Commercial Eucalyptus plantations provide an important source of hardwood for forestry industries, worldwide. Several species of Mycosphaerella are associated with a destructive Eucalyptus leaf disease known as Mycosphaerella Leaf Blotch (MLB). During 2000, a survey was undertaken in several commercial Eucalyptus growing areas of South Africa to determine the identity of the Mycosphaerella spp. contributing to outbreaks of MLB. Symptomatic leaf samples were collected from three major Eucalyptus growing areas and the Mycosphaerella spp. were isolated. Isolates were identified using morphology, ascospore germination patterns and sequence data from the ribosomal DNA operon. Six species, namely M. ellipsoidea, M. irregulariramosa, M. juvenis, M. lateralis, M. marksii, M. nubilosa, as well as a new species, described here as M. fori sp. nov., were recognized. Mycosphaerella nubilosa was the most common species isolated from commercial plantations, particularly on E. nitens, and appears to be the dominant species contributing to MLB. Data obtained in this study show that MLB is caused by a complex of species contributing to disease outbreaks in South Africa.

Fusarium subglutinans has been associated with many hosts. Some isolates of the fungus are responsible for pitch canker disease of pines. A South African population of F. subglutinans from pine seedlings was characterized by comparing it to isolates from various hosts, pine and non-pine. A total of 26 isolates representing six different hosts were selected for the study. Cultural and morphological characteristics were studied. Virulence of isolates was tested on 1-yr-old Pinus patula seedlings. Sexual compatibility of isolates of F. subglutinans with B- and E-tester strains, and crossings of pine isolates in all possible combinations, were performed. Pine isolates were also compared with pitch canker and non-pine isolates using random amplified polymorphic DNAs (RAPDs). All isolates were morphologically characteristic of F. subglutinans, but differed somewhat in cultural features. Only isolates from pine were pathogenic to P. patula seedlings. Virtually no isolates were sexually compatible with the fertile tester strains. An exception was isolate MRC 115 from maize. Some pine isolates (MRC 6211 and MRC 6217) were, however, sexually compatible with South African (MRC 6213) and pitch canker (MRC 6229) isolates. Cluster patterns constructed from RAPD profiles revealed that isolates clustered according to host. South African isolates from pine were related to the pitch canker isolates from other areas, and were similar to non-pine isolates only in morphology. Virulence, genetic analyses and composite RAPD data suggest that pine isolates originate from the same gene pool, and probably represent a distinct new biological species.

Genetic variability among sterile cultured single ascospore isolates of Xanthoria parietina, X. calcicola, X. ectaneoides, X. capensis, X. polycarpa and X. resendei was investigated with RAPD-PCR. If available five out of eight ascospores per ascus were analysed. In some samples multispore and mycelial isolates from ascomata were included in the analysis. Ascospore germination rates and phenotypic features such as growth rate, pigmentation and secondary metabolites were uniform in X. parietina sporelings of the same ascus, but varied among the progeny of meiosis in all other species. Phenotypic features correlated with genetic variability. X. parietina revealed polymorphisms among specimens from different worldwide locations. In contrast nine out of ten sets of sibling spores were genetically uniform, with only 2% polymorphism in the remaining set, indicating that X. parietina might be homothallic. X. calcicola, X. ectaneoides, X. capensis, X. polycarpa and X. resendei revealed 9–66% polymorphic loci and therefore are considered heterothallic.

We conducted a series of experiments to determine if extraradical hyphae of VAM fungi acclimate to low temperatures. Blocks of field soil containing VAM fungi were either slowly cooled or held at room temperature prior to freezing. Infectivity of VAM fungi was greater in soil that was slowly cooled. We hypothesized that greater infectivity following freezing resulted from cold acclimation of extraradical hyphae. This hypothesis was tested using in vitro mycorrhizas cultured in two-compartment Petri plates, in which hyphae grew into a separate compartment. Metabolic activity of these hyphae following freezing was assessed using a vital stain. The majority of cultures that were slowly cooled prior to freezing contained active hyphae, whereas hyphal activity was almost completely eliminated by freezing in non-precooled cultures. Freezing temperature influenced survival and activity of hyphae. To our knowledge this is the first report of acclimation to cold temperatures by VAM fungi.

Somatic compatibility is used to identify clones or vegetative compatibility groups (VCG) in fungal populations. For basidiomycetes, VCGs have normally been associated with either homothallic, non-outcrossing fungi or root decay fungi spreading by vegetative growth. A special instance is provided by the symbiotic relationships between woodwasps and the fungi Amylostereum areolatum and A. chailletii. This association results in clonal propagation, as the wasps (Sirex sp. and Urocerus sp.) transfer arthrospores of A. areolatum or A. chailletii during ovipositing in conifers. Identical isolates of the symbiont are normally carried by all the female offspring of one wasp. The presence of clones of A. areolatum and A. chailletii was tested through somatic compatibility shown in pairings of heterokaryons obtained from wasps, basidiocarps and wood. Isolates found more than 100 km apart within Denmark belonged to the same vegetative compatibility groups. In addition, A. areolatum isolates from Sweden and Lithuania were compatible with a Danish clone, in spite of the barrier of the Baltic Sea. The association between woodwasps and fungi thus creates dispersive clones or VCGs which are stable across time and space. The dispersal through basidiospores may be considered of less importance for A. areolatum, but common in A. chailletii.

Single-copy RFLP markers were developed for the root endophytic fungus Phialocephala fortinii. After an initial screening of 40 probes with four restriction enzymes, EcoRV and HindIII were chosen to analyse 31 strains of P. fortinii and nine strains of a closely related morphotype (Type 1) which have known ISSR-PCR or allozyme phenotypes. Type 1 isolates and a single genetically different isolate with Type 1 like morphology, a representative of a possible third taxon, showed unique alleles with several probes and both restriction enzymes. Consequently, both taxa were easily distinguishable from P. fortinii isolates. Whereas EcoRV in combination with seven probes revealed only eight multi-locus haplotypes among 17 ISSR phenotypes, HindIII in combination with 12 probes was able to distinguish all ISSR and/or allozyme phenotypes except two pairs of strains. Strains of P. fortinii showed a high gene diversity, and up to 15 alleles per locus were observed among the 31 strains of P. fortinii. The 12 probes used in combination with HindIII were (partially) sequenced and BLAST searches showed no similarities with known sequences indicating that they probably are non-coding regions of the genome. Enzyme-probe combinations suitable to indicate the genetic structure of P. fortinii and Type 1 populations were easily identified in the present study. This opens up avenues to study communities of the P. fortinii complex exposed to various environments and management intensities, and will help to promote the understanding of these extremely successful organisms.

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.

Some Armillaria species cause major disease problems in both forestry and gardens. In order to determine routinely and rapidly the identity of an isolate, a PCR-based method was investigated. A fragment, approx. 900 bp, of the IGS region of the ribosomal DNA was amplified from 96 different isolates of six species of Armillaria collected from the U.K. and mainland Europe. Armillaria mellea, A. gallica and A. tabescens produced unique digestion patterns when the PCR fragment was restricted with Alu I. A. ostoyae could be distinguished from A. borealis and A. cepistipes when the PCR fragment was restricted with Bsm I. Previously unreported RFLP patterns were determined for A. mellea and A. gallica. The existence of A. gallica isolates which are putative heterozygotes for the rDNA cluster is also reported. Based on differences in banding patterns. A. mellea, A. gallica, A. cepistipes and A. borealis were subdivided further into groups which correlated with differences in cultural morphology.

Pythium attrantheridium sp. nov. is a new species isolated from cavity spot lesions of carrots as well as apple and cherry seedlings from various locations widely distributed in Canada and the USA. This fungus is closely related to the heterothallic P. intermedium, but is distinguished by: (1) unique molecular characteristics; (2) unique morphological characteristics; and (3) mating incompatibility with P. intermedium. The ITS region of the nuclear rDNA of all strains of P. attrantheridium studied is different from that of all other known Pythium spp. The oogonia attract a large number of antheridia when compatible mating types contact each other. The positive mating type produces zoospores unlike those of P. intermedium. Thus, biological, morphological and molecular data support the recognition of a new species.

Five lignicolous mangrove fungi (Hypoxlon oceanicum, Julella avicenniae, Lignincola laevis, Savoryella lignicola and Trematosphaeria mangrovei) produced extracellular endoglucanase, cellobiohydrolase and β-glucosidase when cultivated under static conditions in a defined liquid growth medium. Agitation of cultures completely repressed enzyme production. Cellulase production by H. oceanicum was 2–5 fold higher compared to other strains. Increasing salinity generally resulted in reduced enzyme production although responses varied, but J. avicenniae appeared relatively unaffected by salinity within the range 0–3·4%.

Pochonia chlamydosporia var. catenulata is a potential biocontrol agent against root-knot nematodes. Diagnosis of isolates has relied on morphological identification, and is both time-consuming and difficult. β-tubulin primers have been developed for the identification of this fungus that were specific enough to distinguish between varieties of the fungus within the same species. Separate primers have been developed for the specific detection of P. chlamydosporia var. catenulata based on ITS sequences, which were able to detect the fungus in soil from various sites in Cuba where the biocontrol agent had been added. When the PCR diagnosis was combined with serial dilution of soil samples on selective medium, colonies were rapidly identified. The fungus was still present, albeit at low densities, in soils inoculated five years previously. The development of a baiting method allowed quick in situ screening of the isolates' ability to infect nematode eggs, and when combined with PCR diagnosis both varieties of the fungus could be detected in infected eggs. RFLP analysis of ITS sequences from P. chlamydosporia provided an extra level of discrimination between isolates.

The entomopathogenic fungus Metarhizium anisopliae var. acridum is registered as a mycopesticide for acridid control in Africa and Australia. Traditionally, identification of M. anisopliae var. acridum infection in grasshoppers and locusts has relied upon development of fungal growth in infected cadavers. Conventional methods of detection of this entomopathogen in the environment and non-target organisms have been based on culture and bioassay. A PCR-based method for the detection of M. anisopliae var. acridum was developed. Sequence data from the distinct ITS rDNA regions facilitated the design of PCR primers that were used in PCR-based diagnostic assays for the detection of fungal DNA. The amplified sequence was 420 bp in length and specific to M. anisopliae var. acridum. Isolates of M. anisopliae var. anisopliae and M. flavoviride produced no PCR product with these primers. Other fungal entomopathogens, plant pathogens, mycopathogens, and soil saprophytes were also not detected by the pathogen-specific primers. The assay was also effective for the detection of M. anisopliae var. acridum DNA in the presence of soil DNA extracts and in infected grasshoppers.

A DNA fragment apparently unique to Verticillium dahliae was found by comparing RAPD profiles of V. dahliae to those of other closely related fungi. RAPD analyses were performed on eight V. dahliae, six V. albo-atrum and three V. tricorpus isolates to identify DNA sequences specific to V. dahliae. RAPD primer E20 (AACGGTGACC) yielded a 567 bp band shared only by V. dahliae isolates. This band from isolate V14 was cloned and sequenced. No significant sequence similarity was found between this amplicon and any other nucleic acid sequence in the databases. Based on the sequence information, a pair of PCR primers, VDS1 (5′-CACATTCAGTTCAGGAGACGGA-3′) and VDS2 (5′-CCTTCTACTGGAGTATTTCGG-3′) was designed. PCR tests showed that VDS1 and VDS2 amplified the expected fragment of DNA from 62 V. dahliae isolates from diverse hosts and geographical origins, but not from any other sources of DNA tested, including DNA from the most closely related species, V. albo-atrum. Southern blot analysis showed that the PCR product specifically hybridized to V. dahliae genomic DNA. An internal control was constructed for competitive PCR and used to develop a detection and quantification assay for V. dahliae, for which the detection limit was determined.

The endophytic mycobiota of healthy twigs and the assemblage of species associated with twig lesions of Eucalyptus globulus and E. grandis planted at the same site in the central west region of Uruguay was examined. Twig segments from E. grandis and E. globulus were plated on 2% malt agar. The diversity of endophytes isolated was low, as was the number of host-specific species. E. globulus endophytes of healthy xylem were different from the colonizers of cracked lesions and healthy bark. Conversely, in E. grandis characterized by black spots, the main difference in species composition was between xylem and bark. Cytospora chrysosperma was isolated from healthy bark and symptomatic twigs of E. globulus, suggesting that symptomatic tissues, such as cracks produced under drought were favourable to saprotrophic expansion.

Responses of Rhizoctonia solani to spatial heterogeneity in sources of carbon, and associated translocation of carbon (C), were studied in a simple microcosm system comprising two discrete domains of agar gels separated on a glass slide and overlain with a porous membrane. Two arrangements of the gel pairs were used, one containing two equally large resources (representing ‘homogeneous’ conditions) and one containing a large and a negligible resource (representing ‘heterogeneous’ conditions). The nutrient sources were a standard mineral salt medium with or without glucose as sole C source. The fungus was inoculated onto one domain and growth responses determined by direct measurement of biomass. Translocation of C was quantified by use of 13C-enriched glucose. This substrate was either added to the agar at the outset, when studying newly developing colonies, or as a pulse into already established colonies. When growing in heterogeneous conditions, the fungus actively translocated C from a glucose-containing domain to sustain growth in the adjacent region lacking such a resource. In homogeneous conditions there was evidence of passive translocation (diffusion), but the fungus preferentially used local resource to maintain growth. Active translocation was only observed in newly growing colonies, whereas passive translocation occurred in both growing and established colonies. When the fungus was pulsed with a 13C-enriched glucose solution after 10 d growth, 2.5 times more 13C was taken up by the fungus grown in heterogeneous than homogeneous conditions, suggesting uptake exceeded local demands. In heterogeneous conditions, the total amount of 13C enriched glucose taken up by the fungus was independent of the location of the enriched glucose in the underlying medium. When the nylon membrane was replaced by Cellophane (an additional C source), degradation of the membrane and an increase in biomass occurred only in the heterogeneous system. The possible implications for these results in soil systems are discussed.

Strains of Hebeloma representative of different climatic zones were grown in axenic culture at either 2°C and 22° or 6° and 22°. Culture filtrates were assayed for proteolytic activity using FITC labelled BSA as a substrate. Assays were run between 0–37°. Growth at low temperature induced greater proteolytic activity (g−1D.W. mycelium). Many of the strains produced protease(s) which retained significant activity at temperatures as low as 0°, and a thermal optimum between 0–6° with a second optimum at higher temperature. The results are discussed in relation the nutrient acquisition potential of ectomycorrhizal fungi at low temperature and the contribution such cold active proteases might make to the soil enzyme pool.

Lactarius angiocarpus sp. nov. is described from miombo woodlands in Zambia. It is the third sequestrate representative of the Russulaceae described from tropical Africa. Morphological characters and DNA sequence data support its placement in Lactarius subgen. Plinthogali. Molecular kinship analyses confirm a multiple origin of sequestrate Russulaceae spp. As none of the previously sequenced sequestrate Russulaceae spp. has been assigned to the Plinthogali clade, the placement of L. angiocarpus indicates an additional point of origin of derivative sporocarp types within the Russulaceae. Within the same subgenus, another tropical African sequestrate species, L. dolichocaulis comb. nov. (syn. Arcangeliella dolichocaulis) is recognized.

Gondwanan vegetation, and the Australian region in particular, is species rich for ectomycorrhizal fungi in epigeous and hypogeous forms with over 100 species recorded in small (1 ha) patches of forests. Distinguishing co-occurring ectomycorrhizal fungi as root associations in native (natural or wildlands) vegetation or plantations and discriminating them from other larger basidiomycetes, e.g. wood and leaf litter decomposer fungi, places large demands on molecular identification, especially if interspecific similarities and intraspecific variation occur in target sequences. One hundred and nine species of larger basidiomycetes from a single forest location were characterised by PCR–RFLP profiles of two genomic regions (nuclear rDNA ITS and mtLSU). Over one-third of the species for which multiple isolates were tested showed intraspecific variation in either one or both genomic regions. This remarkably high variation questions previous assumptions about intraspecific ITS sequence variation and highlights the value of integrated molecular and morphological databases including voucher specimens. It also emphasises the value of molecular investigations that use more than one genomic region. Interspecific similarities were common among the Cortinariaceae, especially in the ITS region. Discrimination of most Cortinariaceae species was achieved using variation in the mtLSU region in conjunction with the ITS. This new information raises the possibilities that the ITS sequence is more conserved and the mtLSU more variable than among species of the other 23 families. In the other families, interspecific ITS variation was greater and the mtLSU profiles grouped species within families. The high variation in the two genomic regions indicated possible differences in the fungal population structure between two adjacent, differently managed blocks of Eucalyptus marginata forest. The significance of this variation to ecology, biodiversity assessment and ecosystem management are discussed.

In vitro, antibiotic effects of the culture filtrates of 29 species of aquatic hyphomycetes against Gram-negative and Gram-positive bacteria, yeasts and hyphomycetes were studied. The culture filtrates of 38% of isolates were active in at least one of our tests for biological activity. Fifteen species inhibited growth of bacteria and four demonstrated antifungal activity. Filtrates of 35% of the isolates were antibacterial. About 50% of the isolates, those with the largest in vitro inhibitory activities (Dimorphosphora foliicola, Flagellospora sp. and Mycocentrospora sp.) were obtained from wood. Filtrates of Articulospora tetracladia were not only active against Gram −ve bacteria, but were also antiviral against bacteriophage T4 and influenza virus.

The secondary metabolites, helvolic acid and cerulenin were detected in liquid culture of Sarocladiumoryzae, causal agent of sheath rot of rice, and also in diseased sheath tissues. These metabolites were examined by thin-layer chromatography (TLC), column chromatography, and by high-performance liquid chromatography (HPLC). Helvolic acid and cerulenin showed the Rf value of 0.49 and 0.61, respectively using TLC. Cerulenin produced two spots in the TLC chromatogram and produced two major peaks in the HPLC chromatogram due to its instability. External application of helvolic acid and cerulenin produced lesions similar to those caused by the fungus. When infiltrated both of these metabolites caused electrolyte leakage in susceptible cultivar, IR 36 and induced lower levels of leakges in White Ponni, the moderately resistant cultivar to S. oryzae. The concomitant production of helvolic acid and cerulenin by S. oryzae in liquid culture and in planta was confirmed and the role of helvolic acid and cerulenin on the virulence of S. oryzae and sheath rot disease of rice is discussed.