Molecular work to clarify major lineages in loculoascomycete fungi is enhanced by sequence data from 18 additional species, confirming the Melanommatales have to be included within Pleosporales, but showing Capnodiales to be separate. Other molecular papers address species concepts in Leveillula, species of Botryosphaeria and related anamorphs, and variation within the species Gaeumannomyces graminis,Phialophora gregata and Pleurotus eryngii. Variation in oospore formation, production and viability in Phytophthora infestans in central Mexico has also been investigated.

Four papers concern aspects of ectomycorrhizal fungi. They compare the fruit bodies found and fungi actually forming mycorrhizas in a single site, Amanita muscaria genotypes in Australian plantations, genotypes of two Pisolithus species associated with native sclerophyll forest in Australia, and glutamine uptake in the same Pisolithus species.

A further four papers concern the detection or visualization of fungal tissues. A PCR-based assay for Biscogniauxia mediterranea is described, an immunoblotting method developed to study growth dynamics in Rhizoctonia solani, microscopic and staining techniques for an endophytic Lophodermium, and epifluoresence microscopy in the study of ascogenous hyphal systems in Arthoniaceae.

Muller's Rachet hypothesis of 1964 is not supported by data from Beauveria bassiana and Metarhizium anisopliae.

The new species Tripospora militaris sp. nov. is described from Argentina and a key to the four known species provided.

Mycological Research105 (1): 41–56 (January 2001).

In the paper by Câmara et al. titled ‘Systematics of Paraphaeosphaeria: a molecular and morphological approach’, three species are incorrectly indicated as belonging to Paraphaeosphaeria in Fig. 1 (p. 43) and Fig. 2 (p. 44). The species epithets nodorum, oryzae and phragmiticola belong in the genus Phaeosphaeria and not in Paraphaeosphaeria; they are correctly assigned in Table 1.

The phylogeny of the Dothideomycetes was investigated using nucleotide sequences of SSU and LSU rDNA. 32 new sequences of these regions from 18 species were aligned with five sequences obtained from GenBank, including two representatives of Pezizomycetes used as outgroup. A combined data set of SSU and LSU rDNA was analysed phylogenetically using neighbour-joining and maximum likelihood. The pseudoparaphysate taxa (Pleosporales incl. Melanommatales) form a monophyletic group. A separation of Melanommatales can be rejected using parametric bootstrapping, and this supports previous results obtained from SSU rDNA studies. The aparaphysate Dothideomycetes form a sister-group to the Pleosporales, but with low bootstrap support. Within these species, two well-supported groups can be distinguished, the Dothideales s. str. and the Capnodiales with Myriangiales as sister-groups. The Myriangiales appear paraphyletic, but this has only poor bootstrap support. A monophyly of Dothideales comprising Capnodiales can be rejected with parametric bootstrapping as well as a subdivision of the loculoascomycetes based on form and opening of the ascomata and ascus. The phylogeny of the aparaphysate taxa requires further studies.

The nucleotide sequences of the nuclear ribosomal DNA, including ITS1, ITS2 and the 5·8S rDNA, were determined for 54 specimens representing 13 Leveillula species on 50 different host plant species. The maximum nucleotide sequence diversity among taxa in ITS1-5·8S-ITS2 was 7·1%. Phylogenetic analyses revealed that Leveillula species formed six clades and three basal taxa. The taxonomic positions of several species that are well characterized by morphology of conidia, especially primary conidia, are supported by the present molecular analyses. This result shows that the morphology of primary conidia mostly provides a good criterion to identify Leveillula species. Twenty-six collections of Leveillula taurica recovered from 14 different plant families formed a distinct clade with L. chrozophorae, L. duriaei and L. elaeagni, and showed high homology in the ITS regions. Eight isolates of L. taurica recovered from the Asteraceae, Balsaminaceae, Fabaceae and Campanulaceae showed high sequence diversity, in contrast with other L. taurica specimens, and clustered separately or with other taxa. The result of Kishino-Hasegawa and Templeton tests demonstrated that the possibility of monophyly of L. taurica s. lat. could be significantly rejected, indicating that L. taurica s. lat. is a species complex composed of several biological species.

Inter simple or short sequence repeat (ISSR) fingerprinting was used to differentiate Botryosphaeria species and related anamorphic fungi. Fifty-one isolates representing 10 different Botryosphaeria species and related taxa, including two RAPD marker groups and one special form of Sphaeropsis sapinea were investigated using five ISSR primers. A total of 171 fingerprint fragments were generated from these isolates, and sizes of fragments were mostly between 200 bp and 1650 bp. Both cluster and principal coordinate analyses were used to examine separately the fingerprint data from the two previously proposed sections Hyala and Brunnea of Botryosphaeria. The results of each analysis indicated that B. dothidea and B. ribis are distinct; B. ribis and B. parva are closely related, but separable; B. mamane is distinct from other Botryosphaeria species with similar morphology or ITS sequences; the A and B RAPD marker groups of S. sapinea and S. sapinea f. sp. cupressi are probably three distinct species; B. obtusa and S. sapinea B group are closely related but distinct; and the name B. stevensii may have been applied to more than one species. This study has shown that ISSR fingerprinting can be a powerful tool for differentiating closely related filamentous fungi with very similar morphologies or ITS sequences, dissecting species complexes, and justifying newly described species.

Isolates of Gaeumannomyces graminis were sampled from 16 cereal crops throughout the cereal belt of southern Australia to determine the extent of genetic diversity and the scale of genetic differentiation among pathogen populations. Data from 13 isozyme and 4 RFLP loci differentiated 79 multilocus genotypes among the 320 isolates analysed. All 17 loci differed significantly in allele frequencies across all populations and significant levels of genetic differentiation were detected between most populations. Genetic differentiation among host groups was high (GST = 0.307) and groups of populations from barley, oats and wheat were significantly different. The average genetic identities among populations were low and populations formed genetically related groups based on similarities in recent cereal cropping histories and not geographical origins. Collectively, these analyses indicate restrictions to inter-population gene flow within G. graminis and imply that population differentiation results from genetic drift and host-mediated selection by different cereal species.

AFLP (amplified fragments length polymorphism) and ISSR (inter simple sequence repeat) analyses were used to detect genetic diversity among 46 Phialophora gregata isolates including 41 from soybean, four from mung bean, and one from adzuki bean. Five AFLP primers amplified 55 fragments, of which 20 bands were polymorphic. Thirteen ISSR primers amplified 66 fragments, of which 45 bands were polymorphic. The ISSR technique detected more polymorphism than the AFLP technique did. A UPGMA (unweighted pair-group arithmetic mean) dendrogram was constructed based on the 121 amplified fragments with AFLP and ISSR primers to depict genetic diversity and relationships among the 46 isolates. Various levels of DNA polymorphism were detected among P. gregata isolates from soybean and mung bean. The estimated average genetic diversity is 0.079 for the population of 45 isolates from soybean and mung bean. The ISSR technique was shown to be more effective and economic than the AFLP technique in detecting genetic variation in P. gregata.

Random amplified polymorphic DNA polymerase chain reaction (RAPD–PCR) was used to assess the genetic diversity in twelve populations (a total of 144 isolates) of the Pleurotus eryngii complex, sampled in Israel. Our results show a higher level of diversity of RAPD polymorphism in the populations, especially in the drier, stressful climates. Twelve primers used in this study amplified 164 scorable RAPD loci, of which 163 (99.4%) were polymorphic and only 1 monomorphic. Out of the 164 loci, 123 (75%) varied significantly (P < 0.05) in allele frequencies among populations. This total proportion (75%) of significant polymorphic loci far exceeds the 5% level expected by chance (binomial test, P < 0.000001). The levels of polymorphism and gene diversity appeared to be highly significantly different between the populations. Sixty-eight per cent of the RAPD diversity was within populations and 32% was between populations. Inter-population genetic distances showed positive association with geographic distance, which was confirmed with spatial autocorrelation analysis of RAPD frequencies. Spearman rank correlation revealed a strong positive association between high polymorphism and the aridity index. In multiple regression, the coefficient of determination of polymorphism and gene diversity was explained by climatic variables linked to temperature and humidity. Our findings further demonstrate the validity of the ‘environmental theory of genetic diversity' hypothesis within P. eryngii populations in Israel. We suggest that natural selection develops a high level of RAPD polymorphism as adaptation to cope with stressful and temporally heterogeneous environments.

The present work reports the development of a PCR assay to detect the ascomycete Biscogniauxia mediterranea in asymptomatic tissues of Quercus cerris. To this purpose, two specific primers (MED1 and MED2) were designed by comparison of sequences comprised between ITS1 and ITS4 generic primers of 21 isolates of B. mediterranea and related species. Specificity of the primers was tested against 19 isolates of B. mediterranea and 20 isolates of related and unrelated genera, and against Quercus cerris DNA. The reliability of the results was confirmed by Southern Blot analysis. The 2 specific primers MED1 and MED2 were able to detect B. mediterranea DNA in the host tissues at picogram quantity of target DNA. This assay would greatly enhance the possibility to study the presence and localization of B. mediterranea during its latent phase in Quercus species.

Spatial distributions and species diversity of ectomycorrhizal fungi were studied. Epigeous fungal fruit bodies and mycorrhizas were investigated in a reforested stand of Pinus densiflora aged 45 yr. Forty species of fruit bodies, including the dominant genera, Russula and Inocybe, were recorded throughout the three-year investigation. A species–area curve of the fruit bodies indicated that mean species number in 1.5 m2 was ca 1.0. Pine ectomycorrhiza, which were randomly sampled throughout the same investigation period, were assigned to 51 morphotypes microscopically. Although the dominant morphotypes were not manifested due to the sampling method used, a species–area curve of the mycorrhizas indicated the mean species number in 1.5 m2 was ca 25. Thus, spatial distributions among fruit bodies and mycorrhizas were quite different, and the mycorrhizas showed more complex distributions than fruit bodies. Several species abundance and diversity coefficients changed in relation to both the calculation method and fungal material used, which suggested the necessity for a uniform method of characterizing ectomycorrhizal fungal community structure.

Basidiomes of Amanita muscaria were collected from sites in three 30–36-yr-old Pinus radiata plantations in New South Wales, Australia. Following DNA extraction from basidiome stipes, inter-simple sequence repeat PCR using the degenerate primers 5′DHB(CGA)5 and 5′DDB(CCA)5, was used to identify the mycelial genotypes from which basidiomes arose. Each site was found to contain 8–10 A. muscaria genotypes, with six genotypes common to all three sites and one common to two sites. The presence of common genotypes at the three sites is taken to indicate that they were introduced as vegetative inocula when seedlings were planted and have persisted for < 36 yr.

Basidiomes of a Pisolithus species were collected from a ca 2500 m2 Australian sclerophyll forest site during 1997 and 1999 and of a second Pisolithus species from a further ca 150 m2 site during 1999. Inter-simple sequence repeat (ISSR) PCR was conducted on DNA extracted from each basidiome using the primers 5′BDB(ACA)5, 5′DDB(CCA)5 and 5′DHB(CGA)5. Thirty-seven genotypes of Pisolithus species I were detected at the North Wilberforce site, with eight genotypes present during both 1997 and 1999. All other genotypes were observed during only one year. Mapping genotype distribution according to location of basidiome collection at the site suggested that most genotypes were present as small (< 2·0 m2) below-ground mycelial genets, however several larger (> 4·0 m2) genets (including one ca 120 m2 genet) were also present. All Pisolithus species II basidiomes at the North Turramurra site were of a common genotype, suggesting the presence of a single large (ca 69 m2) below-ground mycelial genet of this taxon at the site. Both Pisolithus species thus appear to produce large long-lived soil-borne mycelia, but establishment of large genets may be restricted under some circumstances.

Uptake of 14C-labelled glutamine by seven Australian Pisolithus isolates (representing two species) was investigated over the concentration range 0.5 mmol m−3–20 mol m−3. Total uptake did not conform to simple Michaelis-Menten kinetics over this concentration range. At concentrations above 0.5 mol m−3 much of the glutamine uptake appeared to be diffusion-like. At concentrations below 0.5 mol m−3, subtraction of uptake in the presence of 2,4-dinitrophenol from total uptake revealed an active component that followed Michaelis-Menten kinetics. Estimated Km and Vmax values for active glutamine uptake by all isolates were in the ranges 4.0−210 mmol m−3 and 80−637 nmol g−1 d wt. min−1 respectively. pH optima for glutamine uptake were in the ranges 4.5–6.0, 3.7–5.0 or 3.0–4.5, depending on the isolate. While intraspecific variation was observed, there were no apparent relationships between the two Pisolithus species and either kinetic parameters for glutamine uptake or the pH optimum for the process.

Immunoblotting combined with computer imaging and a simple, non-linear mathematical model were used to demonstrate the potential of a technique for non-destructive visualisation and analysis of fungal growth of Rhizoctonia solani over the surface of non-sterile sand. Immunoblotting detected actively growing regions of mycelium enabling visualisation of individual hyphae at the colony edge. A zone of active growth was detected expanding radially over time. Active growth did not continue in the centre of the fungal colony leading to the development of a ring of mycelium surrounding the inoculum. Change in the density of actively growing mycelium with distance from the inoculum unit was summarised for each colony at each time by a Gaussian function, describing a wave of actively growing mycelium, symmetrical in density about its centre but differing amongst replicate colonies. The effectiveness of the immunoblotting technique to detect differences in colony growth was tested by comparing the growth of replicate colonies for two contrasting isolates of R. solani. When both isolates of R. solani were grown at 23 °C the amplitude of the wave increased to a maximum and then decayed over time, the location of the centre of the wave moved outwards at a constant rate, whilst the width of the wave increased. Increasing the temperature to 28 °, accelerated this intrinsic growth process for one isolate, but retarded growth of the other.

Needles of Pinus strobus (white pine) were cleared and stained to survey the occurrence and location of Lophodermium sp., a fungal endophyte. Cytoplasmically dense endophytic hyphae with a pronounced lobed morphology and containing lipid bodies were localized intercellularly between the epidermis and hypodermis. These fungal infections did not appear quiescent, but rather exhibited signs of continual slow growth. A few associated host cells exhibited a hypersensitive response. Material embedded in resin and examined by light microscopy and transmission electron microscopy confirmed the location of hyphae between epidermal and hypodermal cells, and the presence of lipid bodies within the hyphae. In senescing needles, aggressive colonization of needle tissues occurred. Thus, for Lophodermium in white pine, endophytic infection is active rather than quiescent, and displays an alternate hyphal strategy to that seen in the reproductive phase.

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

Ascogenous hyphae of foliicolous members of the Arthoniaceae, including the genera Arthonia, Eremothecella, Amazonomyces, and Cryptothecia, were studied by epifluorescence microscopy. Two additional representatives of Opegrapha were included. The ascogenous hyphae in Arthonia, Eremothecella, Amazonomyces, and Opegrapha arise from a central region of the ascoma and form a dense and branched tree-like structure. The asci of Cryptothecia are not connected by such structures and arise singly. Differences of measurements, structures and branching patterns of ascogenous hyphae indicate that these are species-specific characters. Typical ascogones were not observed. The fusion of tips or croziers of ascogenous hyphae and haploid mycelium were seen in several species. The systematic significance of the observations is discussed.