Methanol extracts from 24 Trichoderma isolates, selected as biocontrol agents and representating different species and genotypes from three of the four taxonomic sections of this genus (T. sect. Trichoderma, T. sect. Pachybasium and T. sect. Longibrachiatum) were screened for antibacterial, anti-yeast and antifungal activities against a panel of seven bacteria, seven yeasts and six filamentous fungi previously used in similar studies. Two different growth media were tested (potato dextrose broth and CYS80), and all isolates included in the antimicrobial tests showed at least one inhibitory activity against one of the target microorganisms in one of the two culture media. No statistically significant differences were detected in the number of active strains between the two culture media, but the highest number of inhibitory strains against bacteria and fungi were found in strains from Trichoderma sect. Pachybasium, whereas strains from T. sect. Longibrachiatum showed the highest anti-yeast values. In all cases, a correlation was found between the strains that were active against yeasts and fungi. However, some degree of variability was detected for strains within the same taxonomic section. In general terms, strains from T. asperellum (mainly in CYS80 medium), and T. longibrachiatum gave the best non-enzymatic antimicrobial profiles.

Eutypa lata, the causal agent of Eutypa dieback of grapevines, is difficult to identify on the basis of colony morphology and is often out-competed by other fungi when isolated from wood. To facilitate diagnosis of the pathogen, we designed SCAR primers capable of amplifying DNA of E. lata and constructed a genomic DNA library from which DNA sequences specific to E. lata were identified and sequenced. SCAR primers were used to identify E. lata directly from culture without the requirement for DNA extraction or prolonged incubation periods and could also detect the pathogen in DNA isolated from grapevine wood. RFLP probes were used in slot-blot assays to detect the pathogen in DNA isolated from 1 yr old cane as well as from mature grapevine trunks. The markers developed in this study have the potential to be used as a research tool to gather information on the epidemiology of the disease and to assess the efficacy of potential control agents against E. lata.

This study aims to clarify taxonomic relationships within the current concept of Xanthoria parietina in northern Europe. For comparison, X. calcicola was also included in the study. Morphological as well as molecular data were utilized. Morphology indicated the presence of three species, Xanthoria parietina, X. calcicola, and X. aureola, the latter of which is resurrected here from synonymy. The most important separating characters involve colour and thickness of the thallus, lobe width, morphology of laminar structures, and the texture of the upper surface. X. aureola, as recognized here, mostly occurs on seashore rocks. Part of the IGS region as well as the complete ITS were sequenced in 70 individual thalli representing ten geographical regions in Europe. In total, 19 different IGS haplotypes and 20 different ITS haplotypes were present in the data set. Owing to indications of possible recombination between the IGS and the ITS, the two data sets were analyzed separately. Haplotype networks were estimated, both of which indicate that X. parietina is distinct from X. aureola and X. calcicola. In our sample, the two latter do not share haplotypes, but are only separated by a few mutational steps.

The phylogeny of the family Micareaceae and the genus Micarea was studied using mitochondrial small subunit ribosomal DNA sequences. Phylogenetic reconstructions were performed using Bayesian MCMC tree sampling and a maximum likelihood approach. The Micareaceae in its current sense is highly heterogeneous, and Helocarpon, Psilolechia, and Scutula, all thought to be close relatives of Micarea, are shown to be only distantly related. The genus Micarea is paraphyletic unless the entire Pilocarpaceae and Ectolechiaceae are included, as also indicated by an expected likelihood weights test. It is suggested that the Micareaceae is reduced to synonymy with the Pilocarpaceae, which also includes the Ectolechiaceae, and that Micarea may have to be divided into a series of smaller genera in the future. Micarea species with a ‘non-micareoid’ photobiont group with Psora and the Ramalinaceae, whereas Micarea intrusa appears to belong in Scoliciosporum. Three species fall inside the paraphyletic Micarea: Szczawinskia tsugae, Catillaria contristans, and Fellhaneropsis vezdae. Tropical foliicolous taxa are nested within groups of mainly temperate and arctic-alpine distribution. A ‘micareoid’ photobiont appears to be plesiomorphic in the Pilocarpaceae but has been lost a few times.

Two specimens of a new species of Hypocrella with large stromata were collected in Bolivia and Costa Rica. The morphology of the new species, H. macrostroma sp. nov., was compared with that of other species with large stromata, i.e. H. africana, H. gaertneriana, and H. schizostachyi. In addition, phylogenetic analyses of partial sequences from three genes, large subunit nuclear ribosomal DNA (LSU), translation elongation factor 1-α (EF1-α), and RNA polymerase II subunit 1 (RPB1), were conducted to determine the relationships of the new species to other species of Hypocrella/Aschersonia. Phylogenetic analyses show that H. macrostroma belongs to a strongly supported clade that includes H. africana, H. schizostachyi, and Aschersonia insperata, whereas other Hypocrella species belong to two sister clades. Hypocrella macrostroma is described and illustrated, and a lectotype is designated for H. gaertneriana.

A 3193 bp contiguous sequence has been cloned from the oleaginous fungus Mucor circinelloides, that contains a full-length gene encoding a putative NADP+[ratio ]dependent malic enzyme (EC. 1.1.1.40). The cloned DNA contains a 2154 bp putative open reading frame containing five introns and encoding a protein of 616 amino acids. The gene encoded what appeared to be an anaerobic isoform of malic enzyme (isoform II); this conclusion is supported by transcript analysis and by the fact that the ORF contains an N-terminal mitochondrial target sequence (a similar cellular location was identified for the anaerobic malic enzyme in Saccharomyces cerevisiae; Boles et al. 1998). The cloned gene did not encode either isoform III (the isoform associated with active growth) or isoform IV (associated with lipid accumulation) previously identified. M. circinelloides therefore must possess (at least) two structural genes for malic enzyme.

To understand the phylogenetic relationships and evolution of the powdery mildew genus Sawadaea (Ascomycota: Erysiphaceae), obligate parasitic fungi of maples, we performed molecular phylogenetic analyses based on 47 ITS and ten 28S rDNA sequences. Seven major clades of Sawadaea, each represented by powdery mildew specimens collected from a single or a small number of closely related sections of Acer (maples), were identified in this study, suggesting that a close evolutionary relationship exists between Acer (host) and Sawadaea (parasite). A 6–11-base insertion/deletion was found in the ITS1 region of Sawadaea, and the presence or absence of the indel was consistent within the respective clades. Because the outgroup genera Podosphaera and Cystotheca have no deletions in these sites, deletion of the sequences may have occurred during the divergence of the respective clades of Sawadaea. The seven clades of Sawadaea were divided into two geographical groups, viz. an East Asian and a global group, based on the countries of collection. Calculation of the evolutionary timing of Sawadaea using molecular clocks showed that the divergence of different species of Acer occurred many millions of years before the radiation of Sawadaea. Thus, the close evolutionary relationship between Sawadaea and Acer found in this study might not be due to a true coevolutionary process. Powdery mildew fungi belonging to Sawadaea may have jumped onto Acer spp. long after the radiation of the major sections of these trees, and then expanded their host ranges according to the phylogeny and geographical distribution of Acer.

The position of several endemic and rare species in Agaricus sect. Duploannulati and the limits of the section were investigated by analysis of sequence data from the ribosomal DNA ITS. The results supported the recognition of two groups, which we treat as subsections Chitonioides and Duploannulati. Most of the species studied proved to belong to subsect. Chitonioides. Species excluded from the section, as well as other potential members of sect. Duploannulati, are considered. Morphological traits deemed important for identification of A. nevoi, A. pequinii, A. gennadii, A. rollanii, and A. padanus are discussed. Taxonomic positions of these species in morphologically-based systems and according to molecular systematics data are compared and analyzed.

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.

Terminalia ivorensis, a tree of central African origin, is planted in several tropical countries for timber and veneer production. During the course of a recent disease survey, an unknown fungus was found associated with basal cankers on dying T. ivorensis in Ecuador. The fungus has orange fruiting structures and septate, fusoid ascospores, similar to those of Cryphonectria, a well-known genus of canker pathogens. The aim of this study was to identify the fungus and to assess its pathogenicity. Identification was based on morphological characteristics as well as DNA sequence data. DNA sequence data from the ITS regions of the rDNA operon and two regions of the β-tubulin gene, were compared with published sequences of Cryphonectria species and the closely related genera Endothia and Chrysoporthe. Pathogenicity tests were conducted on T. superba saplings. Morphological characterisations revealed that the conidiomata of the fungus from T. ivorensis, differed from those typical of Cryphonectria in being superficial and rostrate. Only Cryphonectria longirostris was similar to the fungus from T. ivorensis, but could be distinguished from it based on conidial size. Phylogenetic analyses showed that the fungus from T. ivorensis grouped closely with species of Cryphonectria, Chrysoporthe and Endothia, yet formed a distinct clade. Pathogenicity tests on T. superba provided evidence that the fungus is able to cause distinct stem cankers. We conclude that the pathogenic fungus from T. ivorensis represents a new genus and new species in the Diaporthales and we provide the name Rostraureum tropicale for it. The genus is typified by R. tropicale. Furthermore, C. longirostris is transferred to Rostraureum.

Two new species of the aero-aquatic anamorph genus Pseudaegerita, P. conifera sp. nov. and P. websteri sp. nov. are described and illustrated. Both species were found on submerged plant material in Spain. The new species are compared morphologically with the seven species now accepted in the genus.

The genus Leptographium was described in 1927 and currently includes 48 species, with L. lundbergii as the type species. In recent years, the taxonomic status of L. lundbergii has not been uniformly agreed upon and it has been the topic of considerable debate. The problem was compounded by the absence of a type specimen, and the species was epitypified at a later stage. Unfortunately, the whereabouts of the epitype is now unknown. In 1983, Wingfield & Marasas described L. truncatum, which is morphologically similar to L. lundbergii. Based on DNA comparisons and similarities in their morphology, this fungus was reduced to synonymy with L. lundbergii. The loss of the type specimen as well as variation in the morphology of strains identified as L. lundbergii prompted us to re-examine the taxonomic status of this species. A number of strains from various geographic areas were studied. These include a strain of L. lundbergii deposited at CBS by Melin in 1929 (CBS 352.29) as well as the ex-type strain of L. truncatum. The strains were compared based on morphology and comparison of multiple gene sequences. Three genes or genic regions, ITS2 and part of the 28S gene, partial β-tubulin and partial elongation factor 1-α were compared. Strains currently identified as L. lundbergii, represented a complex of species. Strains initially described as L. truncatum clustered separately from other L. lundbergii strains, could be distinguished morphologically and should be treated as a distinct taxon. L. lundbergii is provided with a new and expanded description based on a neotype designated for it. A third group was also identified as separate from the main L. lundbergii clade and had a distinct Hyalorhinocladiella-type anamorph, described here as H. pinicola sp. nov.

We compared the ability of five strains of the ericoid mycorrhizal fungus Hymenoscyphus ericae to utilise glutamine, ammonium or nitrate at high or low carbon (C) availability. The pattern of intraspecific variation in growth was affected by C availability. When C supply was high, growth differences between strains were explained by the total amount of nitrogen (N) taken up, suggesting variation in uptake kinetics. Under C-limiting conditions, strain differences were linked with their nitrogen use efficiency, implying intraspecific differences in N metabolism. The relationship between growth on glutamine and pH shifts in the media indicated that there was intraspecific variation in glutamine transporters. In addition, the correlation between pH changes and the amount of glutamine-N recovered as ammonium in the media indicated that there were intraspecific variations within the enzymatic pathways involved in glutamine metabolism. Our findings, compared with those of a previous study involving the same ericoid strains, draw attention to the temporal variation in nitrogen source utilisation by ericoid mycorrhizal fungi when maintained in axenic culture.

Downy mildew of sweet basil (Ocimum basilicum) has become a serious disease issue for the producers of sweet basil in Switzerland since it was first recorded in 2001. Reported in Africa in Uganda as early as 1933, major outbreaks of this disease in Europe were first noted in Italy in 1999 and in the USA from 1993. Previous reports have named the pathogen as Peronospora lamii. Its preferential hosts belong to the Lamiaceae family including basils (Ocimum spp.), mints (Menta spp.), sages (Salvia spp.) and other aromatics. This study investigated the taxonomic status of the downy mildew pathogen, using both morphological characters and molecular analysis of the ITS region of the rDNA. The inherent variability of conidial dimensions made species differentiation difficult. Sequence homology and phylogenetic analysis of nine collections of the Peronospora on sweet basil showed unique ITS sequences distinct from those of P. lamii and any other sequenced Peronospora species. This paper describes and illustrates the morphology of this presumably undescribed species of Peronospora. Its taxonomic position and relationships with other related species in the same genus are presented and discussed. In addition to this work, PCR primers for real time PCR analysis have been developed for the specific detection of this downy mildew pathogen from infected tissues or seeds. It is shown that these primers can also be used in classic PCR.

The variability in conidiogenesis of the coprophilous Basifimbria aurea, type species of the genus, is redescribed and illustrated, and is similar to that of B. spinosa. The distinction of the species from Stenocephalopsis subalutacea (syn. Rhinotrichum subalutaceum) is emphasized.

The taxonomic position and properties of an unidentified fungus isolated from wheat roots was investigated. The Sterile Red Fungus (SRF) is characterised by its fast growing habit, red pigmentation, non-sporing nature and mycelial form resembling some Laetisaria and Limonomyces species. rDNA variation was used to study the relationship of the SRF to Laetisaria spp. and Limonomyces spp. Nucleotide sequence obtained from eight Laetisaria and three Limonomyces species representing the ITS 1 region, were analysed by PCR and direct sequencing. Plant growth promoting properties of the five taxa were also determined. The SRF had closest identity (98%) to British material of Limonomyces roseipellis. UPGMA analysis of ITS 1 DNA sequence support a close relationship between SRF and L. roseipellis. The relationship inferred by nucleotide sequence data was supported by phenotypic analysis; both L. roseipellis and the SRF were shown to promote the growth of wheat plants. Unexpectedly, Laetisaria arvalis and material named as Limonomyces roseipellis from New Zealand appeared to be closely related with 98% rDNA sequence identity, suggesting the misidentification of the New Zealand collection.

Traditional taxonomy of aquatic hyphomycetes has been based on conidial morphology and development. Since the predominantly tetraradiate and sigmoid forms are due to convergent evolution, they are often phylogenetically non-informative. The comparison of nuclear small-subunit ribosomal DNA sequences of 30 species (22 new, eight previously published) assigned 22 to Leotiomycetes, four to Dothideomycetes, three to Sordariomycetes, and one to Orbiliomycetes. Eight species of Anguillospora were distributed among the Leotiomycetes, Dothideomycetes, and Orbiliomycetes. All three anamorphs connected with Massarina were assigned to the Pleosporales, however, Clavariopsis aquatica and Tumularia aquatica separated from Anguillospora longissima. The nSSU rDNA sequences of several species were identical (e.g. Anguillospora crassa and A. furtiva), suggesting the need to include less conservative genes for resolving such differences.

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 amplified fragment length polymorphism (AFLP) was used to study genetic variation in Pyrenophora teres causing net blotch of barley in Finland. The mean similarity was 93% between all isolates and a bit higher within two distinct populations based on 175 AFLP markers. Despite the high genetic similarity, 70 unique AFLP genotypes were identified among 72 isolates. Most of the genetic variation (68.5%) was observed within a field population and a smaller portion (30.3%) between them. Significant genetic differentiation (Fst=0.308, P<0.001) was identified between field populations. However only 1.2% of the variation was observed between mating types within a field and a lack of genetic differentiation (Fsc=0.017, P=0.087) was observed. The occurrence of the form of blotch (spot type, f. sp. maculata, or net type, P. teres f. sp. teres) was identified with specific PCR. All isolates were found to be of the net type. The existence of both mating types (MAT1 and MAT2) was identified for the first time in Finland and the ratio of the two mating types was almost 1[ratio ]1 in both locations. The evolutionary potential and the possibility of sexual reproduction of P. teres occurring in Finland are discussed.

105 isolates of Verticillium fungicola from Spanish mushroom crops collected between 1992 and 1999 were tested in vitro for their sensitivities to prochloraz-manganese. Dose response relationships for inhibition of mycelial growth by the fungicide were assessed in radial growth experiments on fungicide-amended malt extract agar. The ED50 values recorded for all 105 isolates studied ranged between 0.8 ppm in 1992 and 8.8 in 1998, with an average of 2.9. 86% of the isolates tested were more sensitive to prochloraz-manganese and had ED50 values below 5 ppm, while the other 14% were slightly tolerant with ED50 values equal or above 5 ppm. Of those tested from 1999, 60% (21 isolates) grew with 50 ppm and 40% (14) also at 100 ppm, although mycelial growth was inhibited at least by 82 and 87%, respectively. The resistance factor calculated ranged from low fungicide resistance (RF=3.0) in 1992 to moderate resistance (RF=12.6) in 1998. These data confirm that the sensitivity of V. fungicola to the prochloraz-manganese gradually diminishes.