Fifty-five Phytophthora sojae isolates were collected from soil samples and diseased soybean plants from Illinois, Indiana, Iowa, and Minnesota in 1994 and 1995. Races for the isolates were determined. DNA of P. sojae isolates was amplified with 16 Operon decanucleotide primers. Twenty-three of 75 amplified fragments were polymorphic. Based on the 23 RAPD markers, a dendrogram depicting the relatedness of the isolates was constructed using UPGMA. The P. sojae isolates clustered into four distinct groups. The isolates of races 3, 4 and 25 clustered into group I. The isolates of races 1, 8 and 13 clustered into group II. The isolates of race 5 clustered into group III, and the isolates of race 7 clustered into group IV. Genetic diversity was detected among isolates of races 1, 3, 4, 5, 7 and 25 but not among isolates of races 8 and 13.

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.

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.

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.

The basidiomycete genus Armillaria causes root rot and death to woody plants in boreal, temperate and tropical regions of the world. Armillaria root rot has been described from various parts of Africa on many different hosts. However, very little is known regarding the evolutionary relationships among Armillaria species in Africa. The aim of this study was to determine the phylogenetic relationships between isolates originating from different regions in Africa using nDNA sequences from two non-coding gene regions. The ITS and the IGS-1 regions of the ribosomal DNA operon were sequenced and analysed using different phylogenetic tree searching methods. Phylogenetic trees grouped the African taxa in two strongly supported clades. One of these represented A. fuscipes and the other an undescribed but distinct species.

The ability of Botrytis cinerea to produce cutinolytic, pectolytic and cellulolytic enzymes was re-examined in vivo and in vitro. In liquid culture, B. cinerea produced both constitutive and inductive forms of hydrolytic enzymes. The constitutive levels were 44 mU, 720 mU, 1820 mU, 12·0 U and 22·4 mU for cutin esterase, endopolygalacturonase (endoPG), exopolygalacturonase (exoPG), pectin methyl esterase (PME) and pectate lyase (PL), respectively. Unlike the other enzymes, however, carboxymethyl cellulase (CMCase) production was found to be entirely inducible. The enzymes secreted at the germination and germ-tube growth stages, and during subsequent disease development, were quantified simultaneously. Complete germination was observed within 9 h of incubation of B. cinerea on the surface of bean leaves and disease symptoms gradually increased during days 1–6 of the incubation. Significant amounts of all hydrolytic enzymes studied in the early hours and monitored during 6 d of incubation were detected, supporting the possibility of a major role for the enzymes produced by B. cinerea in the development of disease. The amount of cutinase was found to be uniform from the beginning, indicating that this enzyme may have an important role in penetrating the impervious cutin layer. The exoPG level increased from the beginning of incubation whereas that of endoPG started to rise later. PL and PME were also detected on the leaf surfaces. The patterns of in vitro production of exoPG, endoPG and PL correlated with the in vivo production of these enzymes, but there was only partial correlation in the production of PME and no correlation in the patterns of cutinase and CMCase production.

Many species formerly listed in Entyloma have been removed and are now placed in several orders. This study aims to clarify the framework of the genus Entyloma and the phylogeny of these plant parasitic smut fungi with molecular data. Analyses of LSU and ITS sequences are presented and support the monophyly of the order Entylomatales. The sequences show higher similarities within the examined species of Entyloma than within other smut families and genera, suggesting a recent radiation. Within the Entylomatales a cluster of anamorphic Tilletiopsis washingtonensis, T. lilacina and T. cremea collections is the sister group to Entyloma. The phylogenetic relationships in the genus Entyloma are a result of joint evolution with their hosts. The analyses of the sequence data show unresolved groups on Ranunculales and a well-supported group on Asteridae.

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.

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.

The performance of isolates of the ectomycorrhizal fungi Coltricia perennis, Laccaria bicolor, Lactarius hepaticus and Paxillus involutus originating from Scots pine stands was studied on solid media with different concentrations of ammonium, glycine and glucose as inorganic nitrogen, organic nitrogen and carbon source. Laccaria bicolor was able to grow on both ammonium- and glycine-containing media. Biomass and growth rate of L. hepaticus and P. involutus on media with ammonium were generally higher than on media with glycine as nitrogen source. Coltricia perennis did not clearly show preference for either ammonium or glycine. Fractal dimensions, calculated from the biomass and radii of the fungi, were in the range 1–3. Fractal dimensions provided extra information about the foraging strategy of mycelia not obtained from growth rate and biomass production measurements, but cannot replace those because the use of fractal dimensions alone may lead to misinterpretation.

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.

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.

Chytridiomycota were isolated into pure culture from cool temperate and warm semi-arid soils of eastern Australia. In pure culture these fungi responded variably to the range of temperatures commonly recorded in their environment. All members of the Blastocladiales, Spizellomycetales and Chytridiales grew in culture at temperatures up to 30 °C. Some isolates from the Blastocladiales and Spizellomycetales continued to grow at or above 37 °. Some isolates of the Chytridiales grew up to but not beyond 35 °. All isolates in the Chytridiales were able to resume growth at 20 ° after brief exposure to temperatures higher than the maximum growth temperature, but were killed by exposure to higher temperatures for 7 d. Because in the natural soil habitat temperature may exceed the maximum for growth it may be a limiting factor that determines the distribution of chytrids in the soil.

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’.

Two distinct and unrelated groups of ophiostomatoid fungi are associated with Protea infructescences. Two species, Gondwanamyces proteae and G. capensis, have unique anamorphs that reside in Knoxdaviesia. This anamorph genus is infrequently found amongst species of Ceratocystis sensu lato. The second group includes Ophiostoma splendens and O. protearum that have Sporothrix anamorphs typical of Ophiostoma. During recent collections of Protea gaguedi infructescences from the Northern Province in South Africa, an apparently new species of Ophiostoma with a Sporothrix anamorph was discovered. It can be distinguished from O. splendens and O. protearum based on its short ostiolar hyphae and its associated plant host. This teleomorph is described as O. africanum and the anamorph as S. africanum. Five species of ophiostomatoid fungi are now known from Protea infructescences and a key is presented for them.

The ability of Peridermium pini and Cronartium flaccidum aeciospores and mycelium to infect known (Vincetoxicum spp., Pedicularis spp., Paeonia spp.) and potential (Melampyrum spp., Pyrola sp., Dactylorhiza sp., Solidago sp., Salix sp., Geranium sp. and Maianthemum sp.) alternate hosts was tested. None of the mycelial cultures and only 10% of the aeciospore samples produced uredinia or telia on the tested species suggesting that most aeciospores in Finland belong to the autoecious P. pini. Aeciospores from three locations in northern Finland, however, produced uredinia or telia on Vincetoxicum mongolicum, V. nigrum, V. fuscatum, Paeonia anomala, three P. officinalis cultivars, Melampyrum sylvaticum and Pedicularis palustris either in vitro or in vivo, indicating that these aeciospores belong to the heteroecious C. flaccidum, which occurs sporadically in Finland. Interestingly, the host-specificity of C. flaccidum encountered in Finland was very low (e.g. one sample produced uredinia or telia on eight species). This, added to the wide distribution of Melampyrum spp. over northern Fennoscandia, suggests that the main alternate hosts for C. flaccidum in Finland may be in Melampyrum rather than Pedicularis.

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.

Cercospora leaf spot of olives is a serious defoliating disease attributed to Pseudocercospora cladosporioides. Although the disease is well distributed throughout olive growing regions of the world, its epidemiology and population structure remains unknown. The aim of this study was to establish the genetic variability of Spanish isolates of P. cladosporioides using DNA sequence data from the ITS region, as well as two protein-coding genes, actin and calmodulin. Phylogenetic data obtained here support P. cladosporioides as closely related to other Pseudocercospora species that cluster within Mycosphaerella. Spanish isolates clustered in two clades: isolates from Catalonia were different from those collected in Andalusia. However, isolates appeared to be genetically relatively homogeneous, suggesting that chemical control of this disease via a managed spraying programme may prove a viable option for controlling the disease in Spain.

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.