This number of Mycological Research News features: In this issue; Fungi associated with insects or used for applied biocontrol; Powdery mildews as invasive plant pathogens: new epidemics caused by two North American species in Europe; and Genetic differences among nuclei in arbuscular mycorrhizal fungi.

This theme issue on fungi associated with insects includes ten papers on this topic. These demonstrate that beetle guts are an amazingly rich source of hitherto unknown yeasts, and a zygosporic stage in Harpellales; report the discovery of the first fossil member of Laboulbeniomycetes, and a new Leptographium associated with a bark beetle; examine genetic diversity at the molecular level in Pandora neoaphidis and Beauveria bassiana; identify the genes expressed during conidiation in Metarhizium anisopliae; report a heterothallic mating system in Termitomyces; and describe two low cost methods for the bulk-culturing of Zoophthora radicans and other entomophthoralean fungi.

Other papers introduce a new molecular fingerprinting method for the biofungicidal Pseudozyma flocculosa; reassesses generic concepts in six genera of smut fungi; reveal that Podosphaera tridactyla is paraphyletic; and demonstrate the production of a N-acetylglucosaminidase by a Neotyphodium endophyte inside its grass host.

Fruit body initiation in Pleurotus ostreatus is found to require a sugar moeity, and the localization and expression of ostreolysin during the fruiting process is also examined.

The following new scientific names are introduced: Leptographium koreanum, and Stigmatomyces succini spp. nov.; and Sporisorium fascicularis (syn. Lundquistia fascicularis) comb. nov.

We isolated over 650 yeasts over a three year period from the gut of a variety of beetles and characterized them on the basis of LSU rDNA sequences and morphological and metabolic traits. Of these, at least 200 were undescribed taxa, a number equivalent to almost 30% of all currently recognized yeast species. A Bayesian analysis of species discovery rates predicts further sampling of previously sampled habitats could easily produce another 100 species. The sampled habitat is, thereby, estimated to contain well over half as many more species as are currently known worldwide. The beetle gut yeasts occur in 45 independent lineages scattered across the yeast phylogenetic tree, often in clusters. The distribution suggests that the some of the yeasts diversified by a process of horizontal transmission in the habitats and subsequent specialization in association with insect hosts. Evidence of specialization comes from consistent associations over time and broad geographical ranges of certain yeast and beetle species. The discovery of high yeast diversity in a previously unexplored habitat is a first step toward investigating the basis of the interactions and their impact in relation to ecology and evolution.

The presence of an ovarian stage in the life-cycle of the Harpellales observed during an ecological study of the Trichomycetes associated with simuliid egg masses in the UK is reported. The ovarian stage was observed to develop zygospores that are morphologically similar to those produced by the midgut species Harpella melusinae in the host larva. Morphological characteristics and the possible developmental stages of this propagule are described using scanning electron microscopy. The discovery of this stage suggests that chlamydospores may play an important role in the life-cycle of the Trichomycetes.

A fossil ascomycete was found attached to the thorax of a stalk eyed fly (Diopsidae: Prosphyracephala succini) in a fragment of Baltic amber. The fungus is assigned to the extant genus Stigmatomyces and described as S. succini sp. nov. This find is the first fossil record of the order Laboulbeniales. At the same time it constitutes the oldest record of a parasitic fungus on an insect. The palaeohabitat is discussed with regard to the find.

During the course of a survey of sapstaining fungi in Korea, a Leptographium was isolated from Pinus densiflora and P. koraiensis logs, infested with the bark beetle Tomicus piniperda. The fungus grew optimally at 25 °C on 2% malt extract agar and showed a high level of tolerance to cycloheximide. The Leptographium has unusually short conidiophores and is morphologically similar to L. pini-densiflorae, L. lundbergii, L. yunnanense, and the Leptographium anamorph of Ophiostoma crassivaginatum. Comparisons of DNA sequence data for three gene regions, as well as morphological characteristics, confirmed that this fungus represents an undescribed taxon. We consequently provide the name Leptographium koreanum sp. nov. for it here.

The entomopathogenic fungus Pandora neoaphidis is an important natural enemy of aphids. ISSR, ERIC (Enterobacterial Repetitive Intergenic Consensus) and RAPD PCR-based DNA fingerprint analyses were undertaken to study intra-specific variation amongst 30 isolates of P. neoaphidis worldwide, together with six closely related species of Entomophthorales. All methods yielded scorable binary characters, and distance matrices were constructed from both individual and combined data sets. Neighbour-joining was used to construct consensus phylogenetic trees which showed that although P. neoaphidis isolates were highly polymorphic they separated into a monophyletic group compared with the other Entomophthorales tested. Three distinct subclades were found, with UK isolates occupying two of these. No specific correlation with aphid host species was established for any of the isolates apart from those in one cluster which contained isolates obtained from nettle aphid, Microlophium carnosum. ERIC, ISSR and RAPD analysis allowed the rapid genetic characterisation and differentiation of isolates with the generation of potential isolate- and cluster specific- diagnostic DNA markers.

110 isolates of Beauveria (104 B. bassiana, 5 Beauveria spp., 1 B. brongniartii) were obtained from Sunn Pests (Eurygaster and Aelia species), litter, and other insect samples at overwintering sites in seven countries in the Middle East and West Asia. DNA was extracted from these isolates, and four techniques were used to characterize and to investigate genetic diversity at the molecular level: ITS-RFLP, ITS sequencing, ISSR-PCR, and AFLP. The ITS-RFLP and ITS sequences did not detect significant genetic variation among the isolates. However, both ISSR-PCR and AFLP analyses gave indications of intraspecific groupings correlated with geographical origin and relative genetic diversity among some isolates, but no obvious association with Sunn Pest hosts. There was no obvious genotypic grouping of B. bassiana isolates from E. integriceps, perhaps suggesting the overwintering populations were infected by generalist native isolates rather than by host-specific ones that might be more suitable for biocontrol purposes.

Metarhizium anisopliae exhibits two different developmental patterns under nutrient-deprived conditions: appressorium formation in early stages and conidiation in late stages of pathogenesis in its insect hosts. In this study we isolated genes enriched during mature conidial production under nutrient-deprived conditions in M. anisopliae by using the method of suppression subtractive hybridization. We sequence-identified seven conidiation-associated genes (cag) in M. anisopliae. One of the genes, cag7, encoded an extracellular subtilisin-like protease, Pr1, that plays a fundamental role in cuticular protein degradation. Reverse-transcription polymerase chain reaction (RT-PCR) analysis confirmed that cag cDNAs are expressed during the development of mature conidia under nutrient-deprived conditions. RT-PCR analysis was also performed for Pr1 during infection of greater wax moth larvae (Galleria mellonella). Results showed up-regulation of Pr1 in the infected insect as the mycelia emerge and produce conidia on the surface of the cadaver. It is well documented that Pr1 is produced during the initial stages of transcuticular penetration by M. anisopliae. Here we show that Pr1 is also up-regulated during the final stages of pathogenesis as the fungus emerges from the dead host and subsequently conidiates on the cadaver.

Fungi of the genus Termitomyces live in an obligate symbiosis with termites of the subfamily Macrotermitinae. Many species of Termitomyces frequently form fruit bodies, which develop from the fungus comb within the nest. In this study, we determined the mating system of a species of Termitomyces associated with the South African termite Macrotermes natalensis. Termite nests were excavated and a Termitomyces sp. was isolated into pure culture from the asexual fruit bodies (nodules) growing in the fungus gardens. For one strain, single basidiospore cultures were obtained from basidiomes growing from the fungus comb after incubation without termites. Using nuclear staining, we show that both comb cultures and single spore cultures have multinucleate cells and that the majority of spores has a single nucleus. However, DNA sequencing of the ITS region in the nuclear RNA gene revealed that the comb mycelium had two different ITS types that segregated in the single spore cultures, which consequently had only a single ITS type. These results unambiguously prove that the strain of Termitomyces studied here has a heterothallic mating system, with the fungus garden of the termite mound being in the heterokaryotic phase. This is the first time the mating system of a Termitomyces species has been studied.

The shelled grains of glutinous broomcorn millet, Panicum miliaceum, were used as solid substrate to prepare granular cultures of Zoophthora radicans, an entomophthoralean biocontrol agent against numerous insect pests. Steamed millet grains were inoculated by mixing 15 g millet grains (D.W.) with mashed pieces of half a 60-mm-dish colony in 3 ml modified Sabouraud dextrose broth and incubated at 15 °C and L[ratio ]D 12[ratio ]12 for up to 24 d. 20 grains were sampled at 3 d intervals from day six onwards and individually assessed for their sporulation capacity using a self-designed device for spore collection. The millet cultures after [ges ]12 d incubation produced 12.0–14.9×104 spores grain−1 during a 7 d period. The maximal sporulation capacity associated with the 21 d-old culture was about half of that of Z. radicans-killed Plutella xylostella larvae (28.7×104 spores cadaver−1), which individually were at least three times larger than the millet grains. Based on the time–concentration–mortality responses of second-instar P. xylostella larvae to Z. radicans in three independent bioassays, the spores ejected from the cultured millet grains, from the mycelial mats from liquid culture, and from larval cadavers displayed insignificant variations in infectivity to the host species, and yielded similar LC50 and LT50 estimates. Conclusively, the millet-based technology for production of granular cultures of Z. radicans was easy, inexpensive and highly efficient, and it could be superior to previous methods used in mass production of mycelium-based preparations of Entomophthorales since this new approach requires no special additives, drying, freezing and milling. This technology may suit to mass production of culturable but nutritionally fastidious entomopathogens from the Entomophthorales.

The entomophthoralean fungi Batkoa sp., Furia sp. and Neozygites floridana have been suggested for biocontrol of insect pests: the first two for control of spittlebug pests of pasture and sugarcane, and the third for mites of agricultural importance. To develop these agents as biopesticides and bioacaricides, it is important to have available culture media that maximize production at low cost. The research reported here evaluates, in different combinations and concentrations, the effect of four complex sources of nitrogen on production of mycelium or hyphal bodies in liquid media of all three species. Yeast extract allowed the highest production of Batkoa sp., with a concentration of 0.5% being the most suitable for vegetative (mycelial) growth. The combination of 0.33% each of yeast extract+beef extract+skim milk allowed the highest production of Furia sp. Mycelium. The combination of yeast extract+skim milk (0.5% of each) allowed the second highest production of Furia sp., and was the most suitable for mass production due to the lower cost. The combination of 1%each of yeast extract+peptone+skim milk was the most suitable for production of N.floridana hyphal bodies.

In an effort to meet the stringent requirements towards registration of microorganisms as biopesticides, several molecular techniques were tested as part of a strategy to develop a quality control system for Pseudozyma flocculosa, the active ingredient of Sporodex, a biofungicide used for the control of powdery mildew fungi. In the first approach, multiplex PCR fingerprints generated by three sets of primers allowed differentiation of several isolates of P. flocculosa from closely related species, or genera such as Tilletiopsis. The same set of primers was used in quality control experiments and revealed fungal contamination that was otherwise not observed by standard culture and microscopy techniques. In addition, the use of random amplified microsatellites generated by (GT)n and (CCA)n primers was applied as a measure of possible genetic variation over 65 repeated subcultures of P. flocculosa. Finally, a novel technique was developed and named reverse intron PCR (RIP), based on the presence of an intron revealed by partial sequencing of mtLSU of P. flocculosa. RIP allowed not only differentiation of P. flocculosa from other species but also separated the isolates of different origins within P. flocculosa. This new technique could find useful applications in phylogenetic studies of closely related fungal species and isolates.

Combined analyses of ITS and LSU rDNA sequences were utilized to resolve the phylogenetic relationships of 98 members of the smut genera Lundquistia, Melanopsichium, Moesziomyces, Macalpinomyces, Sporisorium, and Ustilago (Basidiomycota: Ustilaginales). Minimum Evolution and Bayesian inference of phylogeny resolve three major groups of almost identical composition: Sporisorium, Ustilago, and a basal assemblage of both Ustilago and Sporisorium species. Macalpinomyces deserves generic rank regarding its type species M. eriachnes; all other Macalpinomyces species of our study clearly turn out to be part of Ustilago or Sporisorium. Lundquistia evidently belongs to Sporisorium. Moesziomyces, probably paraphyletic, stands basal to all other genera. Interestingly, Melanopsichium belongs to the Ustilago clade, being the only member of the ingroup not parasitizing on Poaceae. The patchy distribution of commonly used morphological characters along our phylograms points to their variability and dependence on the host's morphological traits instead of being valuable for resolving parasite phylogeny. The new combination: Sporisorium fascicularis comb. nov. (syn. Lundquistia fascicularis) is made.

Podosphaera tridactyla (Ascomycota: Erysiphales) is a morphologically variable species occurring on Prunus s. lat. In order to assess the genetic variation within this species, the rDNA ITS region was amplified from 29 specimens from a range of Prunus species collected in Australia, Switzerland, and Korea. RFLP analysis of the PCR products revealed six groups, and a comparison of sequences from representatives of these groups revealed three clades: Clade 1 contained all specimens from Prunus subgen. Prunus; Clade 2 specimens from a variety of Prunus subgenera, except subgen. Prunus, and Podosphaera longiseta was closely allied to this clade; and Clade 3 contained two specimens, from Japan and Korea. Phylogenetic analysis comparing P. tridactyla with of a range of Podosphaera species suggests that P. tridactyla is paraphyletic.

Fungal endophytes of the genera Neotyphodium and Epichloë are important mutualistic symbionts and pathogens of many cool-season grass species. Here we report the characterization of a secreted N-acetylglucosaminidase from the Neotyphodium sp. endophyte that infects the grass Poa ampla. The enzyme was expressed at low levels within the host, and activity could be detected in the apoplastic protein fraction. Low-level expression could also be detected in endophyte-infected perennial ryegrass (Lolium perenne), Chewings fescue (Festuca rubra subsp. fallax), and tall fescue (L. arundinaceum). The enzyme may function in the recycling of chitin oligomers generated from turnover of the fungal cell wall. This is the first report of a secreted N-acetylglucosaminidase expressed by an endophytic fungus in the infected host plant.

Amphipathic glucose derivatives 3-O-octyl- and 3-O-decyl-D-glucose were synthesized, and the ability to induce fruit bodies on Pleurotus ostreatus was examined. Surfactants such as CHAPS, CHAPSO, MEGA-8, MEGA-9, and MEGA-10 were also assayed. The result was that both glucose derivatives could stimulate fruiting on P. ostreatus, while none of the surfactants assayed in this study stimulated the fruiting of P. ostreatus. These results suggest that sugar moiety is necessary for amphipathic compounds to work as a fruiting signal for P. ostreatus.

In the mushrooms Pleurotus ostreatus and Agrocybe aegerita, expression of the hemolytic proteins ostreolysin and aegerolysin, which belong to the aegerolysin family, has been shown to be initiated specifically during formation of primordia and fruiting bodies. We used rabbit anti-ostreolysin and fluorescent rhodamine-labelled secondary goat antibodies for monitoring ostreolysin and aegerolysin in situ during the mushrooms' development. In parallel, the protein level in developing tissues was monitored with SDS-PAGE and hemolytic assay. Immunolocalization of ostreolysin, visualized by epifluorescence, together with biochemical tests, confirmed specific expression of ostreolysin and aegerolysin in the primordia and fruiting bodies but not in the vegetative mycelia. In the primordia, the proteins were disposed diffusely. In growing and mature fruit bodies they persisted in the lower part of the pileus, in particular in basidia and basidiospores, while in other parts only a few focal remains were observed. Confocal microscopy of immunolabelled sections showed that intracellular ostreolysin was located specifically along the inner edges of hyphae. Since both proteins were found preferentially in the rapidly growing primordia, and in the basidia and basidiospores of maturing fruit bodies, it is suggested that they might play a role in the processes of fructification and/or sporulation.