This issue of Mycological Research News features : Adhering to good cultural practice ; and Mycorrhizas of nonphotosynthetic plants.

Molecular phylogenetic papers in this part concern the position of microspordians in relation to fungi, Entyloma species, and Melampsora epitea populations. Mitochondrial plasmids in Cryphonectria parasitica populations are reported, and a PCR-method for detecting Rhizoctonia solani in soil is described. Incompatability tests and molecular methods address species concepts in the Heterobasidion insulare complex.

An extracellular protein from Colletotrichum coccodes which may be involved in pathogenicity is characterized. A low-cost scale-up method for the production of Clonostachys rosea is described, and Microsphaeropsis isolates with biocontrol potential are compared.

Oidiodendron maius is discovered to be a peat saprobe, not only an endophyte, and probably a Myxotrichum. Metal phosphate solubilization by Rhizoctonia solani has been investigated, as has salt tolerance in Aspergillus nidulans using mutant strains.

No new scientific names are introduced in this part of Mycological Research.

Microsporidia are intracellular parasitic microbes, which were once thought to be among early diverging eukaryotes, mainly because they lack mitochondria. However, this evolutionary scheme has been challenged by protein-based molecular phylogenetic studies, which suggest a more recent origin of the Microsporidia, which clustered Microsporidia with or within ‘true’ fungal lineages. However, most of these studies did not include sequences from basal fungal phyla (Chytridiomycota and Zygomycota), and unrepresentative taxon sampling could have resulted in misleading relationships being suggested. To further investigate the evolutionary origin of Microsporidia relative to Fungi, we performed molecular phylogenetic analyses with newly determined RPB1 (DNA-dependent RNA polymerase II largest subunit) and EF-1α (translation elongation factor I alpha) including sequences from basal fungi. Although the phylogenetic position of Microsporidia in the EF-1α tree still might be misplaced due to the unusually high rate of sequence divergence of the microsporidian EF-1α gene, the phylogenies recovered based on these two protein sequences do not provide strong evidences for a close relationship between Microsporidia and Fungi. Moreover, we have identified within the EF-1α genes a characteristic two amino acid deletion conserved in all fungal sequences currently available, whereas this deletion is absent in microsporidian sequences. These results argue against the view of Microsporidia as highly degenerate fungi, and whether Microsporidia and Fungi are sister taxa remains unresolved.

Many species formerly listed in Entyloma have been removed and are now placed in several orders. This study aims to clarify the framework of the genus Entyloma and the phylogeny of these plant parasitic smut fungi with molecular data. Analyses of LSU and ITS sequences are presented and support the monophyly of the order Entylomatales. The sequences show higher similarities within the examined species of Entyloma than within other smut families and genera, suggesting a recent radiation. Within the Entylomatales a cluster of anamorphic Tilletiopsis washingtonensis, T. lilacina and T. cremea collections is the sister group to Entyloma. The phylogenetic relationships in the genus Entyloma are a result of joint evolution with their hosts. The analyses of the sequence data show unresolved groups on Ranunculales and a well-supported group on Asteridae.

To compare genetic variation among four geographically distant populations of the fungus species Melampsora epitea causing willow rust, a two-primer combination was used for ALFP fingerprinting of 24 isolates from Sweden, along with 10 from France, 24 from Northern Ireland, and 35 from Chile. The banding patterns of all 93 isolates were easily distinguished and revealed polymorphism among individuals within and between the four M. epitea populations. No clustering on the dendrogram could be attributed to country of origin, or almost equivalently, country of origin correlated poorly with genetic distance, suggesting that the geographically distant populations developed collectively as a metapopulation, instead of separately.

A worldwide collection of strains of Cryphonectria parasitica was examined to draw a precise picture of the incidence and diversity of mitochondrial plasmids related to the plasmid pUG1. Amplification by specific PCR of 199 strains showed the presence of pUG1-like plasmids in 22% of the populations examined. The entire plasmid molecules were amplified by multiplex PCR and the products showed different RFLP patterns. The variability was mostly in a non-coding region of the molecule that has been sequenced in some representative strains, enabling the molecular evolution of the molecule to be elucidated. The data show that mitochondrial plasmids of C. parasitica comprise an almost homogeneous family (designated pCp) that can be divided into two clusters based on the presence/absence respectively of a 60 nucleotide region in North American and European plasmids.

Binucleate Rhizoctonia (BNR) was isolated (isolate CFM1) from cabbage grown in soil from pot cultures of the arbuscular mycorrhizal (AM) fungus, Glomus mosseae, grown on subterranean clover, and was identified as AG-Bo by means of morphological and molecular characteristics. A PCR-based assay was developed to detect BNR in roots and soils of pot cultures of AM fungi. Two oligonucleotide primers (CF1f and CF2r) were designed from the ITS region of the rDNA sequence of BNR AG-Bo isolate CFM1. These allowed amplification of a specific fragment (438 bp). Specificity of the primers was tested against several isolates of various anastomosis groups (AGs) of BNR, other related and unrelated fungal species, and roots not infected or infected with fungi. The primers amplified DNA of isolates of both BNR AG-Bo and AG-A, and were used to detect them in roots in the presence or absence of AM fungi. Nested PCR amplification using CF1f and CF2r primers has advantages over microscopic methods for detection of BNR AG-Bo or AG-A on infected roots and soils of AM pot cultures and provides a powerful tool for detection of contamination of these pot cultures.

Extracellular enzymes play an important role in the pathogenicity and virulence of phytopathogenic fungi. Several isolates of Colletotrichum coccodes, causal agent of anthracnose on tomato, were screened to determine the relationship between protease activity and virulence. A direct relationship was observed between extracellular protease activity and the induction of disease symptoms of fruit and mortality in plants. Isolate Cc155 exhibited the highest protease activity after five days of growth in protease induction medium and produced an extracellular serine protease (sp78) that was 78 kDa, auto-degradative, glucose repressible, and non-glycosylated. To determine the role of sp78 in pathogenicity, a UV-induced extracellular protease deficient mutant (np155) was generated from the wildtype isolate Cc155. Np155 maintained growth rates comparable to Cc155 and produced wildtype levels of extracellular cellulase but did not produce extracellular protease. Unlike Cc155, np155 caused no disease symptoms on tomato fruit and 0% mortality on tomato seedlings. These results suggest that extracellular protease activity is required for pathogenicity and virulence of C. coccodes, and that the elimination of protease activity transforms a virulent pathogen to a non-pathogenic endophyte.

The sexual system of Heterobasidion insulare was elucidated and 40 specimens originating from the mainland of China, Taiwan, and Japan were studied with the aid of mating tests. Most of the material was also investigated with DNA fingerprinting using random amplified microsatellite (RAMS) and M13 minisatellite primers. Denaturing gradient gel electrophoresis (DGGE) and sequencing were applied for detecting variation in the internally transcribed spacer (ITS) region of the ribosomal DNA cluster. H. insulare proved to be a species complex with bipolar mating system. Clamp connections occurred in both heterokaryotic and homokaryotic mycelia. Three intersterility groups were found: the ‘T group’ included specimens from Taiwan and Guizhou Province (southern China), the ‘N group’ specimens from northern China and Japan, and the ‘Y group’ specimens from Yunnan (south-west China). The T group seems to be totally intersterile with the two other groups, and was grouped separately in neighbour joining analysis based on DNA fingerprints. As concluded from mating experiments, groups N and Y are partially intersterile, but they are able to produce hybrid heterokaryons, no group specific DNA markers were found, and they were grouped together in neighbour joining analysis. The identity of H. insulare sensu stricto was studied from the type collections. It seems that none of the above-presented taxa can be referred to it, and we propose the term H. insulare sensu lato to be used in future studies.

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.

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.

Oidiodendron maius is accepted widely as an ericoid mycorrhizal endophyte because it is isolated primarily from the roots of ericaceous plants. Reports of the species from other materials are much fewer, suggesting a limited role as a free-living saprobe. We show that assumptions concerning the prevalence of O. maius in a common substrate (i.e. peat) are affected by isolation protocols. Oidiodendron maius was observed on 99.6% of peat samples incubated in moist chambers but only 0–9% of the peat fragments plated on different isolation media. These results, and previous studies showing ability to degrade Sphagnum, indicate that O. maius could inhabit a much broader niche than previously suspected. Sterile ascocarps with peridia morphologically similar to Myxotrichum arcticum were observed on moist incubated peat and in a series of controlled crosses. These observations provide evidence that the teleomorph of O. maius is a species of Myxotrichum.

The effects of different temperatures and pH on the growth and solubilization of insoluble calcium phosphate, cobalt phosphate, manganese phosphate, strontium hydrogen phosphate and zinc phosphate by Rhizoctonia solani on solidified media were assessed. Solubilization of the metal phosphates was monitored by the production of a clear zone around or underneath the fungus. R. solani efficiently solubilized all five metal phosphates except cobalt phosphate when grown on medium at pH 7. Solubilization activity by R. solani decreased with increasing pH on medium containing calcium phosphate but increased on strontium hydrogen phosphate-amended medium. The uptake of metals by the mycelia was unaffected by the pH of the medium or the growth temperature. Small quantities of crystals were produced in the agar when R. solani was grown on calcium phosphate- and strontium hydrogen phosphate-amended media and these were identified as calcium or strontium sulphates respectively: there appeared to be little or no production of insoluble oxalates although a role for oxalate in the overall solubilization process cannot be discounted. These results are discussed in relation to their physiological and environmental significance, and the important roles of fungi in effecting transformations of insoluble metal-containing compounds in the environment.

We report an investigation into the relationship between Aspergillus nidulans mutations sltA1 and agaA50, conferring salt sensitivity and arginase deficiency, respectively, and the ability of spores to swell and commence germination. Spores from both mutant strains exhibited uncontrolled swelling at pH 5.5 in the presence of arginine as the nitrogen source. The controlled swelling of spores from a salt tolerant strain under these conditions was disrupted by the Na+/H+ exchange inhibitor amiloride. Swelling in the presence of amiloride was associated with an elevation in the concentration of arginine within the spores of each strain tested. The possible consequences of alterations in the arginine economy of the spore for colony establishment in osmotically challenged habitats are discussed.