Although Eutypa lata is the main agent of Eutypa dieback of grapevine, another species of Eutypa has been isolated from diseased grapevines in Northern California. Stromata of this recently discovered Eutypa were also collected from Acer macrophyllum, A. negundo, and Fraxinus latifolia in the vicinity of vineyards, and appeared commonly on Umbellularia californica in some mixed-evergreen forests of Napa and Sonoma counties. This second species of Eutypa was distinguished from E. lata because of the sulcate ostiole of the perithecium and smaller ascospores. A morphological comparison with type specimens revealed identical features between the Californian isolates and E. leptoplaca sensu Rappaz (1987). This identification was confirmed through phylogenetic analyses of Eutypa spp. based on the complete sequence of the internal transcribed spacer (ITS) of the rDNA and partial sequence of the β-tubulin gene. These analyses also separated collections of E. maura, E. sparsa, E. lejoplaca, E. tetragona, E. leptoplaca and E. lata, confirming the previously proposed species concepts. The pathogenicity of E. leptoplaca on grapevine was established using isolates collected from Vitis vinifera, U. californica, and A. macrophyllum. The importance of E. leptoplaca in relation to Eutypa dieback and its role as a necrotrophic pathogen are discussed.

Double-stranded (ds) RNA of various types was detected by electrophoresis in 23 of 25 isolates of Helicobasidium mompa. These dsRNAs varied in size from ca. 2 kbp to more than 10 kbp. dsRNAs from an isolate V1 had two distinct nucleotide sequences for putative RNA-dependent RNA polymerase (RDRP). Their complete sequences revealed that V1 dsRNA1 was 2247 bp in length, with a single ORF that encoded a 706-amino acid residue polypeptide with a predicted molecular mass of 82.6 kDa, and that V1 dsRNA3 was 1776 bp in length, with a single ORF that encoded a 538-amino acid residue polypeptide with a predicted molecular mass of 62.6 kDa. RDRP-conserved motifs were identified in both predicted amino acid sequences. Phylogenetic analysis indicated that V1 dsRNA1 was most closely related to Fusarium poae virus 1, while V1 dsRNA3 was most closely related to Helicobasidium mompa 70 virus. These results indicate coinfection of isolate V1 by two distinct partitiviruses.

A search for double-stranded RNA (dsRNA) was conducted among 106 isolates of the pathogenic basidiomycete Heterobasidion annosum. Of these isolates, 47 were tissue isolates from fruit bodies and 59 were isolated from decayed wood. Nucleic acids were extracted from freeze-dried mycelia and dsRNA was separated by cellulose CF-11 chromatography and confirmed by digestions with specific nucleases. dsRNA was present in 19 and 14% of the tissue and wood isolates, respectively. From five of the fruit bodies containing dsRNA basidiospores were investigated and 10–84% of the germinated basidiospores contained dsRNA. On high nutrient media, the germination frequency of basidiospores was reduced by presence of dsRNA in the fruit body (P<0.05); germination frequencies were 34 and 78% for spores from fruit bodies with and without dsRNA, respectively. The same trend was present also on low nutrient media, although not statistically significant; germination was 3 and 10% for spores from infected and dsRNA free fruit bodies, respectively. Transmission of dsRNA in H. annosum from mycelia into basidiospores together with the lowered germination frequency are likely to play a significant role in the life cycle of the fungus. The relative importance of different transmission routes for dsRNA in H. annosum is discussed.

Phenotypic and genotypic variability of 18 endophytic Guignardia strains from different host plants (Anacardiaceae: Anacardium giganteum, Myracrodruon urundeuva, Spondias mombin; Apocynaceae: Aspidosperma polyneuron; Ericaceae: Rhododendron sp.; Fabaceae: Bowdichia nitida; Leguminosae: Cassia occidentalis; Rutaceae: Citrus aurantium) growing in different sites in Brazil was assessed by means of morphometric measurements and inter-single-sequence-repeat-anchored polymerase chain reaction (ISSR-PCR) amplifications of the DNA. Morphology of conidia and ascospores and growth rates of the Brazilian isolates corresponded well with those of G. mangiferae. Multiple correspondence analysis (MCA) of the ISSR-PCR data yielded three groups of strains, which did, however, not correspond either to the host or to the geographic origin. The same individual tree hosted genotypically different strains indicating multiple infections. Phylogeny based on ITS1-5.8S-ITS2 sequence analyses confirmed conspecificity of the Brazilian isolates with G. mangiferae (syn. G. endophyllicola).

Mycosphaerella punctiformis, the type species of the genus Mycosphaerella, is epitypified by material collected on Quercus robur in The Netherlands. The teleomorph is described in planta, and the Ramularia anamorph, for which the new name R. endophylla is proposed, and the Asteromella spermatial state are characterized in vitro. Sequence data of the nuclear ribosomal DNA are presented and analyzed together with other Mycosphaerella spp. with Ramularia and several other anamorphs. Several strains originating from Quercus, Acer and Tilia showed diverging ITS sequences, indicating that the M. punctiformis complex may comprise more than a single phylogenetic species, but this could not be confirmed by the analysis of our dataset. An endophytic phase is established for the first time in the life-cycle of M. punctiformis, as the species was repeatedly isolated from surface sterilized green healthy leaves of Quercus robur in summer at the epitype locality.

Beauveria bassiana is an entomopathogenic fungus with a growing potential for pest control in different agro-ecosystems worldwide. Such potential brings the necessity of developing a strain specific typing system. In a previous study, we reported the identification of molecular variants in mitochondrial DNA (mtDNA) polymorphism in 15 North American isolates. Results indicated a highly conserved mitochondrial genome showing only two mitochondrial genotypes (mitotypes). In this study we used whole genomic DNA from 18 isolates of B. bassiana, two unidentified Beauveria spp., and one each of B. amorpha, B. cylindrospora and B. nivea from more diverse origins. By doing single- and double-restriction enzyme digestion of total genomic DNA with EcoRI, and HindIII and then probing with BbmtE2, the predominance of mitotypes A and B was observed again, along with three newly described mitotypes (C to E). Additionally, by using whole B. bassiana mtDNA digested with HpaII as probe, we further demonstrate up to nine different mitotypes within B. bassiana. With either of the two probes, distinguished between members of the genus Beauveria and from Paecilomyces farinosus and Metarhizium anisopliae. Phylogenetic analysis could not however distinguish B. amorpha and B. nivea isolates from B. bassiana, suggesting a close genetic relation between the three species of the genus. Altogether, these results show high variability in mitochondrial genome, which can be useful as a reliable tool for the biopesticide industry for both species and isolate specific identification.

The taxonomy of the non-yellow species of the genus Rhizocarpon (Rhizocarpaceae, lichenized Ascomycota) occurring in the Nordic countries, with hyaline and muriform ascospores, has been revised. Rhizocarpon amphibium, R. anaperum, R. distinctum, R. furfurosum, R. lavatum, R. petraeum, R. postumum, R. reductum, R. roridulum, R. rubescens, R. subgeminatum, R. sublavatum (reported from the study area for the first time), R. subpostumum, R. suomiense, R. timdalii, and R. umbilicatum are recognized. Their morphology, anatomy, secondary chemistry, ecology, and distribution in the Nordic countries are investigated and discussed. Distribution maps and a key to the species are provided. The most important characters for separating the treated species are pruinose/epruinose thallus, number of ascospores in asci, ascospore size and number of cells per ascospore in optical view, insoluble lichen pigments of the epihymenium and the proper excipulum, and lichen substances. Seven names are lectotypified and two are neotypified.

Penicillium brevicompactum and other isolates with the compact, complex conidiogenous apparatus typical of the species were sequenced in one ribosomal and two protein coding regions. The aligned DNA sequences were analyzed by maximum parsimony and the data from different loci were tested for compatibility using the partition homogeneity test. Analysis of each of the three loci revealed three clades corresponding to P. brevicompactum, P. olsonii and P. biourgeianum. Using the phylogenetic species concept and the genetic isolation of the clades, P. hagemi, P. patrismei, P. stoloniferum, and P. griseobrunneum are all synonyms of P. brevicompactum. P. volgaense is a synonym of Penicillium olsonii, while P. biourgeianum is a distinct species closely related to P. brevicompactum and P. olsonii. Phenotypic distinctions between the species are mostly based on colony characteristics such as colour. P. bialowiezenze, often treated as a synonym of P. brevicompactum, is most closely related to P. polonicum.

A set of aspergillus strains from culture collections and wild-type black aspergilli isolated on non-selective media were used to validate the use of media with 20% tannic acid for exclusive and complete selection of the black aspergilli. The 20% tannic acid medium proved useful for both quantitative and qualitative selection of all different black aspergilli, including all recognized species: A. carbonarius, A. japonicus, A. aculeatus, A foetidus, A. heteromorphus, A. niger, A. tubingensis and A. brasiliensis haplotypes. Even higher concentrations of tannic acid can be utilized by the black aspergilli suggesting a very efficient tannic acid-degrading system. Colour mutants show that the characteristic ability to grow on high tannic acid concentrations is not causally linked to the other typical feature of these aspergilli, i.e. the formation of brown-black pigments. Sequence analysis of the A. niger genome using the A. oryzae tannase gene yielded eleven tannase-like genes, far more than in related species. Therefore, a unique ecological niche in the degradation of tannic acid and connected nitrogen release seems to be reserved for these black-spored cosmopolitans.

Analysis of 44 isolates of Phytophthora cactorum, isolated from strawberry and other hosts, by AFLP showed that the crown rot pathotype is different from leather rot isolates and from P. cactorum isolated from other hosts. 16 of 23 crown rot isolates, including isolates from Europe, Japan, Australia, and New Zealand, were identical in an analysis based on 96 polymorphic bands from seven primer combinations. Leather rot isolates of strawberry could not be distinguished from isolates from other hosts. The pathogenicity test of all 44 isolates on strawberry plants mostly gave unambiguous results, except for three American isolates, which seemed to have reduced aggressiveness compared to the crown rot isolates. These isolates also differed in the AFLP analysis. Comparing information on the origin of the isolates with results from the pathogenicity test, showed that isolates from strawberry fruits or petioles could be either leather rot or crown rot pathotypes. None of the isolates from hosts other than strawberry caused crown rot symptoms in strawberry.

This report describes tests with Evan's Blue and MTT (3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide) for the assessment of the viability of rhinosporidial endospores. MTT stained a proportion of the spherical bodies that we regard as the Electron Dense Bodies (EDBs), and the cytoplasm of freshly prepared endospores or ones that were stored at 4 °. Slow-freezing at −20 °C, exposure to 10% formalin, or 0.1% sodium azide of the endospores abolished MTT-staining in both sites. Evan's Blue stained the EDBs and cytoplasm of fresh endospores or those stored at 4 °, and of sodium azide-treated or frozen (−20 °)-thawed endospores. TMRE (tetramethyl-rhodamine ethyl ester) specifically labelled the spherical bodies, supporting the conclusion that these spherical bodies have a mitochondrial-like structure. TMRE-staining was however retained in endospores after their treatment with formalin, sodium azide and slow-freezing while MTT-staining was abolished in all these treated endospores.

These results indicate that EvB and TMRE were capable of revealing the morphological integrity of endospores but failed to identify the metabolic inactivation of the endospores after treatment with formalin, sodium azide or slow-freezing. Only MTT was capable of identifying metabolically active endospores and hence their viability and could prove to be of value in standardizing models of infection.

Pythium attrantheridium sp. nov. is a new species isolated from cavity spot lesions of carrots as well as apple and cherry seedlings from various locations widely distributed in Canada and the USA. This fungus is closely related to the heterothallic P. intermedium, but is distinguished by: (1) unique molecular characteristics; (2) unique morphological characteristics; and (3) mating incompatibility with P. intermedium. The ITS region of the nuclear rDNA of all strains of P. attrantheridium studied is different from that of all other known Pythium spp. The oogonia attract a large number of antheridia when compatible mating types contact each other. The positive mating type produces zoospores unlike those of P. intermedium. Thus, biological, morphological and molecular data support the recognition of a new species.

A survey of corticolous crustose lichen collections in connection with the Flora Neotropica project revealed a number of undescribed species of Trichothelium. While a few typically foliicolous taxa may occasionally be found on bark, five species seem to be exclusively corticolous, all with a Trentepohlia photobiont. Three of them are new species: T. angustisporum, T. caudatum, and T. kalbii spp. nov. A key is provided to all five corticolous species, accompanied by data on their distribution and ecology. A considerable range extension is reported for T. horridulum, with new collections from Costa Rica and north-eastern Brazil.

Potebniamyces pyri (anamorph Phacidiopycnis piri) is the causal agent of Phacidiopycnis rot of apples and pears. The disease has recently been recognized in pears in the USA. Little information on the basic biology of the fungus is available. In this paper, we report the effects of culture media, temperature, water potential and pH on mycelial growth, and the effects of media and light on pycnidia production. Prune juice agar was the best for rapid mycelial growth. Pear juice agar, apple juice agar, potato dextrose agar, and oatmeal agar (OMA) also favoured mycelial growth. Czapek-Dox agar was not suitable for mycelial growth. The fungus was able to grow at temperatures from −3 to 25 °C. Optimal mycelial growth occurred between 15 and 20 °. The average radial growth rate on OMA was 3.9 mm d−1 at 15 ° and 4.4 mm d−1 at 20 °. Mycelial growth was not observed after 10 d at 30 °, but growth resumed at 20 °. The fungus failed to resume growth at 20 ° after being incubated for 10 d at 35 °. The fungus was able to grow at water potentials as low as −4 MPa but no growth took place at −7.3 MPa. Active mycelial growth was observed on OMA at pH between 3.2 and 6.1. Optimal growth was observed at pH around 4 and no growth was observed at pH 7.1. No pycnidia or very few formed on the nine media at 20 ° in the dark. Fluorescent light significantly stimulated formation of pycnidia. OMA was the best medium for production of pycnidia and macroconidia. Pycnidia that formed on 8-wk-old OMA cultures incubated at 20 ° under 12 h dark/12 h light produced abundant macroconidia and the technique is recommended for inoculum production of conidia for research.

Randomly amplified polymorphic DNA (RAPD) markers were used to assess the genetic diversity of isolates of grey snow mould (‘gray snow mold’), Typhula incarnata, taken from infected turfgrasses from 40 different locations in the northern USA. Data from 115 markers using 37 RAPD primers showed 48% polymorphism. The distance coefficients between isolates indicate the wide genetic diversity of T. incarnata across the sample area. Dendrograms generated using neighbour-joining (NJ) bootstrap analyses showed three clades and suggest possible recent colonization from common founder groups. Partitioning of the genetic variance using analysis of molecular variance (AMOVA) of four groups based on geographic locations (Michigan, lower and upper peninsula; Minnesota; Wisconsin) showed that genetic variation attributable among groups and within groups was 12.67 and 87.33%, respectively. No correlation was found between geographic distance and pairwise genetic distance of the groups. High outcrossing and sexual recombination of T. incarnata may well be key factors explaining the genetic variability as shown with the low Fixation index (FST) and high average of genetic diversity per locus within groups.

A toxic protein, bassiacridin, was purified from a strain of the entomopathogenic fungus Beauveria bassiana isolated from a locust, using chromatographic methods. The final toxic fraction contained between 0.1 and 0.3% of the proteins of the crude extract. Bassiacridin showed no affinity for ion exchangers, was characterised as a monomer with a mol. wt of 60 kDa and an isoelectric point of 9.5, and exhibited β-glucosidase, β-galactosidase and N-acetylglucosaminidase activities. Injection of fourth instar nymphs of Locusta migratoria with the pure protein at relatively low dosage (3.3 μg toxin g body wt−1) caused a rate of mortality near to 50%. The effects of the crude and pure fractions were characterized at tissular and cellular levels. The formation of melanised spots on tracheae and air sacs and of melanised nodules in contact with the fat body was observed in injected locusts. Alterations of the fine structure of epithelial cells of tracheae, air bags, and integument were also revealed. The insecticidal protein showed a specific activity against locusts. Bassiacridin is different from the other macromolecular toxins of entomopathogenic fungi already described. Microsequencing of peptides generated by trypsic digestion of bassiacridin confirmed that it is a novel molecule and showed that it exhibits a probably limited similarity with a chitin binding protein from yeast.

The European Union (EU) has introduced statutory limits for patulin in fruit products. Species definitions need to be unambiguous for this capability, especially to determine ‘weak spots’ in food, drink and feed production. Fungi were analysed for the isoepoxydon dehydrogenase (IDH) gene of the patulin metabolic pathway to indicate potential patulin production. In several cases inhibition of PCR product formation was indicated. Dilution as a means of overcoming PCR inhibition and to determine optimal concentrations was assessed and the ramifications for nucleic acid analysis in general are highlighted. The inhibitors may be secondary metabolites. However, inhibition was not involved obviously for Penicillium brevicompactum. The gene was detected frequently from Aspergillus section Clavati and Penicillium subgenus Penicillium species. Some strains within certain species were negative for the gene. Detection of the gene product in Byssochlamys is reported.

The diversity and host specificity were studied of a collection of Colletotrichum strains derived from endophytic colonies in leaves of 12 tree species in the Iwokrama Forest Reserve, Guyana. Analysis included ISSR-PCR and RAPD molecular fingerprinting techniques, rDNA ITS sequencing and morphological and cultural characterization. Most strains belonged to one of two species, C. gloeosporioides and a further taxon which is probably referable to C. boninense. Almost no strains were found to be genetically identical, indicating that clonal reproduction does not play a prominent role. No degree of host specificity could be detected even at molecular fingerprint level. The implications for estimation of fungal diversity in closed tropical forests may be profound.

The production of fumonisins, fusaproliferin and beauvericin by Gibberella fujikuroi different mating populations isolated from maize in Argentina was evaluated. From 203 strains of Fusarium verticillioides (G. fujikuroi mating population A), 193 were fumonisin producers. Among members of mating population A, female fertile strains produced 20% more toxin than female sterile ones. Among 78 Fusarium proliferatum strains (G. fujikuroi mating population D) 65 produced fumonisins. The percentage of strains that were high, intermediate and low level toxin producers varied according to the species evaluated and the area from which the strains were isolated. Fusarium subglutinans (G. fujikuroi mating population E) strains produced low levels or were no fumonisin producers. Strains from both G. fujikuroi mating populations D and E were able to produce fusaproliferin and beauvericin. Among the members of F. subglutinans (G. fujikuroi mating population E) the fusaproliferin production was more constant. Co-production of fumonisin, fusaproliferin and beauvericin among the strains belonging to G. fujikuroi D and E was also observed. The co-production of fumonisin, beauvericin and fusaproliferin in maize need to be considered, since from the toxicological point of view interactions between these toxins could occur. The toxigenic ability of the strains evaluated prompt us that is necessary to determine the natural occurrence of fusaproliferin and beauvericin in Argentinean maize.

A violet root rot fungus recently found in Japan was identified as Helicobasidium brebissonii. The fungus was compared with another violet root rot fungus, H. mompa, which is distributed throughout Japan, in terms of morphological characteristics of the basidiospores and the conidial state, sequences of rDNA ITS regions, and pathogenicity on carrot, sweet potato and apple rootstock. The two species were clearly discriminated by these features and the taxonomic status of both species is discussed.