. bassiana isolates, 8.9 (1–13) markers per primer were scored, and a total of 123 fragments were amplified, in which 102 (82.9%) were polymorphic. Among the 168 polymorphic bands, 7 bands were considered to be specific for B. brongniartii isolate Bbr06, 14 bands for B. amorpha isolate Ba08, and 18 bands for B. velata isolate Bv01. Within 36 B. bassiana isolates, genetic similarity ranged from 0.651 to 0.972. However, the genetic similarity values among different Beauveria species ranged from 0.411 to 0.720, suggesting that ISSR technique was successful in differentiating the three closely related species from B. bassiana. The results also indicated that there was a certain association between B. bassiana isolates and their geographical origins, but no clear correlation between those isolates and their insect hosts. The present study suggested that ISSR markers can be used as robust molecular markers for the population genetics, epidemiological and ecological studies of entomopathogenic fungi.

The ability of six fungi to solubilize natural-occurring gypsum was tested in vitro. The solubilization process was monitored by the production of a clear zone (halo) around or underneath the growing colony on Czapek–Dox agar containing 0·5% (w/v) gypsum (CaSO4.2H2O). Aspergillus niger, Penicillium bilaii, P. simplicissimum and Paxillus involutus displayed differential solubilization activities depending on the supplemented nitrogen source. Colonies grown on nitrate-containing medium showed the ability to solubilize gypsum, but when ammonium (at equivalent nitrogen) was used there was a significant reduction in solubilization. It was found that liquid cultures of nitrate-grown fungi produced substantial amounts of oxalic acid, whereas in ammonium-containing medium oxalic acid was only detected in small amounts. The production of citric and gluconic acid under these experimental conditions was low in both media, although the involvement of citric acid in gypsum solubilization is possible. Coriolus versicolor and Phanaerochaete chrysosporium did not exhibit any solubilization activity in nitrate- or ammonium-containing medium. Additionally these two fungi excreted small quantities of oxalic acid in both media with no citric acid being produced in liquid medium. Concomitant with the solubilization process and inside the clear solubilized zone, A. niger, Pax. involutus and P. bilaii produced crystals of differing shapes and abundance depending on the fungal strain. No crystals were produced by P. simplicissimum. These crystals were identified as calcium oxalate based on HPLC analysis and energy-dispersive X-ray microanalysis. Abundance of these crystals was found to be correlated with both oxalic acid production and the acidity of the medium. It is concluded that gypsum solubilization was predominantly achieved by both oxalic acid, which was accompanied by formation of calcium oxalate crystals, and citric acid production rather than the acidity of the medium. The results are discussed in relation to the significance of such an activity in agricultural applications, e.g. land reclamation, as well as the possible roles played by these fungi in mineral cycling.

The diversity and host specificity were studied of a collection of Colletotrichum strains derived from endophytic colonies in leaves of 12 tree species in the Iwokrama Forest Reserve, Guyana. Analysis included ISSR-PCR and RAPD molecular fingerprinting techniques, rDNA ITS sequencing and morphological and cultural characterization. Most strains belonged to one of two species, C. gloeosporioides and a further taxon which is probably referable to C. boninense. Almost no strains were found to be genetically identical, indicating that clonal reproduction does not play a prominent role. No degree of host specificity could be detected even at molecular fingerprint level. The implications for estimation of fungal diversity in closed tropical forests may be profound.

Black mould lesions were caused by Alternaria alternata in 76% of 228 tomato fruit with characteristic sunken black lesions collected from fields of processing tomatoes in California. Analysis of 29 RAPD primers revealed a high level of genetic diversity among the 69 isolates tested. Two major phenetic groups (Group 1 with 55 isolates and Group 2 with 14) were identified independently by PCA and by UPGMA of Jaccard similarity coefficients. Only 34 of 137 RAPD markers were monomorphic for all isolates and the genetic similarity between the two groups was 50%. Co-infection of black mould lesions by genetically distinct strains of A. alternata occurred in two of 10 isolates tested. There was no evidence for geographic clustering of isolates with high levels of genetic similarity, suggesting that isolates are widely dispersed across California. Only one isolate was identified which also caused stem canker disease on a susceptible tomato cv., suggesting that these strains play a minor role in causing black mould on processing tomatoes in California. This isolate and two other known stem canker isolates were clustered together with 11 other isolates in Group 2. Group 2-specific bands were also identified in a survey of seven additional isolates known to produce host-specific toxins.

Phylogenetic relationships among Phytophthora species were investigated by sequence analysis of the internal transcribed spacer region I of the ribosomal DNA repeat unit. The extensive collection of isolates included taxa from all six morphological groups recognized by Waterhouse (1963) including molecular groups previously identified using isozymes and mtDNA restriction fragment length polymorphisms. Similar to previous studies, the inferred relationships indicated that molecular groups of P. cryptogea/drechsleri-like and P. megasperma-like taxa are polyphyletic. Morphological groups V and VI, which are differentiated by the presence of amphigynous or paragynous antheridia, are not monophyletic: species of the two groups are interspersed in the tree. Species with papillate and semi-papillate sporangia (groups I–IV) clustered together and this cluster was distinct from those of species with non-papillate sporangia. There was no congruence between the mode of antheridial attachment, sporangial caducity, or homo- or heterothallic habit and the molecular grouping of the species. Our study provides evidence that the antheridial position together with homo- or heterothallic habit does not reflect phylogenetic relationships within Phytophthora. Consequently, confirming studies done previously (Cooke & Duncan 1997), this study provides evidence that the morphological characters used in Phytophthora taxonomy are of limited value for deducing phylogenetic relationships, because they exhibit convergent evolution.

Maple, linden and oak leaves were immersed in a stream for 1–21 d. Cumulative mass loss, ergosterol content, and species richness of released aquatic hyphomycete conidia increased with time. Numbers and richness of attached conidia were highest on days 1 and 2. Denaturing gradient gel electrophoresis revealed up to seven fungal phylotypes on the leaves before their immersion in the stream and after one day of stream exposure. After 5 d of immersion the contribution of these terrestrial fungi decreased and that of aquatic hyphomycetes increased. The dominant phylotypes belonged to Anguillispora filiformis, Articulospora tetracladia and Flagellospora curvula, which also dominated the community of released spores. The molecular diversity was highest on day 2 and 3 on all substrates. This may be due to a few species of terrestrial fungi, later outcompeted by aquatic hyphomycetes, and to many different conidia of aquatic hyphomycetes, some of which may germinate but are unable to establish themselves and reproduce.

The genetic relationships between several morphological groups recognized within Colletotrichum acutatum sensu lato were investigated using RAPD analysis and vegetative compatibility analysis. Isolates were examined from fruit rots originating in New Zealand and Australia, from Lupinus spp. in New Zealand, Canada, France and the U.K., and from Pinus radiata in New Zealand and Australia. The genetic distinctness of the groups recognized morphologically is confirmed. Two genetically distinct groups of C. acutatum-like pathogens are recognized from lupin, one comprising isolates from New Zealand and the U.K., the other isolates from Canada and France. C. acutatum f. sp. pineum isolates from New Zealand and Australia form two genetically distinct groups.

Conidia of Metarhizium anisopliae need water activity (Aw)>0·98 for germination irrespective of whether they are formulated in an aqueous or oil carrier. The presence of nutrients in the carriers or culture medium accelerated germination but did not increase the range of Aw (or humidities) over which conidia could germinate. Insect extract stimulated more conidia to germinate than yeast extract. Oils appeared to extract fungistatic and stimulatory compounds from the insect cuticle. The ratio of these compounds depends on both the insect species and the carrier. Conidia of M. anisopliae formulated in oils were observed with SEM to flow over the surface of insect and plant cuticles. The conidia were probably deposited at sites which are conducive for germination and infection and consequently increased the overall mortality of insects. In contrast, aqueous formulations of conidia remain as drops on the leaf surface immediately after application with a pipetteman or sprayer.

The relationship between teleomorphs of Cordyceps spp. and their presumed anamorphs have been investigated by analysis of 5.8S and ITS rDNA sequences. The morphological and sequence data confirm that Paecilomyceshawkesii is the anamorph of Cordyceps gunnii, while Cordyceps hawkesii is a synonym of C. gunnii, and P. gunnii is a synonym of P. hawkesii. The following presumed connections are also confirmed: Beauveria brongniartii is the anamorph of C. brongniartii, Metarhizium anisopliae var. majus is the anamorph of C. brittlebankisoides, Beauveria sobolifera is the anamorph of C. sobolifera, Mariannaea pruinosa is the anamorph of C. pruinosa, Paecilomyces militaris is the anamorph of C. militaris, and Hirsutellasinensis is the anamorph of C. sinensis. The other isolates sequenced are unlikely to be anamorphs of the teleomorphs from which they were isolated because the sequences from the culture and the teleomorph are quite different. 5.8S and ITS sequences provide useful information for establishing anamorph–teleomorph connections and assisting in the delimitation of species within Cordyceps.

Wheat bran, cassava bagasse and sugar cane bagasse were shown to be adequate substrates for the growth and aroma production by the mould Ceratocystis fimbriata. Among the nutritive media tested, sugar cane bagasse complemented with a synthetic medium containing glucose (200 g l−1) gave a fruity aroma while the leucine or valine-containing medium gave a strong banana aroma. Aroma production was dependent on growth and the maximum aroma intensity was detected at about the time of the maximum respirometric activity. Twenty-four compounds have been separated by GC headspace analysis and 20 were identified, among them: 1 aldehyde, 7 alcohols, 4 ketones and 8 esters. It was clearly demonstrated that the chromatographic profile of the headspace of the culture was dependent on the substrate used and on the eventual precursor added. When leucine or valine was added to the substrate, the production of total volatiles in the headspace reached values up to tenfold higher than that for ripe bananas. The Gompertz model, a logistic-like equation, was used to fit the integrated CO2 and volatiles production data.

Rhizophlyctis rosea was found in 44% of 59 soil samples from national parks, urban reserves and gardens, and agricultural lands of eastern New South Wales, Australia. As some of the soils are periodically dry and hot, we examined possible mechanisms that enable survival in stressful environments such as agricultural lands. Air-dried thalli of R. rosea in soil and pure cultures of R. rosea, two isolates of Allomyces anomalus, one isolate of Catenaria sp., one of Catenophlyctis sp. and one of Spizellomyces sp. recovered following incubation at 90 °C for two days. Powellomyces sp. recovered following incubation at 80 °. Sporangia of all seven fungi shrank during air-drying, and immediately returned to turgidity when rehydrated. Some sporangia of R. rosea released zoospores immediately upon rehydration. These data indicate that some Chytridiomycota have resistant structures that enable survival through periodic drying and high summer temperatures typical of soils used for cropping. Eleven Chytridiomycota isolated from soil did not survive either drying or heat. Neither habitat of the fungus nor morphological type correlated with the capacity to tolerate drying and heat.

Spatial characteristics of Suillus variegatus populations, including the size, distribution and number of genets, were measured in four naturally regenerated stands of Scots pine, Pinus sylvestris in Sweden that were more than 100 years old. In the oldest forest, a continuous tree-layer had been present at least since the last forest fire in 1647. Genets were identified based on somatic incompatibility reactions performed on mycelial cultures from sporocarps. In total, 38 genets were identified from 120 tested sporocarps. The maximal extension of a genet, as reflected by its outermost sporocarps, was 27 m in the oldest stand and ranged between 10 and 17 m in the other stands. On average, genet size was 20 m in the oldest stand and 10 m in the other stands. Other genets were not found within the domains of established genets. The closest detected distance between genets was 1·5 m, and the average distance was 4 m. The number of genets ranged between 56 and 74 ha−1 at the studied sites. Possible reasons for the high degree of resemblance in characteristics in old forests between S. variegatus, the dominant bolete in mature forests, and S. bovinus when present, more a characteristic for younger forests and only scarcely occurring in older forests, are discussed.

Alternaria core rot of red apple cultivars is a serious post-harvest disease in South Africa. Thirty isolates of Alternaria spp. previously isolated from apple, together with reference isolates of A. alternata and A. infectoria, were characterised and grouped according to their sporulation patterns and conidial morphology. Isolates were identified as belonging to A. arborescens, A. infectoria and A. tenuissima species-groups. The isolates were also analysed for production of mycotoxins and other secondary metabolites and their cultural characteristics on DRYES medium were recorded. Apple fruit were artificially inoculated with the thirty-two Alternaria isolates and the resulting lesion types were recorded. A data matrix was constructed using all these characters and subjected to cluster analysis to show the similarity between different isolates. Isolates classified as A. infectoria species-group based on sporulation patterns, cultural and biochemical data could be easily differentiated from isolates classified as A. arborescens and A. tenuissima species-groups, which clustered close together. Isolates were further subjected to DNA sequence analysis of the internal transcribed spacers (ITS) 1 and 2 of the nuclear rRNA gene. A phylogeny estimated from the ITS data set delineated two clades, one being typified by the A. infectoria species-group, and the other representing both A. tenuissima and A. arborescens species-groups. The ITS data set clearly separated isolates of A. infectoria species-group from the other species-groups, as all isolates had a distinction of 35 base pair insertions and 6 base pair deletions in the ITS regions. The results obtained in the present study showed that the major pathogens associated with core rot disease of Top Red apples in South Africa belong to the A. tenuissima species-group.

Parental and progeny isolates of the endophytic fungus Epichloë festucae were tested for compatibility with five grass species: Lolium perenne, Festuca arundinacea, F. longifolia, F. pratensis, and F. rubra subsp. rubra. One parental and some progeny isolates adversely affected the growth of plants, causing increased mortality of inoculated seedlings, stunted growth and chlorotic leaf symptoms. Growth of hyphae from surface-sterilized leaf sheaths and blades was more vigorous and concentrated from stunted than from symptomless plants. In addition, the growth of isolates was influenced by the host species, whereby estimates of hyphal concentration were consistently highest in meadow fescue associations and lowest in tall fescue associations. Light microscopic and transmission electron microscopic examination of stunted plants indicated no changes in host cells. Intercellular hyphae were observed within vascular bundles of leaf blades and sheaths of many of the endophyte–grass associations. The frequency of infected vascular bundles and the concentration of hyphae within them was highest in plants infected with the stunting parental isolate and lowest in plants infected with the symptomless parental isolate. Hyphae within vascular bundles were typically in close contact with sieve-tube elements and appeared to be functioning as powerful sinks, diverting assimilates and restricting growth.

The fungus Pochonia chlamydosporia is a biocontrol agent with commercial potential for root knot and cyst nematodes. It produces an alkaline serine protease, VCP1, during infection of nematode eggs. The gene encoding VCP1 was sequenced and the sequences of cDNAs from six isolates from different nematode hosts were compared. The gene encoding VCP1 was similar to PR1 from Metarhizium anisopliae with similar regulatory elements. Comparison of translated cDNA sequences revealed two amino acid polymorphisms at positions 65 and 99, indicating a difference between isolates from cyst and root nematodes. The positions and nature of the polymorphisms indicated that the two forms of VCP1 might have different properties and this was tested with five chromogenic polypeptide substrates. Enzyme assays revealed the two forms differed in their abilities to utilise Succ-Ala-Ala-Pro-Phe-pNa and Succ-Ala-Val-Pro-Phe-pNa, suggesting different amino acid affinities at the S3 binding region. This indicates host related genetic variation in VCP1 between isolates of P. chlamydosporia isolated from different nematode hosts, which might contribute to host preference. Such differences may be important in future exploitation of P. chlamydosporia as a nematode biocontrol agent.

Tropical xylariaceous taxa in the genera Biscognauxia, Hypoxylon and Xylaria were evaluated for their ability to produce wood-decay enzymes and effect mass loss and lignin solubilization in angiosperm and gymnosperm wood. All xylariaceous taxa were capable of cellulose and xylan hydrolysis, but few produced enzymes involved in lignin breakdown. Xylariaceous fungi were incapable of causing mass loss in gymnosperm wood, but several caused significant mass loss in angiosperm wood during a six month in vitro exposure. Mass loss values obtained were low at approx. 20% of those obtained for basidiomycetes. Lignin was solubilized at similar rates to mass loss by Hypoxylon and Xylaria species, resulting in indices of lignin solubilization between 0.88 and 1.11, which were similar to those obtained for white-rot basidiomycetes and higher than those previously reported for any other xylariaceous fungi.

Degenerate PCR primers were designed based on EF-1 alpha (EF-1α) gene sequences of several filamentous fungi retrieved from sequence databases. These primers were used to isolate a partial sequence, approximately 830 bp in length of the EF-1α from isolates of Microdochium nivale obtained from various geographic locations across the world. Two distinct groups of isolates were evident among those isolates examined. Sequence homology for comparisons within group was 99.7% for group A and 99.8% for group B. Primers specific to either group A or group B sequences were designed and tested on isolates from around the world. Comparisons were made with primers previously reported for the two varieties of M. nivale and revealed that Group A type isolates correlated with M. nivale var. majus and group B isolates with M. nivale var. nivale.

The primers from this study and those previously reported were in agreement for all isolates with the exception of one isolate (NRRL 3289) which failed to amplify with previously published M. nivale primers. Sequence analysis of NRRL 3289 suggested that it was an isolate of M. nivale var. nivale as indicated by the EF-1α based primers developed in this study. This study provides sequence based phylogenetic evidence of two species and when taken together with biological differences reported, leads to the recognition of M. majus comb. nov. (syn. Fusarium nivale var. majus). Descriptions of the two species are provided.

Conocybe lactea was examined as part of a larger study on the distribution of amatoxins and phallotoxins in fungi, and the taxonomic relationships between these fungi. As amatoxins are present in the congener C. filaris, the locally abundant C. lactea was examined using HPLC and mass spectroscopy. Amatoxins were not found in C. lactea, but the related phallotoxins were present in small quantities making it the first fungus outside of the genus Amanita in which phallotoxins have been detected. Despite the presence of a related toxin, C. lactea was found not to be taxonomically close to C. filaris. Phylogenetic analyses using nuclear ribosomal RNA genes indicated that North American specimens of C. lactea were conspecific with North American specimens of C. crispa in Conocybe sect. Candidae. European C. crispa was a distinct taxon. The implications of the use of the name C. albipes for these taxa are discussed. Nucleotide data confirmed placement of the sequestrate taxon Gastrocybe lateritia in sect. Candidae, but as a distinct taxon. It is hypothesized that the unique sequestrate morphology of G. lateritia may be caused by a bacterial infection.