Dry core rot of apple (DCR) and Alternaria black rot of citrus (ABR) have in the past respectively been ascribed to Alternaria alternata and A. citri. In recent years, however, it has been speculated that several other species of Alternaria could also be associated with these diseases. In an attempt to elucidate the identity of these taxa, 25 isolates associated with DCR, and 26 isolates associated with ABR were selected for molecular characterisation. Nucleotide sequences of 1116 sites including the histone gene section and the internal transcribed spacers (ITS 1 and 2) of the rRNA gene were determined for these isolates. The gene trees generated from the individual and combined data sets using maximum parsimony, maximum likelihood and neighbour-joining analysis methods distinguished five clades with strong bootstrap support, namely Alternaria sp., A. arborescens, A. infectoria, A. tenuissima, and a clade containing isolates variable in morphology, referred to as the Alternaria group. In the alignment of the combined ITS and histone data set, unique transition/transversion substitutions, as well as positional insertions and deletions were observed for each of the above clades. In addition, key sequences in the form of serial composing nucleotides in both the ITS and histone sections of the alignment were also discovered for the molecular identification of A. arborescens, A. infectoria and A. tenuissima. The final phylogeny also indicated that no host specificity existed among the species associated with these two post-harvest disease complexes. Contrary to the host specificity observed on leaf diseases of these hosts in the field, it appears that the post-harvest diseases are the result of adverse storage conditions and opportunism of different small-spored Alternaria spp.

Species delimitation within the core group of Peziza is highly controversial. The group, typified by P. vesiculosa, is morphologically coherent and in previous analyses of LSU rDNA sequences it formed a highly supported clade. Phylogenetic diversity and species limits were investigated within the group using sequences from the complete ITS region (ITS1-5.8S-ITS2). Eighty-three specimens were selected for molecular study from a larger sample of material studied morphologically to explore the intra- and interspecific variation of each putative species. The sister group taxon, P. ampelina was used as the outgroup and two specimens of P. subcitrina were additionally included. Seven independent lineages of rDNA were identified (I–VII), each representing one to several species. These lineages form two larger clades, A (II, and I or III) and B (IV–VII), supported by macromorphology: small (generally <2 cm), shallowly cup- to disc-shaped apothecia (A) and large (up to 15 cm), deeply cup-shaped to expanded apothecia (B). The overall exciple structure (a stratified or non-stratified medullary layer) and to some degree spore surface relief, likewise support the groupings. Clade A contains taxa with smooth or nearly smooth spores (except for P. lohjaënsis), while clade B contains taxa with a range of spore ornamentations, from smooth, finely warty to distinctly warty, and spiny. The position of groups I (P. vesiculosa and P. ammophila) and III (P. lohjaënsis) are uncertain, and these taxa also deviate morphologically from the other clade A members. The following species are recognized based on morphology and ITS rDNA analyses: P. ammophila and P. vesiculosa (I); P. alcis, P. ampliata, P. domiciliana, P. fimeti, P. nivalis, and a number of putative species or intraspecific entities (II); P. lohjaënsis (III); P. sp. c (IV); P. arvernensis (V); P. echinispora and P. sp. d (VI); and P. varia (VII). The nomenclature of these species is analyzed and taxa are typified as necessary. Based on ITS and morphology, we found no justification for recognizing more than one species in the ‘P. varia complex’, including 27 specimens that have been referred to under the names P. cerea, P. micropus and P. repanda, from an array of substrates and different geographical areas. Morphological characters previously used to delimit species within this complex, such as colour variation of the apothecia, presence or absence of a stipe, stratified or non-stratified medullary exciple (or thickness of the excipular layers), cell types in the outermost exciple and moniliform vs filiform paraphyses were not correlated with the subgroups supported by ITS analyses and appeared to be plastic. Therefore, P. cerea and P. micropus are placed in synonymy with P. varia. The name P. repanda is rejected. Levels of sequence divergence were low within group II, comprising 33 small apothecial specimens. Twelve fine-scale lineages were identified, but the analyses did not resolve relationships among these. P. granulosa sensu Boudier is considered a synonym of P. fimeti. These have previously been distinguished mainly by occurrence on various soil types, including burnt soil and soil mixed with sawdust or woodchips vs on dung. The substrate and habitat have been much emphasized in the taxonomy of Peziza, but the results obtained here indicate that populations on a diverse array of substrates may be closely related, or indeed, conspecific.

In several studies of oak decline in Europe, one semi-papillate (Phytophthora psychrophila sp. nov.) and two non-papillate homothallic Phytophthora species (P. europaea and P. uliginosa spp. nov.) were isolated, together with other Phytophthora species, from rhizosphere soil samples which could not be assigned to existing taxa. P. psychrophila differs from other semi-papillate species of Waterhouse's morphological Group IV, like P. ilicis and P. hibernalis, by its uniform, dome-shaped and cottonwool-like colony growth pattern on V8 juice agar and malt extract agar, the occurrence of sympodially branched primary hyphae, the high variation in size and shape of the sporangia, shorter pedicels, lower optimum temperature for growth, and ITS sequences. P. europaea is distinguished from related non-papillate Group V and VI species, namely P. fragariae, P. cambivora, and the ‘alder phytophthora’, by producing oogonia with tapered bases, irregular walls and exclusively paragynous antheridia, its cardinal temperatures for growth, and ITS sequences. P. uliginosa differs from related Group V and VI species by its large oogonia with exclusively paragynous antheridia, the predominant occurrence of ellipsoid sporangia with markedly wide exit pores, its slow growth, low cardinal temperatures, its colony growth patterns, and ITS sequences. P. uliginosa is separated from P. europaea by its larger oogonia without tapering bases, lower cardinal temperatures and growth rates, different colony growth patterns, and greater aggressiveness on Q. robur.

Internal transcribed regions (ITS1 and ITS2) of 11 different fungi, i.e. 8 species of Alternaria, Nimbya gomphrenae, Stemphylium vesicarium, and Ulocladium botrytis, were sequenced. Their phylogenetic relationships with another 36 species in Pleosporaceae in GenBank were analyzed by parsimony and distance methods. These two methods positioned filament-beaked Alternaria as a monophyletic group discrete from the other members in genus Alternaria. A hypothesis proposing that the filament-beaked speices of Alternaria evolved a particular evolutionary route distinguishable from other species based on molecular evidence, the unique traits of morphological adaptation for liberation and their subsistent strategies has been discussed.

Intraspecific heterogeneity among 27 Asian monoconidial isolates and four ATCC ex-type cultures of Monascus purpureus from red rice and sufu was revealed as mycelial compatibility groups (MCGs) using mycelial reactions of 496 mycelial parings. Among 88 pairs, at least three kinds of mycelial incompatible reactions were found at the interaction zone of confronting colonies as: (1) closely forming but not overlapping colonies; (2) sparsely forming aerial mycelia; and (3) thick mycelial band followed by mycelial deterioration of one partner. A mycelial-free zone was not found in any interaction. Compatible interactions were evidenced by freely intermingled hyphae with no apparent inhibition of growth of either partner. At least six MCGs were found based on compatible reactions. MCG1 and MCG2 consisted of 11 isolates, mostly from Thailand. Also, MCG1 contained two ATCC ex-type cultures, ATCC16365 (from Indonesia) and ATCC26264 (from Taiwan). In addition to nine Thai isolates, MCG2 included T9·1 isolate from China and isolate ATCC16427. Furthermore, four small MCGs of M. purpureus were identified. MCG3 (isolates A4·8 & TT4·2) and MCG4 (isolates 10 & M6) were comprised of isolates from Thailand. MCG6 consisted of Japanese isolate M22, and ATCC16360. Isolate ATCC16360 was of unknown origin and previously had been identified as M. anka. MCG5 consisted of Japanese isolate M20 and two Thai isolates, M13 & M14. The result indicated the Thai isolates of MCG1 (16·1, T8·6, TT2·3, TT5·1, M1, M2, M3, M7 & SR) were the same clone as those isolates from Indonesia and Taiwan. Also, the Thai isolates of MCG2 (4, 8, 11, TT1·1, M4, M8, M15, M17 & M18) along with the Chinese isolate T9·1 and ATCC16427 were from the same genetic origin. This study was correlated to those of RAPD analysis of genetic variation within a collection of Monascus spp. isolated from red rice and sufu. M. purpureus isolates used as a starter culture of red rice production among Asian countries were from a rather restricted genetic pool, e.g. China, Taiwan and Japan, although the isolates of M. purpureus studied were moderately genetically diverse.

The phylogeny of Nephroma was studied by nucleotide sequences of the mitochondrial ribosomal small subunit (mtSSU rDNA) and the internal transcribed spacers of the nuclear ribosomal repeat (ITS), together with chemical characters. The biological material included both bipartite and tripartite species and all Nephroma species native to northern Europe. Phylogenetic analyses demonstrated that all Nephroma species form a monophyletic group and that Peltigera constitutes the sister group to Nephroma. The two gene regions revealed qualitatively similar relationships within Nephroma and chemical characters had a minor impact on tree topologies. The results demonstrated that tripartite Nephroma species do not form a monophyletic group within the genus, this being in agreement with previous findings from bi- and tripartite Peltigera species. The results also indicated that N. resupinatum does not form a monophyletic group with all other bipartite Nephroma species but form a sister group to the studied Nephroma taxa. Furthermore, N. helveticums. lat. is highly variable and seems to represent aggregates of closely related taxa. Also N. laevigatum and N. resupinatum are genetically variable. All European Nephroma species can be rapidly and accurately identified on the basis of their fungal ITS sequences. This will prove useful in ecological and environmental studies.

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.

The level of molecular variability in the arbuscular mycorrhizal fungus (AMF) Glomus intraradices was assessed. We established several monoxenically growing single-spore cultures of the G. intraradices collected from a long-term soil tillage experiment in Tänikon (Switzerland). They were grown in symbiosis with Ri T-DNA transformed carrot roots. The ITS region was amplified from genomic DNA of fungal spores using ITS1 and ITS4 primers. The amplified ITS region of three of the fungal isolates was cloned and sequenced. All sequences obtained from our samples closely resembled those previously reported for G. intraradices, and no evidence for genetic exchange was found on the interspecific or intergeneric level. Phylogenetic analysis showed the presence of at least two different sequence families within a single-spore isolate. The single strand conformation polymorphism (SSCP) method detected different sequence types in a single PCR sample. This technique enabled us to detect several distinct ITS sequences within each of the single-spore isolates of the AMF. Estimates of intra- and intersporal sequence diversity were obtained by comparing diversity within and among the single-spore isolates. A higher level of diversity was found among spores than within a single spore.

The phylogenetic relationships of several smut fungi parasitic on dicotyledons were analysed. Parsimony analysis was performed based on the sequences of the ITS regions of the rDNA genes. Three genera were considered: Microbotryum, Sphacelotheca and Ustilago. The cladogram showed a dichotomy: the species of Microbotryum and Ustilago parasitic on dicotyledons (dicot Ustilago) were found to be divided into two independent taxa among the Microbotryaceae. A divergent mechanism of evolution of the species of each of these two clades with their respective hosts could be involved in this dichotomy. According to our results, Microbotryum appears monophyletic and restricted to the anthericolous smuts on Caryophyllaceae. However, no morphological characters have yet been found to support this distinction, and we refute the denomination Bauhinus as defined by Moore to describe the group of ‘dicot Ustilago’ as it leads to controversial determinations. Sphacelotheca belongs to Microbotryales, but could not be synonymised with Microbotryum as S. polygoni-persicariae is in an independent clade. Lastly, U. duriaeana is independent of the ‘dicot Ustilago’ clade; the position of this species among Microbotryales is still uncertain.

Genetic and physiological diversity was assessed in isolates of the lichen-forming fungus Xanthoria elegans originating from a range of geographic localities and climatic regimes including polar and temperate regions. In vitro cultures were established and isolates examined by production of multi-locus RAPD markers and by sequence comparison of the ITS region of the rDNA tandem repeat unit. Both molecular techniques revealed significant variability. Phylogenetic analysis of RAPD profiles (226 markers) clustered isolates from particular locations together and Mantel's test showed a correlation between genetic divergence and extent of geographic separation. Phylogenies based on ITS sequence divergence were not well supported and did not correlate with the geographic origin of samples, suggesting a single locus approach to be less informative than RAPD data in a study of this nature. Physiological investigations also revealed significant variability between isolates with different geographic origins. Temperature had a significant effect on relative growth rate (RGR) such that X. elegans originating from sites with lower mean annual temperatures had significantly higher RGRs at all test temperatures between 2 ° and 18 °C. Enhanced metabolic activity might be an adaptation for growth in colder climates. The results demonstrate high genetic diversity within this morphologically variable species in response to geographic and environmental factors, and are discussed in relation to data obtained for non-lichenised fungal species and the possibility of cryptic speciation.

Alternaria alternata, A. radicina and A. dauci are important seed-borne fungi on carrot, with the first two species having a high toxigenic potential, for which a specific and sensitive detection method is required. Because both the traditional deep-freeze-blotter method and plating on selective medium are time consuming and laborious, a PCR-based assay was developed. Sequences of the internal transcribed spacer regions of the ribosomal gene repeat (rDNA) from 45 different Alternaria isolates were determined, a restriction fragment length polymorphism analysis was performed and a phylogenetic tree was constructed. Based on the sequences, specific primers for detection and identification of the three Alternaria species on carrot seeds and roots were designed. The primers were highly sensitive and were shown to be able to differentiate between the three Alternaria species. A. alternata and A. radicina could be detected in DNA isolated from carrot material applying the specific primers, even at low infection levels. The PCR-assay was compared to the deep-freeze-blotter method (DFBM) and plating on Alternaria radicina Selective Agar (ARSA, for A. radicina) by testing naturally infected seed samples and root material. Results of the PCR-assay were similar to those of the blotter method and plating on ARSA for the detection of A. alternata and A. radicina. A positive correlation was found between the percentage of seed infection established by the blotter method and the intensity of the amplified, specific product. The PCR-assay based on the specific primers developed seems to be a good alternative for the deep-freeze-blotter method and plating on ARSA, especially when time is an important issue.

The ear rot severity of 15 maize hybrids and the accumulation of fumonisin B1 (FB1), fumonisin B2 (FB2), beauvericin (BEA) and fusaproliferin (FP) after artificial inoculation in the field with a toxigenic strain of Fusarium proliferatum has been investigated in Poland during the seasons 1996, 1997 and 1999. The year of inoculation proved a significant influence on ear infection degree and mycotoxin accumulation. Inoculated ears contained 11–71% Fusarium-damaged kernels. Mycotoxins were detected in all hybrids at levels of 18–231.9 μg g−1 for FB1, 0.4–26.5 μg g−1 for FB2, 0.2–19.6 μg g−1 for BEA and 0.3–6.4 μg g−1 for FP. Mycotoxin concentrations were higher in Fusarium-damaged kernels (up to 361.5, 41.1, 44.3 and 10.0 μg g−1 for FB1, FB2, BEA and FP, respectively) than in healthy-looking kernels (up to 26.0, 2.3, 1.9, 0.3 μg g−1 for FB1, FB2, BEA and FP, respectively). All hybrids showed high susceptibility to the fungal infection and high toxin content in kernels during the three years of investigation.

A method for the early detection and diagnosis of common bunt, caused by Tilletia tritici, has been developed. The technique is based on a nested PCR amplification of DNA from T. tritici in infected plant inflorescence tissue at the stage of internode elongation. Primers amplifying a 212 bp fragment in T. tritici were designed on the basis of sequences in the ITS2 region in the ribosomal RNA genes. Disease diagnosis obtained with nested PCR amplification correlated well with the conventional disease diagnosis, which involves manual observation of disease symptoms at maturity. With the present method, wheat varieties can be screened for resistance towards common bunt at the first node stage. There was no variation between the ITS2 region from T. tritici, T. laevis, T. controversa, and T. bromi and the primers are therefore not species specific. The PCR diagnosis method has only been validated for common bunt caused by T. tritici, but it is likely also to be applicable to T. laevis and T. controversa.

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.

The objectives of this study were to examine genetic variation, physiological dissimilarities and diversity in pathogenicity between Verticillium fungicola var. aleophilum and var. fungicola and within a population of six V. fungicola var. fungicola strains responsible for dry bubble outbreaks on mushroom farms. Genetic variability was investigated using random amplification of polymorphic DNA (RAPD). Mycelial growth rate, extracellular enzyme production and susceptibility to hydrogen peroxide were used to examine physiological dissimilarities. Variation in pathogenicity was studied both in vitro and during mushroom cultivation. All the physiological properties studied indicated that var. aleophilum isolates were potentially more efficient than var. fungicola isolates for rapid colonisation of the mushroom cultivation medium. They could then interact more efficiently with Agaricus bisporus to produce dry bubble disease. RAPD analysis confirmed that all the French isolates belonged to var. fungicola, and two isolates were distinguishable from the homogeneous group constituted by the others. These isolates had a higher mycelial growth rate and lower extracellular enzyme activities in liquid media, except for chitinases. Their spores were more susceptible to germination inhibition by hydrogen peroxide, and they were responsible for higher levels of affected mushrooms. The two varieties might be regarded as pathotypes that are geographically isolated, and variation in isolates of var. fungicola might have consequence for mushroom growers.

A new order is introduced (Jahnulales) to accommodate ascomycetes with stalked/sessile and dimorphic ascomata, hyphal stalk cells that are ca 40 μm wide, and ascospores that are unequally 2-celled with or without various types of appendages or sheaths. The order includes a single family, the Aliquandostipitaceae, and has affinities with the Pleosporales within the Dothideomycetes. Studies on freshwater ascomycetes in Egypt and Thailand yielded one new genus Patescospora, and Jahnula siamensiae sp. nov., and the new combination J. sunyatsenii (syn. Aliquandostipite sunyatsenii) is made based on molecular results.

The foliage of Pinus strobus (eastern white pine), as that of all other conifers examined, is occupied by endophytic fungi, the most frequent of which is Lophodermium nitens. The number and extent of endophytic infections and the genetic relationship of individual isolates within living needles as well as their relationship to isolates from forest floor needles is unknown. To examine these and related questions, forest floor isolates and foliar endophytes from needle segments were obtained for ribosomal DNA sequencing and randomly amplified polymorphic DNA (RAPD) analysis. Molecular and morphological data were compared and infection frequency determined as a function of position along the needle. Ribosomal DNA sequences of foliar and ascospore isolates showed high levels of genetic similarity (>97% identity) for the internal transcribed spacer region. RAPD profiles were able to distinguish ascospore siblings from non-siblings, and also revealed that many needle isolates belonged to the same genotype as adjacent neighbour isolates, as would be expected from mycelial spread within the needle. Morphotype evaluation and RAPD profiles showed similar patterns: identical morphotypes grouped together and showed little or no genetic difference under RAPD analysis. Both morphological and molecular data indicated that the majority of infections were contained within 1 mm needle segments but could extend to about 4 mm in length. Infection frequency increased along the length of the needle from the proximal (shoot) end to the distal tip, with markedly higher rates in the distal quarter. Thus, endophytic infections of L. nitens in white pine needles consist of many localized, discrete infections, originating from ascospores and differentially distributed along the length of the needle. In the course of this work, it was found that GenBank accession no. AF203470 under the name Meloderma desmaszieresii appeared not to be that species but L. nitens on the basis of the ITS sequences.

Using degenerated primers for conserved regions of RecA homologs we have isolated a gene from Pleurotus ostreatus that shows characteristic features of RAD51 homologs. The encoded amino acid sequence of P. ostreatusRAD51 (PoRAD51) shows greatest sequence similarities with RAD51 from Coprinus cinereus (89% identity). Furthermore the genomic organisation of PoRAD51 is almost identical to that of RAD51 from C. cinereus. Northern analysis shows that the expression of PoRAD51 is found in vegetative mycelium, and fruit body tissue, and that it is expressed at elevated levels in lamellae/basidia and following DNA damage. A sporulation deficient mutant strain of P. ostreatus (ATTC 58937) showed expression patterns of the RAD51 gene that are similar those of the normal sporulating strain.

The lichens Usnea florida and U. subfloridana have since long been recognised as distinct species. They show many similarities in morphology, but have different reproductive strategies. Usnea florida is always provided with many apothecia and produces no specialised asexual propagules. Usnea subfloridana has soralia, isidiomorphs and occasionally apothecia. Phylogenetic analyses based on continuous sequences of the ITS and LSU regions of the nuclear ribosomal DNA and the gene coding for β-tubulin, show that specimens of the two species form one monophyletic group of intermixed specimens, and not two groups corresponding to morphology, which would have been expected if two species were at hand. The ‘species pair’ concept in lichenology is discussed. Other Usnea species included in the study are: U. articulata, U. barbata, U. ceratina, U. filipendula, U. hirta, U. rigida and U. wasmuthii.

Correct morphological identification of Alternaria is important and demands a combination of modern standardised methods and up-to-date literature. The production of secondary metabolites has previously been used as a means of identification and classification. In this study, 153 fungal isolates belonging to the genus Alternaria were examined. They were grown under standardised conditions and subjected to morphological and chemical examination. All isolates were grouped according to their three-dimensional sporulation pattern on potato carrot agar and their colony colour on dichloran rose bengal yeast extract sucrose agar (DRYES). After extraction, all isolates were analysed by a high performance liquid chromatograph equipped with a diode array detector and the resulting metabolite profiles were subjected to multivariate statistic analyses. The analyses of metabolite profiles showed that the isolates could be divided into three major species-groups that were morphologically identifiable as the A. infectoria species-group, the A. arborescens species-group and the A. tenuissima species-group. The A. infectoria species-group is chemically very different from both the A. arborescens and the A. tenuissima species-groups with only a few metabolites in common. None of the 35 A. infectoria species-group isolates produced any known metabolites and all had white or greyish white colonies on DRYES. The A. arborescens species-group and the A. tenuissima species-group, shared most of the known metabolites and had colonies of various shades of green on DRYES. One cluster of isolates belonging to the A. tenuissima species-group was able to produce tentoxin, which has not been reported previously from any A. tenuissima isolate. The results suggest that each species-group contains several taxa and these taxa need to be formally described before species specific metabolite profiles can be established.