A technique based on the polymerase chain reaction (PCR) for the specific detection of Phytophthora medicaginis was developed using nucleotide sequence information of the ribosomal DNA (rDNA) regions. The complete IGS 2 region between the 5 S gene of one rDNA repeat and the small subunit of the adjacent repeat was sequenced for P. medicaginis and related species. The entire nucleotide sequence length of the IGS 2 of P. medicaginis was 3566 bp. A pair of oligonucleotide primers (PPED04 and PPED05), which allowed amplification of a specific fragment (364 bp) within the IGS 2 of P. medicaginis using the PCR, was designed. Specific amplification of this fragment from P. medicaginis was highly sensitive, detecting template DNA as low as 4 ng and in a host-pathogen DNA ratio of 1000000: 1. Specific PCR amplification using PPED04 and PPED05 was successful in detecting P. medicaginis in lucerne stems infected under glasshouse conditions and field infected lucerne roots. The procedures developed in this work have application to improved identification and detection of a wide range of Phytophthora spp. in plants and soil.

Using the mating type genes (MAT-1 and MAT-2) from Cochliobolus heterostrophus (a pathogen of maize) as probes, MAT genes from C. carbonum (also a maize pathogen) and C. victoriae (an oat pathogen) were cloned. Sequence analyses and functional tests showed that C. carbonum MAT-1 and C. victoriae MAT-2 are structurally similar to their C. heterostrophus homologues, and all three genes function comparably when expressed in C. heterostrophus. Gel blot analyses of DNAs from C. heterostrophus or C. carbonum, probed with C. heterostrophus MAT-specific fragments, revealed that field isolates from diverse geographical locations contained either MAT-1 or MAT-2, but never both. The entire currently existing collection of C. victoriae isolates, however, contained only MAT-2, and all members of the collection were found to be either female sterile or completely infertile. Thus, crosses between isolates of C. victoriae were not possible, although crosses between C. victoriae and C. carbonum (but not C. heterostrophus) were readily made, confirming previous reports that these two species are interfertile. These observations, combined with careful scrutiny of the literature, have led to the hypothesis that MAT-1 female fertile isolates of C. victoriae have never existed. All available evidence is consistent with the notion that the entire C. victoriae field population was derived from a single progenitor strain, which arose as the result of horizontal transfer of genes for pathogenicity to oats into a strain of C. carbonum that was MAT-2 and female sterile.

Amplification of the internal transcribed spacer (ITS) of ribosomal RNA genes using PCR and subsequent RFLP analysis offers promise for improved species-level identification of ectomycorrhizal fungi. Intrageneric homogeneity and intraspecific heterogeneity of these characters are, however, frequent. A diverse group of ectomycorrhizal fungi with worldwide distribution was characterized by ITS-RFLP analysis. DNA of 64 identified and five unknown ectomycorrhizal fungi was extracted and their ITS regions were selectively amplified with ITS1-F and ITS4 ribosomal RNA gene primers. Each PCR product was digested with seven restriction enzymes. The resulting restriction fragments were compared by distance matrix analysis. Restriction enzyme analysis of amplified fragments was sufficient for distinguishing all but two closely related species. Intraspecific variation was observed in Cenococcum geophilum and Pisolithus arhizus, providing further evidence that these taxa represent species complexes.

A total of 500 strains of the thermophilic fungus Thermomyces lanuginosus were screened for their ability to produce proteases. A selected strain, T. lanuginosus P134, showed high enzymatic activity for proteases. The maximal yield of proteases was obtained with 4% casein, 4% glucose and 4% yeast extract in submerged culture. The protease system was shown to contain at least two protease isoenzymes by SDS–PAGE with gels containing gelatin as protease substrate. The effects of inhibitors imply that the two enzymes appear to be serine proteases. The crude proteases of T. lanuginosus P134 displayed unusual properties. The proteases were shown to be one of the most thermostable proteases so far isolated in fungi. The proteases were 100% stable at 50 °C. The half-life at 60 and 70° was approximately 160 min and 60 min, respectively. Temperature optimum of activity was 70°. The crude proteases were stable over a broad pH range (4–11). Optimum pH of activity was at 5·0 and 9·0.

A RAPD-PCR assay was developed and used to test for competitive variability in growth of the nematode biological control fungus Pochonia chlamydosporia. Saprophytic competence in soil with or without tomato plants was examined in three isolates of the fungus: RES 280 (J), originally isolated from potato cyst nematode (PCN) cysts; RES 200 (I) and RES 279 (S), both originally isolated from root knot nematode (RKN) eggs. Viable counts taken at 70 d indicated that I was the best saprophyte followed by S, with J the poorest. RAPD-PCR analysis of colonies from mixed treatments revealed that there was a cumulative effect of adding isolates to the system. This suggested that the isolates did not interact and that they may occupy separate niches in soil and the rhizosphere. To investigate parasitic ability, soils were seeded with two isolates of the fungus: J and S, singly or in combination. Tomato or potato plants were grown in these soils; free of nematodes, or inoculated with PCN or RKN, and incubated for 77 d. The abundance of the PCN isolate J in PCN cysts was significantly greater than that of the RKN isolate S but in RKN egg masses, S was significantly more abundant than J. RAPD-PCR analysis of colonies from mixed treatments confirmed that J was more abundant than S in PCN cysts whereas the converse was observed on RKN egg masses. This substantiates the phenomenon of nematode host preference at the infraspecific level of P. chlamydosporia and highlights its relevance for biological control of plant parasitic nematodes.

Phylogenetic analyses of the ITS1-5.8S-ITS2 region of the ribosomal RNA gene cluster were carried out with two short-cycled (aecial/telial) European rusts on Asteraceae, Puccinia distincta causing the current pan-European epidemic on Bellis perennis, and P. lagenophorae causing a similar disease on Senecio spp., as well as the macrocyclic P. obscura which alternates between B. perennis (pycnial and aecial host) and Luzula spp. (main host). All three species formed a well-resolved cluster when compared with the ITS sequences of a range of other rust fungi, using both parsimony and distance methods. The sequences of P. distincta and P. lagenophorae differed from each other at three positions whereas P. obscura differed from P. distincta at 37 points. Together with consistent morphological and epidemiological differences across Europe, these data support the recognition of P. distincta as a separate species from P. lagenophorae. Both may be derived from P. obscura, although the precise evolutionary history remains obscure.

The endophytic behaviour of Sclerotinia pseudotuberosa, cause of black rot of fruits of Castanea sativa, was investigated in asymptomatic tissues of the host, including nuts, buds and bark. A nested polymerase chain reaction (PCR) assay was developed for the specific detection of S. pseudotuberosa. Two specific primers, RAC1 and RAC2 were designed within the ITS1-5.8S-ITS2 region of rDNA by comparison of sequences of 24 isolates of S. pseudotuberosa and related species. Specificity of primers was tested against isolates of related and unrelated genera and the total DNA of C. sativa. The assay proved to be highly sensitive and capable of detecting picograms of S. pseudotuberosa DNA. Our study showed that S. pseudotuberosa was present asymptomatically in all the chestnut tissues tested. It is discussed that latency of S. pseudotuberosa in chestnut tissues may represent a adaptive strategy of the pathogen for rapid and massive host colonization in favourable environmental conditions.

A piece of Baltic amber (Tertiary, Eocene) contains an inclusion of a springtail (Collembola) which is overgrown by an Aspergillus species. The fossil fungus is described as A. collembolorum sp. nov. The excellent mode of preservation of the numerous conidiophores is remarkable and can be explained by sporulation in liquid resin. This is the second report of a fossil Aspergillus, the first being from Dominican amber.

Aspects of the ecology of oospores of Phytophthora infestans were studied in the highlands of central Mexico. From an investigation of a random sample of strains, it was found that isolates differed in their average capability to form oospores when engaged in compatible pairings. Most crosses produced large numbers of oospores but a few yielded none and some yielded only a few oospores. The results reveal that oospore production and fecundity is dependent on both isolates and the combining ability of a specific combination of parental strains. On average, 14% of the oospores produced were viable as determined by the plasmolysis method. Viability ranged from a low 1% in one cross to a high of 29% in another cross. Oospores were found in 10–20% of naturally infected Solanum demissum leaves from two different collections, and leaflets with two lesions per leaflet produced more oospores than did leaflets with 3–5 lesions per leaflet. There was no consistent trend for preferential mating between isolates from the same location or host.

Host-parasite interactions of Agaricus bisporus and Verticillium fungicola were studied in paired-cultures. Different growth reactions were found according to the culture medium used, but no apparent alterations could be detected by light and electron microscopy in A. bisporus vegetative mycelia. In vitro, different hydrolytic enzymes are produced by V. fungicola in cultures with A. bisporus vegetative mycelial cell walls as the only carbon source. In vitro, this enzyme preparation is able to partially digest isolated A. bisporus vegetative mycelial cell walls. These results suggest that although apparent attachment of the mycoparasite to the host cell surface seems to occur, there is no in vivo A. bisporus vegetative mycelial cell wall degradation.

Recombination between two strains of Metarhizium anisopliae, subcultured on artificial medium and passed through an insect host was investigated. When grown on agar culture, or passed through a host, strains did not show detectable genetic changes. When passed through an insect in the presence of the other strain, genotypic changes in the isolates, as detected by esterase electrophoresis, restriction digestion of total genomic DNA and RAPD-PCR, were apparent. The instability of molecular markers suggests recombination between strains.

Mycosphaerella tasmaniensis is newly described from Mycosphaerella leaf blotch symptoms occurring on Eucalyptus nitens in Tasmania, Australia. Single ascospore cultures produced a Mycovellosiella anamorph, described here as M. tasmaniensis. Both states occurred together, as well as separately on leaf spots. Phaeophleospora epicoccoides (=Kirramyces epicoccoides) is commonly associated with leaf spots of Eucalyptus spp. in Australia. The teleomorph, Mycosphaerella suttoniae, previously known only from Indonesia, was also collected on E. grandis leaves from Australia. A Cylindrocladium leaf blight disease of young E. grandis trees in Indonesia was found to be associated with a new species of Calonectria. Calonectria multiseptata and its anamorph Cylindrocladium multiseptatum is newly described and distinguished from other species based on their larger, multi-septate ascospores and conidia.

In this investigation, we have determined the life span of the fruit bodies of Agaricus bisporus grown in a protected cultivation environment as 36 d. We describe the morphological changes occurring in ageing mushrooms and find that signs of senescence become visible around day 18; they steadily increase in amplitude towards the end of the fruit bodies' life. Cytologically, karyolysis and focal cytoplasmolysis were the first signs followed by indications of an increased permeability of the cytoplasmic membranes and by structural changes of the cell wall. These changes result in extracellular aggregations of the lytic cell remains encapsulating or bridging the hyphal cells. Cells of the stipe tissues were transformed to empty cylinders or had irregularly collapsed. Most basidia and subhymenial cells remained alive even on day 36. When the mushrooms were cultivated according to our usual growth procedures, about 50% of the fruit bodies were infected by Trichoderma harzianum and/or Pseudomonas tolaasii on day 18; all fruit bodies died on day 24 due to diffuse bacterial and mycotic infections manifesting themselves by tissue necrosis and discoloration of the caps and stipes. When none of the fruit bodies was harvested at the time of the first flush they soon formed a canopy of pilei and their growth ceased. On day 14, all these fruit bodies showed widespread mixed infections followed by a diffuse liquefaction necrosis of all tissues including the hymenium. Post-harvest fruit bodies kept at ambient temperature or at 2 °C at low r.h., or at 20° at high r.h. revealed a diffuse cell wall destruction which was followed by cytoplasmic degeneration in due course. Fruit bodies kept refrigerated had the least gross and cell structural changes during our observation period of 7 d. We conclude that the morphological changes occurring in post-harvest and in senescent fruit bodies of Agaricus bisporus are different.

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’.

Currently, mycopesticide development for locust and grasshopper control depends on aerial conidia or submerged spores of entomopathogenic fungi. In our study, the production of submerged conidia of Metarhizium anisopliae var. acridum (IMI 330189) was investigated in a liquid medium containing 3% biomalt and 1% yeast extract (BH-medium). The effects of freeze and spray drying techniques on the quality of submerged conidia were determined. The influence of different additives on the viability of fresh submerged conidia and their suitability for oil flowable concentrate formulation development was assessed. In a BH medium maintained at 180 rev min−1, at 30 °C for 72 h, IMI 330189 produced a green pigmented biomass of submerged conidia whereas in Adámek medium it produced a yellowish biomass of submerged spores. The spore concentration was high in both media; however, the size of the spores produced in the BH medium was significantly lower than those produced in Adámek medium (P<0.001). Submerged conidia can be effectively dried using either freeze or spray drying techniques. The viability and speed of germination were significantly affected by the drying and pulverizing process (P<0.001). The initial viability was significantly higher for spray-dried submerged conidia than for freeze-dried spores. Pulverizing of freeze-dried submerged conidia reduced the speed of germination and the viability by 63–95%. Dried submerged conidia can be stored over 45 wk at low temperatures (<10°) without suffering a significant loss in viability. Furthermore, we have identified carriers that are suitable for oil flowable concentrate formulation development.

The phylogenetic relationship between Epicoccum nigrum and Phoma epicoccina has been assessed by means of sequencing of the ITS regions of rDNA. The analysis of the sequences from five isolates of E. nigrum and four of P. epicoccina suggests that both entities represent the same biological species.

The present work reports the development of a PCR assay to detect the ascomycete Biscogniauxia mediterranea in asymptomatic tissues of Quercus cerris. To this purpose, two specific primers (MED1 and MED2) were designed by comparison of sequences comprised between ITS1 and ITS4 generic primers of 21 isolates of B. mediterranea and related species. Specificity of the primers was tested against 19 isolates of B. mediterranea and 20 isolates of related and unrelated genera, and against Quercus cerris DNA. The reliability of the results was confirmed by Southern Blot analysis. The 2 specific primers MED1 and MED2 were able to detect B. mediterranea DNA in the host tissues at picogram quantity of target DNA. This assay would greatly enhance the possibility to study the presence and localization of B. mediterranea during its latent phase in Quercus species.

Characteristics of hyphal septation, cellular interaction, sporulation, cultures and/or nucleotide sequences from the 5′ terminal domain of the nuclear large subunit rRNA gene of some species of Exobasidium, Microstroma and Muribasidiospora were examined and compared. Our analyses show that the order Microstromatales comprises Microstroma, Muribasidiospora triumfetticola and the anamorphic species Rhodotorula bacarum, R. phylloplana and Sympodiomycopsis paphiopedili, whereas Muribasidiospora hesperidium and Muribasidiospora indica are members of the Exobasidiales. Because of the high degree of morphological divergence between Muribasidiospora triumfetticola and Microstroma, a new genus, Volvocisporium, and a new family, Volvocisporiaceae, of the Microstromatales are proposed. Apart from the phylogenetic implications, the most interesting observations in the present work are the unusual development of the basidia, especially of the basidiospores, and the nutrient transport system from the host attacking hyphae to the basidia in Muribasidiospora triumfetticola.

Two-dimensional gel electrophoresis was used to analyse stage-specific proteins from Phytophthora palmivora, a pathogen of cocoa and other economically important tropical crops. Approximately 1% of proteins appeared to be specific for each of the mycelial, sporangial, zoospore, cyst and germinated cyst stages of the life-cycle. Three proteins excised from protein gels of P. palmivora were identified as isoforms of actin by database searches to public libraries of Phytophthora infestans. The protein profiles of parallel samples of P. palmivora and P. infestans demonstrated that 30% of proteins precisely co-migrated suggesting that proteomics may be used to examine changes in the specific stages in the life cycles of Phytophthora spp.

In an in vitro study, interactions between ectomycorrhizal fungi, saprotrophic basidiomycetes and soil microfungi known from pine forests were examined in relation to different ammonium concentrations. The saprotrophic basidiomycetes Clitocybe marginella and C. vibecina and the soil microfungi Penicillium pinetorum, Oidiodendron flavum and Torulomyces lagena were grown against the ectomycorrhizal Cenococcum geophilum, Laccaria bicolor and Rhizopogon luteolus. Interactions were species specific and overgrowth was the most common (43%), followed by inhibition at distance (26%) and contact inhibition (16%). Ammonium concentrations affected the type and strength of the interactions. The competitive strength of the ectomycorrhizal fungi was relatively high and only the growth of the mycelia of L. bicolor was clearly suppressed by the Clitocybe spp. The interactions are discussed in relation to the ecology of the fungi and potential future studies.