Rust fungi cause economically important diseases of cereals, and their ability to rapidly evolve new virulent races has hindered attempts to control them by genetic resistance. PCR-based molecular tools may assist in understanding the genetic structure of pathogen populations. The high multiplex DNA fingerprinting techniques, amplified fragment length polymorphisms (AFLP), selectively amplified microsatellites (SAM) and sequence-specific amplification polymorphisms (S-SAP) were assessed for their potential in investigations of the genetic relationships among isolates of the wheat rust pathogens, Puccinia graminis f. sp. tritici (Pgt), Puccinia triticina (Pt), and P. striiformis f. sp. tritici (Pst), the oat stem rust pathogen P. graminis f. sp. avenae (Pga), and a putative new P. striiformis special form tentatively designated Barley grass yellow rust (Bgyr). Marker information content, as indicated by the number of species-specific fragments, polymorphic fragments among pathotypes, percentage of polymorphic loci, and the marker index, was highest for the SAM assay, followed by the AFLP and S-SAP assays. UPGMA analysis revealed that all marker types efficiently discriminated the five different taxa and Mantel tests revealed significant correlations between the marker types. Within pathogen groups, the marker types differed in the amount of variation detected among isolates; however, the major differences were consistent and polymorphism was generally low. This was reflected by the AMOVA analysis that significantly partitioned 90% of the genetic variation between taxa. Of the three marker types, SAMS were the most informative, and have the potential for the development of locus-specific microsatellites.

Species of Rhizoctonia cause a blight of Lotus corniculatus, a perennial forage legume. We characterized genetic variation and virulence in populations of R. solani and binucleate Rhizoctonia's associated with diseased L. corniculatus in field plantings over several years. Isolates of anastomosis groups AG-1 and AG-4 accounted for the R. solani recovered from diseased leaf and shoot tissues. Isolates of binucleate Rhizoctonia were recovered predominantly from soil and associated plant debris. Isolates of R. solani were more virulent on leaves and shoots of L. corniculatus than were binucleate Rhizoctonia isolates. Numerous unique DNA restriction patterns were observed among binucleate isolates and anastomosis groups of R. solani. Variation in restriction patterns was greater among isolates of AG-1 from the lower plant canopy than from the upper canopy. No restriction pattern was shared by any isolate from AG-1 and AG-4. Allelic and genotypic heterogeneity of AG-1 isolates were also greater in the lower plant canopy. Binucleate isolates exhibited greater heterogeneity than AG-1 isolates from either canopy region. L. corniculatus offers significant opportunities for investigating temporal and spatial dynamics of genetic structure of Rhizoctonia populations in perennial plant systems.

Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis of the first intergenic region (IGR1) of nuclear ribosomal DNA was used to clarify the relationship between IGR1 variations and six Japanese biological species of Armillaria: A. gallica, A. nabsnona, A. ostoyae, A. cepistipes, A. mellea and Nagasawa's E (Nag. E: taxonomically unknown species). The procedure was then used to identify Armillaria species associated with Wynnea species (W. americana and W. gigantea) and Entoloma abortivum. By combining the RFLP patterns obtained using three endonucleases, HaeIII, HinfI and MspI, the IGR1s from 18 isolates of six Armillaria species were assigned to nine different RFLP phenotypes and the six species were distinguished from each other. Each of the RFLP phenotypes from the Armillaria isolates associated with Wynnea species or E. abortivum matched a corresponding phenotypes observed among the six Armillaria species. Based on this, all four isolates from W. gigantea were identified as A. mellea, two from W. americana as A. cepistipes, and all three from E. abortivum as Nag. E. These results provide new information on the biological species of Armillaria associated with Wynnea and E. abortivum.

In order to evaluate substrate dependence of the symbiotic fungal associations in leafy liverworts (Jungermanniopsida), 28 species out of 12 families were investigated by transmission electron microscopy and molecular methods. Samples were obtained from the diverse substrates: from naked soil, from the forest floor on needle litter, from between peat moss, from rotten bark of standing trees, and from stumps and rotten wood. Associations with ascomycetes were found in most of the specimens independent from the substrate. Seven species sampled from soil were found to contain basidiomycete hyphae. Ultrastructure consistently showed dolipores with imperforate parenthesomes. Molecular phylogenetic studies revealed that three specimens belonging to the Jungermanniales were associated with members of Sebacinaceae, while Aneura pinguis (Metzgeriales) was associated with a Tulasnella species. These taxa are so far the only basidiomycetes known to be symbiotically associated with leafy liverworts. The probability that the associations with Sebacinaceae are evolutionary old, but the Tulasnella associations more derived is discussed. The sebacinoid mycobionts form a similar interaction type with the jungermannialian leafy liverworts as do the associated ascomycetes. The term ‘jungermannioid mycorrhiza’ is proposed for this distinctive symbiotic interaction type.

Leptosphaeria maculans (anamorph Phoma lingam), the ascomycete causing stem canker of crucifers, is a species complex that can be separated into at least seven distinct subgroups using a combination of biochemical and molecular criteria. In the present study sequences of the entire ITS region, including the 5.8S rDNA, of 38 isolates representing the seven subgroups, along with specimens from culture collections, were analysed, compared to those of closely related Leptosphaeria species, and the phylogeny inferred using parsimony and distance analyses. A well-supported clade encompassed all isolates of the seven subgroups along with L. conferta, a known saprobe of dried crucifer stems. The L. maculans isolates were further separated into two well-supported clades corresponding to L. maculans s. str. and the recently named L. biglobosa. Parsimony and distance analyses further separated groups within both species, usually corresponding to specific host plants or geographic origin, e.g. L. maculans ‘brassicae’ from cultivated Brassica, L. maculans ‘lepidii’, from Lepidium sp., L. biglobosa ‘brassicae’, from various Brassica species, L. biglobosa ‘thlaspii’ from Thlaspi arvense, L. biglobosa ‘erysimii’ from Erysimum sp., and L. biglobosa ‘canadensis’ mostly found in central Canada. The oldest L. maculans specimens maintained in international collections clustered with either L. maculans ‘brassicae’, L. biglobosa ‘brassicae’, or a still different group closely related to L. biglobosa ‘thlaspii’. The evolutionary relationships between the seven infraspecific groups are discussed in terms of phytopathological relevance and species isolation linked with specific life cycle, geographic isolation or host specificity.

24 isolates of Epulorhiza were obtained from the roots and protocorms of orchids in Singapore. Two groups were distinguished based on morphological and cultural characteristics. Group 1 comprised 20 isolates which were identified as E. repens, while those in group 2 were identified as E. calendulina-like Rhizoctonia. The ITS-5.8S rDNA sequence identity ranged from 88–100% among isolates of E. repens and six sub-groups were further delineated. The sequence identity was 98–100% among E. calendulina-like Rhizoctonia isolates. The sequence identity between E. repens isolates and E. calendulina-like Rhizoctonia isolates ranged from 18–44%. Apparently, isolates from both groups 1 and 2 were genetically distinct. Phylogenetic analysis showed that the distribution of the isolates correlated with the sites and the hosts from which the fungi were isolated. No matching sequences have been found in the GenBank database for the ITS region of E. repens and E. calendulina-like Rhizoctonia.

Three Cryptosporiopsis species have thus far been isolated from roots of woody plants. A fourth species, which was recently isolated from roots of Calluna vulgaris, Erica tetralix, Vaccinium vitis-idaea, and V. myrtillus in The Netherlands, is described here as new. Sporulation on the natural substratum has not been observed and the morphological description of this fungus is therefore based on characters expressed on oatmeal and malt extract agars. The phenotypic characters indicated a close relationship with the other root-inhabiting species of Cryptosporiopsis and species of the associated teleomorph genus Pezicula. This relationship was confirmed by phylogenetic analyses using sequence data of the 5.8S nuclear rDNA and flanking internal transcribed spacers. In order to facilitate recognition of this possibly under-recognized category of root inhabitants, a key to the root-inhabiting Cryptosporiopsis species based on characters in vitro is given.

We report the development of two new transformation systems, polyethylene glycol (PEG)-mediated transformation of protoplasts and Agrobacterium tumefaciens-mediated transformation of mycelium, for the filamentous ascomycete Venturia inaequalis. New binary vectors have been created for the latter. Although transformation was initially achieved using a PEG-mediated method, this was superseded by the A. tumefaciens-mediated approach. The advantages of the latter include: ease of the protocol, no requirement for protoplasts; higher transformation efficiency; and single-site integration. A comparison between the two transformation methods is presented.

Inter-simple sequence repeat PCR (ISSR-PCR) was used to develop markers for simple sequence repeat-rich (SSR) regions for investigation of genetic relatedness of Pisolithus isolates collected from eastern mainland Australia. Primers were designed to amplify ten SSR-rich regions and these were used to screen 14 Pisolithus isolates. Two amplified loci showed size polymorphisms among the isolates (regarded as polymorphic), two were monomorphic for all isolates, while the remainder amplified alleles for only some isolates. UPGMA analysis of the alleles for each isolate at each locus together with ITS-RFLP analysis, separated the isolates into two groups. These two groups appear to correspond to isolates that ITS sequence data have previously separated as P. albus and P. microcarpus.

Isolates of Pleurotus cystidiosus and P. smithii were studied to verifie the occurrence of P. cystidiosus instead of P. smithii in South America. The two species are mainly separated by the growth rate of the anamorph in culture, the morphology of the anamorph and teleomorph, intercompatibility tests, and genetic variability. In order to see if the isolate found in Brazil and previously identified as Antromycopsis macrocarpa (the anamorph of P. cystidiosus) belongs to P. cystidiosus, a species with a world wide distribution, or to P. smithii which is restricted to Mexico and South America, or if P. cystidiosus and P. smithii are the same species, isolates of different geographic origins were studied. Growth rate in culture, mono-dikaryotic matings, and genetic variability determined by RADP were investigated. The results show that the criteria used to separate the two species are unsatisfactory, and that P. smithii should be considered a synonym of P. cystidiosus; this extends the distribution of this later species to Central and South America.

During a phylogenetic study of ophiostomatoid fungi, a group I intron-like sequence was noted in the SSU rDNA gene of Gondwanamyces proteae. Secondary structure and sequence characteristics assigned the intron to the I E class. We then examined 27 related Group I-like sequences deposited in GenBank, and as a result 15 additional and previously uncategorized I E rDNA introns were identified. This study, with other recent publications, suggests that the I E class might represent a major family of group I introns that are located within the nuclear SSU and, to some extent LSU, genes in fungi.

Genetic relationships of the complex of Fusarium species associated with crown rot and head blight in cereals and some species associated with plant diseases in general were examined by distance and maximum parsimony algorithms of their internal transcribed spacer sequences. The analysis clustered the complex of Fusarium species that causes root and crown rot and head blight of cereals and three other clusters of F. sambucinum, F. venenatum and F. poae into one clade. This group of Fusarium species was also found in this study to correspond to the group defined by the presence of the tri5 gene. The tri5 gene was recently reported to co-segregate with the locus governing the type of trichothecene produced, and probably maps in the trichothecene gene cluster. The other clusters of F. avenaceum, F. tricinctum, F. torulosum, F. oxysporum, F. verticillioides and F. solani did not have the tri5 gene. Although, F. pseudograminearum was phylogenetically close with the cluster of F. graminearum, F. culmorum and F. cerealis, it could be distinctly separated from them. The distinct genetic status of F. pseudograminearum from F. graminearum corroborated with other published molecular data and isozyme findings. The molecular analysis provided a simple diagnostic tool to differentiate fungi causing crown rot from those involved in head blight.

Sphaeropsis sapinea is an opportunistic pathogen causing serious damage to conifers, pre-disposed by adverse environmental conditions or mechanical damage. Three different morphological forms of the fungus have been described and are commonly referred to as the A, B and C morphotypes. Isolates of the different morphotypes have also been separated based on differences in pathogenicity and molecular characteristics. These differences, however, overlap and have not been considered sufficiently robust to justify the description of separate taxa. The aim of this study was to consider relationships between isolates representing different S. sapinea morphotypes, using multiple gene genealogies inferred from partial sequences of six protein-coding genes and six microsatellite loci. Genealogies generated for the protein-coding genes and microsatellite loci were not congruent but both consistently grouped isolates representing the A and C morphotypes in separate but closely related clades. In contrast, isolates of the B morphotype grouped together in a clade that was equally different to the A and C morphotypes as it was to the clade encompassing isolates of Botryosphaeria obtusa. These results provide strong evidence to show that the B morphotype isolates are distantly related to S. sapinea and represent a discrete taxon, which we describe here as Diplodia scrobiculata sp. nov.

The interaction between Puccinia recondita f. sp. tritici (now widely referred to as P. triticina) and endophytic fungi in wheat was examined in laboratory experiments to determine whether the presence of fungal endophytes suppresses leaf rust disease caused by this fungus. Endophytes and cell-free washings from culture plates of the endophytes reduced the density and size of pustules in a susceptible cultivar when inoculated 3, 7 and 14 d prior to the pathogen. Disease at 12 d was reduced when the endophytes were inoculated simultaneously up to 50 mm from the fungus. Interactions between endophytes and this Puccinia are most probably mediated by defence mechanisms induced in the host plant.

Ascospore appendages of two species of Linocarpon and three species of Neolinocarpon are illustrated using electron microscopy. The structure of these appendages can be used to distinguish between species. The ascospore appendages of species in both genera, however, were found to have similar structure. Thus, species of Linocarpon and Neolinocarpon could not be delineated at the generic level based on ascospore appendage structure.

Diversity in trichothecene mycotoxin production by 167 isolates of Fusarium graminearum was examined by chemical and molecular methods. Isolates from barley, corn, and wheat grown in Korea produced either deoxynivalenol (DON) or nivalenol (NIV), whereas isolates from corn grown in the United States produced DON only. Southern blotting of MseI-digested genomic DNA's from these isolates was performed using a 0.6-kb fragment of Tri5, a key enzyme for trichothecene production, as a probe. This technique revealed a single-band polymorphism between these isolates, with 1.8- and 2.2-kb bands arising from DON and NIV producers, respectively. The same set of isolates was subjected to previously developed PCR assays using primers derived from Tri7 or Tri13. These assays also revealed a single-band polymorphism between NIV- and DON-producing chemotypes. The polymorphisms at Tri5, Tri7, or Tri13 in all of the US isolates were consistent with their chemotypes as identified by GC–MS. However, for seven Korean isolates, chemical and molecular analyses yielded seemingly inconsistent results. This issue was resolved by Southern blot analysis with the Tri5 probe using two other restriction enzymes and sequence comparison of a 3.8-kb region spanning Tri5. In addition, one of these exceptional isolates was found to carry both DON and NIV chemotype-specific regions, possibly resulting from recombination between the two chemotypes.

The illudins are a family of fungal sesquiterpenes that have been studied as anti-tumor agents, and they also have antibacterial activity. Over a four-year period, 25304 fungal isolates (approximately 97% ascomycetes and 3% basidiomycetes), were screened for antibacterial activity against methicillin-resistant Staphylococcus aureus. Illudin-like compounds with antibacterial and cytotoxic activity against tumor cell lines were observed in 10 basidiomycete strains. The isolates were recovered from different types of substrata using indirect methods and only formed sterile mycelia in pure culture. The isolates were genetically related but not identical, based on PCR-based fingerprinting techniques. DNA sequencing of the ITS1-5.8 S-ITS2 region of the strains revealed that nine had identical sequences, indicating that they were conspecific. The sequence of the remaining isolate was 96.34% similar, suggesting that it was a closely related species. The D1–D2 region of the 25 S rRNA gene of the two strain types was also sequenced. Both sequences were 99.39% similar, and Coprinopsis gonophylla (syn. Coprinus gonophyllus) was the closest match for both. Strains were grown in pure culture on a rice-based medium that allowed the development of basidiomata from one culture of the main strain type, which was identified as C. episcopalis, a close relative of C. gonophyllus. Both species (or strain types) produced different types of illudin-like compounds. Three novel illudins (I, I2 and J2) were found to be produced by the cultures identified as C. episcopalis, while only illudinic acid was produced by the other Coprinopsis sp. The taxonomical relationships of the Coprinopsis species identified in this study with other illudin producers previously reported in the literature are discussed.

We evaluated the possibilities of exploitation of several mycoparasitic fungi in the biocontrol of cucurbit powdery mildew (Sphaerotheca fusca) in melon greenhouses. To simplify the screening, an in vitro biological control test on detached leaves of melon has been used and a detailed microscopic analysis of the interactions between mycoparasites and S. fusca conducted. In this context, the effect of mycoparasitic fungi on the formation of infection structures of S. fusca such as haustoria, conidia and conidiospores has been quantified. On the basis of the microscopic data, effect of mycoparasites on severity and incidence of cucurbit powdery mildew has been discussed. Our results show that, under controlled environmental conditions, Acremonium alternatum, Ampelomyces quisqualis and especially Lecanicillium lecanii, when applied in early stages of infection, were able to significantly reduce cucurbit powdery mildew symptoms and S. fusca development on melon leaves. These results indicate that these mycoparasites are promising candidates for the biocontrol of cucurbit powdery mildew in melon greenhouses.

Conocybe lactea was examined as part of a larger study on the distribution of amatoxins and phallotoxins in fungi, and the taxonomic relationships between these fungi. As amatoxins are present in the congener C. filaris, the locally abundant C. lactea was examined using HPLC and mass spectroscopy. Amatoxins were not found in C. lactea, but the related phallotoxins were present in small quantities making it the first fungus outside of the genus Amanita in which phallotoxins have been detected. Despite the presence of a related toxin, C. lactea was found not to be taxonomically close to C. filaris. Phylogenetic analyses using nuclear ribosomal RNA genes indicated that North American specimens of C. lactea were conspecific with North American specimens of C. crispa in Conocybe sect. Candidae. European C. crispa was a distinct taxon. The implications of the use of the name C. albipes for these taxa are discussed. Nucleotide data confirmed placement of the sequestrate taxon Gastrocybe lateritia in sect. Candidae, but as a distinct taxon. It is hypothesized that the unique sequestrate morphology of G. lateritia may be caused by a bacterial infection.

Different isolates of a foliar endophytic species of Phyllosticta were isolated from different tropical tree species in India to examine genetic variation among the isolates. Internal transcribed spacer-restriction fragment length polymorphism (ITS-RFLP) analysis did not detect any variation among the isolates, suggesting that they all belong to the same species. Sequence data of the ITS region of ribosomal DNA (ITS1 and ITS2, including 5.8S rDNA) supported the identity of the present fungal isolates as P. capitalensis. These results show that P. capitalensis (teleomorph Guignardia endophyllicola?) is an ubiquitous foliar endophyte that can infect tree hosts from different families and habitats.