This month Mycological Research News features a report of an extraordinary richness of undescribed truffle-like fungi in Australia, and an explanation of why mycologists should always deposit voucher specimens and cultures to enable their work to be validated, co-authored by 18 mycologists.

Amongst the 21 papers included in this issue are three on the biocontrol of insect pests by Erynia and Metarhizium species, including methods of application and longevity of conidial preparations. Biomass estimations by ergosterol in decomposing leaves in the Everglades, and by chitin in Crinipellis infections on cacao are described. Variation in Phytophthora infestans provides evidence of heterokaryons, conidial production in Colletotrichum acutatum is favoured by monsoon conditions, and the conidia of Pestalotiopsis neglecta inhibit other fungi. Six papers on mycorrhizas address competition with saprobic fungi and other ectomycorrhizal species, the characterization of a chitin synthase, variation at the molecular level in two endomycorrhizal fungi, a culture technique, and the early response of tobacco roots to colonization. Amongst other papers are ones characterizing a protease in Schizophyllum, describing incompatability groups in Pleurotus tuberregium, and showing different Venturia species attack different pears.

The following new scientific names are introduced: Pseudohelicomyces gen. nov.; Anthostomella acuminata, A. applanata, A. caffrariae, A. colligata, A. meerensis, A. palmae, A. raphiae, A. spiralis, Pseudohelicomyces albus, Ramulispora cerealis, and Trichoderma stromaticum spp. nov.

Experimental preparations (alginate granules and mycelial mats) of the entomopathogenic Erynia neoaphidis were assessed for their capacity to cause infections in populations of Macrosiphum euphorbiae on pepper plants, in a greenhouse trial performed over 14 d. The dried preparations contained hyphal cells, which upon rehydration produced conidiophores and ejected ballistospores. Applications of alginate granules and mycelial mats on soil were more rapid in causing infections in the host population compared with foliar applications. On the last sample date, infection rates of 10–14% were obtained with foliar applications of E. neoaphidis as alginate granules and mycelial mats, respectively. With soil applications of E. neoaphidis granules and mats, infections were 23–36% after 14 d. From all sample dates, 183 cadavers were found on pepper plants treated with E. neoaphidis and 37 (= 20%) exhibited external sporulation after overnight incubation.

Comparisons were also made with single foliar sprays of Beauveria bassiana (Mycotrol WP®) and the systemic insecticide pymetrozine (Chess WP®), the latter being the only treatment to significantly reduce aphid numbers during the trial period. Treatment with B. bassiana resulted in good initial infection rates (50–60%) which declined over the period and reached the same infection level as E. neoaphidis treatments at the end of the trial. With B. bassiana, a total of 81 cadavers were found, of which 62 (= 77%) sporulated.

The effect of simulated rain on the persistence of oil and water formulations of conidia of the entomogenous fungus Metarhizium anisopliae when applied to oilseed rape foliage was investigated, using third instar larvae of the mustard beetle (Phaedon cochleariae) as the target host. Rain significantly (P < 0.01) reduced the susceptibility of the beetle larvae to M. anisopliae but the amount of inoculum removed was influenced by the formulation. Larvae exposed to plants treated with conidia formulated in aqueous Tween, Shellsol T, or sunflower oil/Shellsol T resulted in 55, 82.5 and 72.5%, mortality, respectively. The mortality for these respective formulations was reduced by 42, 57 and 51% if the plants were exposed for 1 h to simulated rain. Laboratory and field studies showed that more inoculum collected beneath plants sprayed with conidia formulated in Shellsol T or aqueous Tween than in the more viscous sunflower/Shellsol T mixture. Mortality studies on leaves taken from field plots suggested that conidia on leaf surfaces could be replenished by repeated application. The number of conidia isolated from field plots was greater where inoculum was applied bi-weekly than once weekly.

Conidia of the entomopathogenic Metarhizium flavoviride were harvested 8, 10, 12 or 15 d after inoculation at 25 °C and then (as conidiated rice) dried rapidly (10–12% r.h. and 17–20° for 17 h to about 15–22% moisture content) or slowly (50–60% r.h. and 27° for 5 d to about 27–32% moisture content initially). The subsequent survival of these conidia in air-dry storage at 50° with 8·1% moisture content was then assessed. Conidia longevity (assessed by the duration of storage until conidia viability was reduced to 50%, p50) was maximal when conidia were harvested 10 d after inoculation, and was much greater following slow rather than rapid drying. The substantial beneficial effect of slow desiccation to subsequent conidia survival is consistent with that detected in other propagules in anhydrous biology, and is also of considerable practical utility for the biological control of insects by entomopathogenic fungi.

Fungal biomass was detected in peat soils from throughout the Everglades based on the presence of ergosterol. Ergosterol concentrations in soils were not detectably affected by the dominant plant, Cladium jamaicense or Typha domingensis, or phosphorus content of soils. In situ decomposition of decaying leaves, measured by respiration, was high (maximum 484 μl O2 h−1 g−1 dry biomass). Approximately 30% of respiration was by bacteria, and the rest was by fungi and other eukaryotes. Respiration rates were essentially the same for decomposing leaves of both plant species, with higher rates early in the decomposition process. Respiration rates were relatively unaffected by the nutrient status of the site, except for eukaryotic respiration on Cladium, which was usually higher at a high nutrient site. Ergosterol concentration increased in decaying leaves through time and was unrelated to the nutrient level except for Cladium, where it was higher at the high nutrient site. Eukaryotic respiration was not correlated with ergosterol concentration in decomposing leaves of Typha, but was positively correlated with ergosterol for Cladium.

Crinipellis perniciosa is the cause of witches' broom disease of cocoa, a serious problem in South America. The aim of the project was to develop a measure of fungal biomass in cocoa tissue infected with C. perniciosa using chitin as a marker. Axenic cultures of primary and secondary mycelium of C. perniciosa were exposed to strong hydrochloric acid to hydrolyse the chitin. The glucosamine so formed was converted to hexamitol by sodium borohydride and the acetylated and derivatized products were separated by gas chromatography and identified by mass spectrophotometry. As a proportion of the mycelial D.W., chitin was found to be 14% in primary phase mycelium and 17% in the secondary phase. An average figure was used to calculate the amount of fungal biomass from estimates of chitin in dried and fresh cocoa broom tissue infected with C. perniciosa. The green brooms, which represented an early stage of infection, contained 81 μg fungal biomass mg−1D.W., whereas brown brooms from a late stage in infection contained 161 μg fungal biomass mg−1D.W. of broom. In a detailed analysis of a single fresh broom most fungal biomass was found at the base (215 μg mg−1D.W.) but declined towards the shoot tip (10 μg mg−1D.W.). The assay was sufficiently sensitive to measure a minimum fungal biomass in cocoa of 40 ng mg−1D.W. of plant tissue. The method developed was specific to chitin, and significantly more sensitive than previously described methods.

Isolates from potato in an isolated garden in North Wales in 1995 consisted of essentially two distinct phenotypes. Phenotypes P1 and P2, of A1 and A2 mating type respectively, had distinctive multi-locus (RG57) and telomeric RFLP fingerprints. In addition, about 6% of isolates forming abundant oospores were self-fertile and showed a combination of both fingerprints. Among a total of 40 single-hyphal-tip and single-sporangial propagations from two self-fertile isolates, 16 were P2 whereas only one propagation was P1; four propagations were still self-fertile and still carried the molecular markers typical of both P1 and P2. Most of the remainder were A1 but otherwise carried both P1 and P2 molecular markers. Uninucleate zoospore lines from two of these were also of A1 mating type but had segregated molecular markers typical of P2; a zoospore line from a third segregated both A2 mating type and markers typical of P2. The original self-fertile isolates appeared to have heterokaryotic hyphae and sporangia with P1 and P2 nuclei and also nuclei of A1 mating type but P2 markers. Tetraploid nuclei within a heterokaryon, resulting from fusion of P1 with P2 nuclei, could have segregated to yield the latter.

Colletotrichum acutatum is cosmopolitan and causes diseases in many crops including Hevea brasiliensis. It sporulated freely on PDA at 10–40 °C with peaks at around 15° and 25°. Self-inhibition of spore germination occurred at concentrations above 8×106 spores ml−1. Ultraviolet radiation (especially 254 nm) inactivated the spores. Spore germination was around 90% between 15–35°. Free water promoted spore germination but was not essential; high relative humidity (95%) was sufficient. Spores could also withstand temperatures up to 35°. It is concluded that the most favourable conditions for the spread of C. acutatum are prevalent in the major rubber growing areas in Sri Lanka throughout the monsoons.

A germination inhibitor in the spore matrix of Pestalotiopsis neglecta was strongly inhibitory to other mitosporic fungi, especially coelomycetes. Germination of conidia of P. neglecta was enhanced by an exogenous source of certain carbohydrates, especially pectin but also some sugars. Light was not important for germination or growth, and was not essential for reproduction, although the number of acervuli trebled when light intensity was increased from 210 μmol m−2 s−1 to 300 μmol m−2 s−1. The possible significance of these adaptations in the ecology of P. neglecta is discussed.

In an in vitro study, interactions between ectomycorrhizal fungi, saprotrophic basidiomycetes and soil microfungi known from pine forests were examined in relation to different ammonium concentrations. The saprotrophic basidiomycetes Clitocybe marginella and C. vibecina and the soil microfungi Penicillium pinetorum, Oidiodendron flavum and Torulomyces lagena were grown against the ectomycorrhizal Cenococcum geophilum, Laccaria bicolor and Rhizopogon luteolus. Interactions were species specific and overgrowth was the most common (43%), followed by inhibition at distance (26%) and contact inhibition (16%). Ammonium concentrations affected the type and strength of the interactions. The competitive strength of the ectomycorrhizal fungi was relatively high and only the growth of the mycelia of L. bicolor was clearly suppressed by the Clitocybe spp. The interactions are discussed in relation to the ecology of the fungi and potential future studies.

The cultivation and competitiveness of Tuber borchii was investigated. Seedlings of Pinus pinea were infected either with T. borchii, Laccaria bicolor (strain S238), Hebeloma sinapizans, T. borchii and L. bicolor, or T. borchii and H. sinapizans. Uninoculated seedlings weakly infected with Sphaerosporella brunnea were used as controls. In 1990 the seedlings were transplanted to a coastal site at Marina di Ravenna, Italy. Examination of seedling roots in 1994 and 1995 showed that L. bicolor, H. sinapizans and S. brunnea ectomycorrhizas were replaced by T. borchii and other ectomycorrhizal fungi. The DNA sequence of the ITS region was analysed using the specific pair of primers, ITS1–ITS4, to type the ectomycorrhizas of Tuber borchii. The ectomycorrhizas were also identified by digestion of the PCR products using different restriction enzymes. T. borchii truffle production began in 1994 and for the next four years an average of 7 kg of truffles ha−1 were harvested. This study describes a simple method for the cultivation of T. borchii.

Chitin synthase genes of Tuber magnatum were sought in order to investigate the molecular bases of its growth. Primers designed from highly conserved Chs domains were used to identify a chs gene portion. A genomic library was used to obtain the full gene sequence (TMchs4) with an open reading frame which encodes a predicted protein of 1230 amino acids. The sequence is similar (62%) to the class IV chitin synthase from Neurospora crassa. The putative protein shows hydrophobicity pattern that suggests the presence of several intramembranous domains and other peculiar features common to the other chs of class IV. Northern experiments demonstrated that the gene is expressed in ascomata sampled at different maturation steps. These data suggest that ascomata growth requires new chitin deposition, which is based on a chs gene activation and not only on an enzymatic activity.

To study the genetic variability in Glomus mosseae and Gigaspora margarita sequence similarities of ITS regions of rDNA were analysed from spores collected from a permanent pasture and from pot cultures. PCR amplification with the primers ITS1 and ITS4 was performed and products were cloned and sequenced. The sequences from single spores of G. mosseae and Gi. margarita confirmed that there is variation in the ITS region in a single spore. Phenetic analysis of sequences from both species supported the morphological identification which placed the species into two separate groups. Through the analysis of these sequences an estimate of genetic diversity was derived which clearly showed that the three field spores of G. mosseae were at least 2–5 times more genetically diverse than one single spore (field) and a pool of spores (pot culture) of Gigaspora margarita. This demonstrates that a high degree of variation exists in this natural population of G. mosseae.

Arbuscular mycorrhizal (AM) fungi are obligate biotrophic organisms. Root organ culture (ROC) can be used to grow these fungi under in vitro conditions. The ROC technique was used with Gigaspora margarita and Ri T-DNA transformed carrot roots to examine the fungal growth and physiology under long-term axenic symbiosis. The fungus formed spores inside the host roots under in vitro conditions. Sporulation was a temporal phenomenon found in dual cultures more than 18–20 mo old. The spores were formed singly or in rare cases clusters of two or three. No preferential zone of formation was found. The spores formed intraradically were 10–15% of the total spores formed in a single culture. These intraradical spores were analysed for their morphology and DNA polymorphism pattern with spores which had formed conventionally in the medium. Both analyses showed no detectable variation. This is the first report of spore formation by Gigaspora inside the roots of a host.

Catalase and ascorbate peroxidase enzymatic activities were examined during the interaction between Nicotiana tabacum and the arbuscular mycorrhizal Glomus mosseae. Transient enhancements of both enzymatic activities were detected in the inoculated plant roots coinciding in time with the stage of appressoria formation in the root surface. The analysis of free salicylic acid content in roots revealed that the increases in enzymatic activities were coincident in time with the accumulation of SA in inoculated roots. These data indicate that the first reaction of the root cells to the invasion of arbuscular mycorrhizal fungi is a defence response.

Schizophyllum commune produces a variety of mycelial proteolytic enzymes. The specific functions of many of these enzymes are unknown, but several have elevated activity when the mycelium is grown in nitrogen-limiting conditions, suggesting a role in mycelial autolysis. We have purified one of these nitrogen-limitation induced enzymes, a small serine protease, ScPrI, from S. commune mycelial extracts. ScPrI has an apparent molecular mass of 22 kDa and is active against classical substrates for chymotrypsin and subtilisin proteases. The pH optimum for activity is neutral to slightly alkaline and the protein denatures above 50 °C. The enzyme is inhibited by PMSF, TPCK and chymostatin, and it shows little dependence on metal ions. Hydrolysis of oxidized insulin B-chain peptide by ScPrI demonstrated cleavage following aromatic amino acids and leucine. Kinetic analysis of hydrolysis of N- succinyl-AAPF-pNA and N-succinyl-AAPL-pNA revealed similar Kms for both substrates but the Vmax was nearly 3-fold higher for the substrate with phenylalanine in the P1 position.

Genetic relationships were investigated among several populations of Pleurotus tuberregium from Nigeria, Papua New Guinea and New Caledonia. Intrastock mating compatibility studies using progeny from two collections demonstrated a tetrapolar mating system for P. tuberregium. Interstock matings among the geographically distinct populations were compatible. All isolates were found to be intersterile with tester strains of other Pleurotus species, showing that P. tuberregium represents a unique intersterility group in Pleurotus. Nucleotide sequences of the ITS region of the rDNA gene were determined for 30 isolates and used to infer phylogenetic structure of populations. Phylogenetic analysis shows that African and Australasian-Pacific isolates form at least two distinct evolutionary lineages. Higher genetic divergence was observed among ITS sequences from the Australasian-Pacific region than among African isolates, which suggests a possible origin of P. tuberregium in the Australasian-Pacific region.

Pseudohelicomyces albus gen. et sp. nov. is described. It is characterised by the presence of hyaline, cylindrical arthroconidia originating from either spiral vegetative hyphae or from spiral, branched conidiophores. The hyphae and conidiophores may or may not possess clamp connections. The characteristics of the basidiocarps formed in culture were identical to material collected from nature. P. albus is the anamorphic stage of Psilocybe merdaria and shows similarities to Helicomyces.

Eight new species of Anthostomella, A. acuminata, A. applanata, A. caffrariae, A. colligata, A. meerensis, A. palmae, A. raphiae and A. spiralis, are described from South Africa. They are compared with similar species and illustrated with differential interference contrast photomicrographs.

The relationship of Venturia nashicola, the scab fungus of Japanese and Chinese pears, to the European pear scab fungus, V. pirina, was reexamined. Ascospores of V. pirina were longer and wider than those of V. nashicola and the conidia of V. nashicola significantly shorter than those of V. pirina. Results from mating experiments in culture clearly showed sexual isolation between the species. V. nashicola was only pathogenic on Japanese and Chinese pears and V. pirina was only pathogenic on European pear. It is concluded that V. nashicola is distinct from V. pirina.