The mitosporic fungus Pochonia chlamydosporia is a potential biocontrol agent for cyst (Heterodera spp. and Globodera spp.) and root knot (Meloidogyne spp.) nematodes, which are important agricultural plant pests. 54 isolates from diverse geographical regions and several nematode hosts were used in this study. Genetic variation was examined using enterobacterial repetitive intergenic consensus (ERIC) primed PCR and sequences from the internal transcribed spacer (ITS) rRNA region. ERIC PCR yielded 35 scorable binary characters from all the fungi tested and cluster analysis of the data showed that isolates from cyst nematodes were more genetically variable than those from root knot nematodes. The ITS regions were highly conserved, the only significant difference being an extra thymidine in isolates from Meloidogyne spp. Assays with nematode eggs indicated that isolates differ in their ability to infect different nematode genera. The results indicate host related variation in P. chlamydosporia. This finding has significant implications for the application of P. chlamydosporia as a biocontrol agent.

The association of ascomycetes with Fucus serratus was investigated by comparing the broad-based molecular and cultural diversities of healthy and dead fronds. Four PCR primer pairs were used to amplify the 18S (primers NS1-FR1; NS1-EF3) or 28S rRNA (primers NL209-NL912; NL359-NL912) genes directly from the DNA of algal thalli. Two novel primer pairs, NL209-NL912 and NL359-NL912 giving product sizes of 700 and 559 bp respectively, were designed to amplify the 28S rDNA from ascomycetes specifically. All primer combinations amplified DNA from 33 reference taxa isolated from Fucus serratus, and the products generated by primers NS1-FR1GC and NL359-NL912GC were separated in 18–38% and 38–60% denaturant gradients respectively after DGGE. The 18S rDNA DGGE system resolved eight bands from algal DNA, but many of the sequences separated were not fungal, whereas the 28S rDNA system resolved seven bands that were all identified as ascomycetes. Phylogenetic analysis and BLAST search results of environmental sequences revealed the presence of four main ascomycete groups: (1) the Halosphaeriales, (2) the Hypocreales, (3) an unidentified Lulworthiales complex, and (4) the Pleosporales. Few fungal isolates were detected molecularly suggesting that fungal colonisation of fronds was limited, mainly to species in dead casts.

We hybridized the telomere-associated DNA sequence pTel46 isolated from Coprinus cinereus with Helicobasidium mompa genomic DNA. The hybridized fragments were more sensitive to Bal31 nuclease than those that were not hybridized, suggesting that they were located at the ends of chromosomes in H. mompa. The hybridization profile can be used to estimate chromosome number, since the number of chromosomes in a single basidiospore isolate is about half that in putative parent strains. Thus, single basidiospore and field isolates might be homokaryons and heterokaryons respectively. We found telomere-linked restriction fragment length polymorphisms (RFLPs) in strains of H. mompa isolated from field and individual basidiospores. Thus, this marker appears to be an excellent tool with which to reveal the considerable polymorphism of H. mompa and to identify strains. The RFLP was not found among several strains of the same mycelial compatibility group (MCG) isolated from the same field, suggesting that strains belonging to an MCG group are identical.

Corky root of tomato caused by Pyrenochaeta lycopersici is a disease of concern in Italy and for many tomato growing areas in the world. Isolates of the fungus were characterized at both the physiological and molecular level. The optimal in vitro growth temperature for all isolates was 23 °C. All Italian isolates of P. lycopersici showed similar RAPD and esterase banding patterns. No relevant polymorphisms were detected after enzymatic digestion of PCR-amplified ITS and IGS regions. The overall results indicate a low degree of genetic variability within a collection of 43 Italian isolates. These data are of interest in breeding programs for resistance against corky root of tomato and they provide useful information for the development of molecular diagnostic tools for the rapid identification and detection of P. lycopersici.

Genetic variation of 42 isolates of Corynespora cassiicola, a destructive fungal pathogen of many economically important crop plants including rubber, was investigated using RAPD–PCR analysis. Five genetic groups were identified using RAPD–PCR profiles generated by eight random primers. Results indicate that there is a significant genetic variation among C. cassiicola isolates collected from different host plants. These results should facilitate the development of rubber clones with enhanced resistance against all genetic groups of C. cassiicola.

A species of Pseudocercospora causing foliar lesions on Hibbertia aspera (Dilleniaceae) is morphologically indistinguishable from a species of Pseudocercospora causing foliar lesions on Platylobiumformosum (Fabaceae) from the same locality. In order to assess the degree to which these fungi were related, we sequenced the ribosomal DNA ITS region of cultures derived from single conidial isolates. Cultures were obtained from four specimens of each respective host. The ITS sequences for cultures derived from the same host were identical, with the exception of one isolate that had a 4 bp insert, while the Pseudocercospora species from Hibbertia consistently differed at three bases from the species isolated from Platylobium. These data suggest that two distinct species of Pseudocercospora are represented, and that they probably result from a recent evolutionary divergence. P. platylobii and P. hibbertiae-asperae spp. nov. are described and illustrated.

Sequence analysis of the ITS region of rDNA was used to investigate the level of genetic diversity occurring within Hyaloperonospora parasitica, and to show the relationship between phylogenies of these fungi and their hosts (Brassicaceae). Maximum parsimony and neighbour-joining analyses were performed using sequences from 32 isolates of Hyaloperonospora and Perofascia, which infect core brassicaceous hosts. For comparison, five isolates of Peronospora were also studied. The constructed phylogenetic trees showed trichotomy, showing that Hyaloperonospora, Perofascia, and Peronospora have different evolutionary histories. Although isolates from Peronospora and Perofascia clearly formed respective clades, the Hyaloperonospora group allowed separation of the isolates into four distinct clades, which shared significantly low sequence similarities. We suggest that H. parasitica infecting brassicaceous hosts should be divided into a number of distinct species. The comparison of the phylogeny of H. parasitica and that of the Brassicaceae suggests that this fungus is closely related with tribes Arabidae and Brassiceae within this host family, illustrating the potential of downy mildews for co-evolution with their hosts.

An innovative ex situ fungal baiting method using soil collected from field sites which allows the simultaneous detection of mycorrhizal fungi for multiple terrestrial orchids is presented. This method demonstrated that coarse organic matter (>2 mm) in the litter and topsoil was the most important reservoir of inoculum of these fungi. A new in situ seed baiting method using multi-chambered packets to simultaneously assess germination for different orchid species within soil is also introduced. These in situ and ex situ methods are compared using seed of orchids in the genera Monadenia, Microtis, Caladenia, Pterostylis and Diuris, using urban Banksia woodland sites with high or low weed cover. Both these seed baiting methods detected compatible fungi for these orchids, but common orchids germinated more frequently than those which were uncommon at the field sites. Germination rates were not significantly affected by weed cover even though adult orchids were rare in areas with high weed cover. The two new seed baiting methods vary in efficiency and applicability depending on the situation where they are used. However, the ex situ method allowed the time-course of germination to be observed, resulting in the production of more protocorms and facilitation of the isolation of mycorrhizal fungi. These techniques provide valuable new tools for detection of compatible mycorrhizal fungi to assist orchid research and conservation.

We describe a 16 yr dataset of ectomycorrhizal fruit bodies under Scots pine (Pinus sylvestris), starting from seedlings, and explore the effects of a 50% thinning treatment imposed in year 12. The over-riding pattern in the data was of successional development, with Paxillus involutus and Laccaria proxima in the earliest years, followed by Suillus species, while in later years Amanita and Cortinarius species became prominent. The typical pattern was for each species in turn to increase to a maximum count then gradually decline without actually disappearing from the community (an addition succession). For nine out of the 30 species recorded the time profiles were fitted significantly by a log-normal curve, although the best-fit lognormal models consistently under-estimated the peak count. Thinning increased counts of Suillus bovinus, Gomphidius roseus and Cortinarius semisanguineus, but overall its effect on the community was minor. Rainfall in September was weakly correlated with successional advancement (measured as DCA first axis scores). Mean fruiting date increased significantly as the stands aged.

The morphology of two soil-borne Verticillium species, V. dahliae and V. tricorpus, was studied on two semi-selective agar media, in the absence and presence of soil. Morphology of the fungi differed considerably between the media, with respect to presence and shape of microsclerotia, dark hyphae (i.e. short melanised hyphae attached to the microsclerotia) and dark mycelium (i.e. melanised mycelium throughout the colony). On modified soil extract agar (MSEA), a pectate based agar, V. dahliae always had globose to elongate microsclerotia, without dark hyphae or dark mycelium, whereas V. tricorpus always had dark hyphae or dark mycelium, and microsclerotia, whenever present, were globose to irregular in shape. On ethanol agar (EA), V. dahliae had large microsclerotia and abundant dark hyphae, whereas V. tricorpus did not form microsclerotia, but always abundant dark mycelium. For the first time we observed the formation of dark hyphae by V. dahliae to a great extent. In the presence of soil, most characteristics were less pronounced, and V. dahliae microsclerotia were smaller, but V. tricorpus produced large microsclerotia, even when they were absent in pure culture. Morphological characteristics suitable for discrimination between the two species on MSEA plates in the presence of soil were selected and tested with fresh isolates from agricultural fields. The two fungi could be distinguished using qualitative characteristics and microsclerotial size. Molecular analysis and morphology on potato dextrose agar confirmed all identifications made on soil dilution plates.

Consumption of locoweeds, legumes endemic in arid western USA, has long been associated with locoism, a disease of ruminant animals. To explore the relationship between fungi associated with locoweed and locoweed toxicity, 11 locoweed populations from various sites in New Mexico were assessed for endophytic fungi. Endophytes were isolated from the leaves, stems, seeds, and flowers of eight populations of the toxic locoweeds Astragalus mollissimus, Oxytropis lambertii, and O. sericea. Fungal cultures grew very slowly and sporadically produced subcylindrical conidia with very dark transverse septa. All cultured endophytes produced the alkaloid swainsonine, which causes locoism. Endophyte-infected locoweed populations produced swainsonine, and the swainsonine level of endophyte strains in vitro was highly correlated with the swainsonine level of their host plant populations. The rDNA ITS from mycelia from four endophyte isolates and β-tubulin encoding regions from mycelia of 18 fungal endophyte isolates were amplified using PCR and the nucleic acid sequences were analyzed. The nucleic acid sequences of the β-tubulin encoding regions were essentially identical among all the endophytes regardless of plant genus and locations. Morphological evidence and sequence analysis of the ITS region suggest that the endophytes are most closely related to Embellisia. However, with the paucity of Embellisia species represented in sequence databases, precise taxonomic placement will await further study.

Tuber borchii mycelium (strain 1BO) is able to utilise glucose, fructose or mannitol in the culture medium as a carbohydrate source. Since sugars not only function as a metabolic resource and structural constituent of cells, but also act as important regulators of various processes, we investigated if high sugar concentrations could influence fungal growth and development. The studies performed in this paper revealed that fructose or mannitol used at high concentration (50 g l−1) in the culture medium do not influence the growth and the biochemical responses of fungus but the growth of T. borchii mycelium is subject to glucose repression. In experiments with a high glucose concentration (50 g l−1) and with 2-deoxyglucose, a non-metabolisable glucose analogue, the growth of T. borchii was halved with respect to the control (10 g l−1 of glucose). The morphological and biochemical analyses revealed that the hyphae were metabolically and functionally active, but the activity of mannitol dehydrogenase was reduced to one-third in the high glucose treatment. This is the first evidence of glucose repression of growth and activity in the ascomycetous ectomycorrhizal fungus T. borchii.

Pulse-field gel electrophoresis (PFGE) was used to determine the genome size and characterize karyotypic differences in isolates of the cacao biotype of Crinipellis perniciosa (C-biotype). The karyotype analysis of four isolates from Brazil revealed that this biotype could be divided into two genotypes: one presenting six chromosomal bands and the other presenting eight. The size of the chromosomes ranged from 2.7 to 5.3 Mb. The different genotypes correlate with telomere-based PCR analysis. The isolates with six chromosomal bands had two that appeared to be doublets, as shown by densitometric analysis, indicating that the haploid chromosome number for this biotype is eight. The size of the haploid genomes was estimated at approximately 30 Mb by both PFGE and Feulgen-image analysis. DNA hybridization revealed that the rDNA sequences are clustered on a single chromosome and these sequences were located on different chromosomes in an isolate dependent manner. This is the first report of genome size and chromosomal polymorphism for the C-biotype of C. perniciosa.

The presence of some fungi on a variety of food products, like cheeses or cured meat products, is beneficial for the ripening of the product and for the development of specific flavour features. The utilization of these fungi as starters, which are inoculated normally as asexual spores on the food products at the beginning of the ripening process, is becoming a usual procedure in the food industry. The starter culture also prevents undesirable fungi or bacteria from growing on the product. Penicillium nalgiovense is the most frequently used starter for cured and fermented meat products, but the fact that this fungus can secrete penicillin to the meat product makes it important to get strains unable to synthesize this antibiotic. In this work we report that P. nalgiovense strains impaired in penicillin production can be obtained by disruption of the pcbAB gene (the first gene of the penicillin biosynthetic pathway). When applied as starter on cecina (a salted, smoke-cured beef meat product from the region of León, Spain), the pcbAB-disrupted strain showed no differences with respect to the parental penicillin-producing strain in its ability to colonize the meat pieces and to control their normal mycoflora. Both strains exerted a similar control on the presence of bacteria in cecina. A similar proportion of penicillin-sensitive and penicillin-resistant bacteria were isolated from pieces inoculated with the penicillin-producing or the non-producing P. nalgiovense strains. The decrease of the bacterial population on the surface of cecina seems to be due to the higher competition for nutrients as a consequence of the inoculation and development of the P. nalgiovense mycelium and not due to the production of penicillin by this fungus. Penicillin production was less affected than growth in a solid medium with high NaCl concentrations; this suggests that the high salt concentration present in cecina is not a limiting factor for penicillin production by P. nalgiovense.

Trichoderma harzianum is a ubiquitously distributed asexual soil fungus that produces a variety of antibiotic compounds. Colonisation of soil inhabited by competing microbiota is facilitated by the antibiotic activity of these compounds. In addition, T. harzianum produces hydrolytic enzymes that degrade the cell wall components of many microorganisms, which can then be used as a source of nutrients. Recently, biotypes of T. harzianum differing morphologically from those originally described by Rifai were isolated on commercial mushroom (Agaricus bisporus) farms. These ‘aggressive’ biotypes cause devastating crop loss on mushroom farms. The aggressive biotype in North America was originally known as ‘Th4’ but has been recently renamed Trichoderma aggressivum f. aggressivum. In contrast, ‘non-aggressive’ biotypes, have no noticeable effect on the crop, are similar to T. harzianum and are commonly found on mushroom farms. The mechanism of disease establishment is unknown. We have identified a metabolite produced by T. aggressivum isolates in vitro that inhibits growth of A. bisporus and other fungi. This antifungal compound is not produced by ‘non-aggressive’ T. harzianum isolates under the culture conditions tested and is identified as 3,4-dihydro-8-hydroxy-3-methylisocoumarin. Another compound was isolated from both liquid culture and infested compost. Although its chemical structure could not be precisely determined, this compound also inhibits A. bisporus growth, is predominant in infested compost and likely has a inhibitory effect on the mycelia present in mushroom compost, resulting in devastating crop loss.

The ability of the preferentially saprotrophic fungus Armillaria cepistipes and the pathogenic A. ostoyae to capture fresh stump resources was investigated in managed Alpine Norway spruce (Picea abies) forests where both species occur sympatrically. The incidence of Armillaria species and genets as rhizomorphs in the soil as well as external and internal (heart rot) stump colonisation were determined in three comparable 1 ha plots. The results indicate that A. cepistipes and A. ostoyae have a very similar strategy to capture fresh stump resources. Both species produce dense networks of rhizomorphs in the soil enveloping living trees. After the felling of the trees, the stumps are rapidly captured through spread of mycelial fans in the cambial zone. Our study suggests that A. ostoyae is slightly more competitive than A. cepistipes in primary resource capture.

Beauveria bassiana is a popular biocontrol agent used as ‘green’ pesticide in crop insect pest management. Chromosome number has been variously reported as five, six, seven and eight in this species. The range of chromosome number and the minimum chromosome number in this economically important fungus were assessed through telomere fingerprint analysis of a sample of 17 isolates from different and similar hosts and distant and same geographic origin. Genomic DNA digested with EcoRI, which has no cutting site in the telomere repeat sequence arrays was probed with a radioisotope-labelled (5′-TTAGGG-3′)8 oligonucleotide. The probe-hybridised regions appeared as discrete bands – each representing a telomere. The number of bands in each lane was counted and halved to arrive at the chromosome number of that isolate. The chromosome number varied from 5 to 10 in the different isolates. The telomere probe hybridised bands were also scored for presence or absence in a 0–1 matrix and a dendrogram based on similarities between the isolates was constructed using the NTSYS-pc ver. 2.02i software. The isolates showed very little similarity; the overall similarity was 14%. Only two isolates which were of diverse host and geographic origin showed 100% similarity. Isolates from the same epizootic that showed 43% similarity in their telomere fingerprints had 96% similarity in their RAPD (Random amplified polymorphic DNA) fingerprints with 10 primers. The genetic distances computed from any one DNA fingerprinting method thus do not reflect the true genetic similarities of the isolates. The frequency distribution pattern of the pair-wise similarities computed from telomere fingerprints hinted at the occurrence of recombination in this fungus. Telomere fingerprinting proved very useful in typing isolates since each of them was found to have a unique fingerprint. Isolates with the same chromosome number neither showed a distinct morphology or virulence character nor a close similarity in telomere or RAPD fingerprints to merit their subgrouping into a taxonomically relevant or practically useful unit.

Auxarthron conjugatum (Onygenaceae) and Myxotrichum deflexum (Myxotrichaceae) are distantly related cleistothecial (gymnothecial) ascomycetes that form ascomata with strikingly similar peridia in which rigid, branched and anastomosed, thick-walled hyphae create a cage- or mesh-like enclosure (reticuloperidium). We tested the hypothesis that the reticuloperidium plays a role in dispersal mediated by arthropods by enclosing ascomata of these fungi together with flies from the family Sarcophagidae. Gymnothecia of both fungi were picked up easily when the stiff hairs of the flies impaled the ascomata by passing through the interhyphal spaces of the reticuloperidium. Ascospore release from the gymnothecia then occurred during grooming activities during which the limbs of the flies caught the ascoma appendages causing the peridium to be torn apart. This adaptation to arthropod morphology and behaviour is interpreted as the driving force behind the evolution of reticuloperidia in unrelated groups of cleistothecial ascomycetes.

DNA sequence analysis of the internal transcribed spacer region 1 (ITS1) and random amplified polymorphic DNA (RAPD) markers were used to survey genetic variability in relation to agronomic and regional factors among 60 isolates of Thanatephorus cucumeris (anamorph Rhizoctonia solani) collected from lesions on potato stems or sclerotia of potato tubers. Based on comparative sequence analysis it was shown that all isolates belonged to anastomosis group 3 subgroup Potato Type (AG-3 PT). ITS1 sequence polymorphisms were found within 45 of the 60 isolates showing that different types of the ITS-region are present in individual isolates. Cloning and sequence analysis of the ITS1 region from three selected isolates with sequence polymorphism showed that two different ITS1-types were present in each isolate. RAPD analysis identified 51 RAPD-phenotypes among the 60 investigated isolates indicating a high level of diversity within the subgroup AG-3 PT. Putative clonal isolates with identical RAPD- and ITS1-types were identified within fields, and in one case the same phenotype was found in two different fields separated by several hundred kilometers. Population subdivision analysis based on phenotypic as well as genotypic diversities showed differentiation among populations from different fields when isolates were sampled from tubers, indicating restricted gene flow among soil populations. Low differentiation was seen among field populations sampled from stems, indicating that gene flow is taking place. The population structure was not influenced by the previous crop in the rotation nor by the two cultivars ‘Sava’ and ‘Bintje’.

A method for visualization of fungal nuclear structures using the DNA specific fluorochrome SYTOX Green and confocal laser scanning microscopy is presented. The staining procedure detects nuclei in different cell types with high contrast. Furthermore, the method permits generation of high-resolution confocal image stacks using optimised scanning conditions. This approach significantly improved visualization of stained fungal nuclear structures. The technique was applied to some lichenized fungi, but can also be used to study similar detail in non-lichenized fungi. In certain cases, incompletely separated chromosome-like structures, heterochromatic material as well as weakly stained nucleolar regions can be seen. In Xanthoria parietina, variation in the nuclear division was observed in developing ascospores. Delayed division after the formation of endospore thickening resulted in the exchange of nuclear material through a narrow isthmus.