The life-cycle of many plant-pathogenic fungi, especially those infecting aerial plant organs, contains several specific developmental stages. If these are sufficiently distinct in their physiology from vegetative hyphal growth, they present potential targets for non-fungitoxic plant protectants. The present review identifies such targets especially in the pre-penetration stages of the infection cycle of Magnaporthe grisea and other fungi infecting from air-borne spores. Examples of non-toxic natural products with activity against spore germination, attachment, appressorium formation, appressorium maturation and penetration of the host surface are given. In contrast, no substances selectively active against in planta growth or sporulation appear to be known. The selective activity of numerous secondary metabolites against specific infection stages without accompanying toxicity against vegetatively growing hyphae indicates a direction for the development of future natural product-derived fungicides which are more easily degraded in the environment and possess fewer non-target effects. Such substances are produced by many saprotrophic and endophytic fungi in pure culture. The paucity of data on the production of biologically active substances in natural situations limits the interpretation of their ecophysiological significance for the producer.

Scleroderris canker of conifer is caused by Gremmeniella abietina var. abietina, which comprises several taxa, including races, varieties, and biotypes. The European race of G. abietina var. abietina was introduced into North America early in the century, most likely on asymptomatic infected pine seedlings, and is currently widespread in eastern North America. In order to detect latent infections and differentiate the North American and European races of the fungus, we developed oligonucleotide primers to amplify by PCR portions of the ITS of the ribosomal DNA of G. abietina var. abietina. The 417 bp amplified DNA fragment comprises two Msp I restriction sites in the NA race but only one in the EU race. DNA extractions directly from infected asymptomatic needles, or from single fruiting bodies, followed by PCR amplification and Msp I digestion allowed the detection and race identification of both races of G. abietina var. abietina from seedlings and branches of Pinus resinosa and P. banksiana. A nested PCR assay was sensitive enough to detect the equivalent of a single infected seedling in a bulk sample of 1000 healthy seedlings. Validation tests were conducted by comparing PCR and isolation assays with 104 fascicles. All samples for which the fungus was isolated yielded a positive PCR assay and there was no false negative, i.e. samples for which the fungus was isolated but not detected by PCR. Among the samples from which the fungus was not isolated, most yielded a negative PCR assay (71%), but a proportion (29%) yielded positive PCR assays. In several of those cases, aggressive contaminants had apparently overgrown the pathogen. The method described here can lead to the detection and race identification of the NA and EU races of G. abietina var. abietina directly from infected tissues without the need to culture the fungus and should find applications in nursery inspection and quarantine.

The mountain pine beetle, Dendroctonus ponderosae, and its fungal associates are devastating the lodgepole pine forests in British Columbia, Canada. During our fungal survey, an unknown Leptographium species has been consistently isolated from both D. ponderosae and infested lodgepole pine (Pinus contorta var. latifolia). This Leptographium species has similar morphology with the Leptographium anamorph of Ophiostoma clavigerum whose association with the D. ponderosae is well known. However, thorough morphological comparisons showed that this fungus is distinct from all the other Leptographium species described in the literature, and especially from O. clavigerum. Comparison of DNA sequences of multiple loci and the profiles by the PCR-RFLP marker also confirmed that this Leptographium species represents an undescribed taxon. Based on its distinct morphological, physiological characteristics and phylogenetic position, we describe it as L. longiclavatum sp. nov.

Conserved primers were used in a polymerase chain reaction to amplify the ITS region of the rDNA of 100 Microdochium nivale isolates collected from different turfgrasses in southern Ontario. The profile of the restriction digestion of the amplified ITS region revealed that all the M. nivale isolates analysed belonged to var. nivale. RAPD profiling and RFLP analyses of the IGS regions of rDNA revealed extensive genetic diversity within var. nivale. With RAPD markers, the average similarity coefficient was 66% and the estimate of genotypic diversity was 0·179. Population subdivision analysis showed that 92·2% of the total genetic diversity was found among individuals within populations compared to 7·8% among populations. In dendrograms derived from genetic distances using RAPD and IGS-RFLP markers, there was some evidence for host specialization. Most RAPD markers were shared by individuals from different turfgrasses, and the populations were not highly differentiated. The high level of genotypic diversity detected within populations and the low level of genetic differentiation among populations show that recombination and migration are likely playing important roles in the population biology of M. nivale var. nivale.

The ectomycorrhizal fungus Hebeloma velutipes consists of two biological species (BSP 16 and 17). Within BSP 17 a dikaryon was found with two divergent types of the ribosomal Internal Transcribed Spacer (ITS1 and 2). The two ITS types segregated in monokaryotic progeny of that dikaryon, showing that these different ITS types represent different alleles at homologous rDNA loci in the two nuclei. RFLP analysis of a number of strains of BSP 17 showed that the polymorphism is widespread in Europe. There was no deficiency of the heterokaryotic type, demonstrating that ITS divergence in this species is not correlated with reduced intercompatibility. A strain from North America, not assigned to a biological species, showed the same polymorphism. Cladistic analysis of the two ITS sequences showed that they were not sister groups. One of the ITS types formed a monophyletic group together with the ITS type of BSP 16, the other type formed a clade with the ITS type of H. incarnatulum (BSP 18). BSP 16 and 17 showed partial intercompatibility. However, several lines of evidence suggest that the polymorphism of BSP 17 is not the result of frequent and continuing hybridisation with BSP 16. Instead, we give arguments for the hypothesis that the polymorphism evolved in allopatry and that the two types have come together relatively recently. The results of the polymorphism indicate a potential problem for molecular identification of fungal species based on ITS fingerprinting. The results also show that no generalisations are possible about the relation of speciation (the formation of BSP) and nuclear ITS divergence.

Needles of Pinus strobus (white pine) were cleared and stained to survey the occurrence and location of Lophodermium sp., a fungal endophyte. Cytoplasmically dense endophytic hyphae with a pronounced lobed morphology and containing lipid bodies were localized intercellularly between the epidermis and hypodermis. These fungal infections did not appear quiescent, but rather exhibited signs of continual slow growth. A few associated host cells exhibited a hypersensitive response. Material embedded in resin and examined by light microscopy and transmission electron microscopy confirmed the location of hyphae between epidermal and hypodermal cells, and the presence of lipid bodies within the hyphae. In senescing needles, aggressive colonization of needle tissues occurred. Thus, for Lophodermium in white pine, endophytic infection is active rather than quiescent, and displays an alternate hyphal strategy to that seen in the reproductive phase.

Almost 900 species were encountered during a seven year survey of fungal fruit bodies in 13 forests and grasslands plots measuring about 1 ha. The data were comparable to those from a Swiss forest plot monitored for 21 years. Species richness and abundance were almost linearly correlated on a log/log scale. Abundance and yearly frequency of species were tightly correlated. The geographic range of species may also be related to these two parameters. Rare species scored low on abundance, yearly frequency and geographic range. About half of the species were rare, and occurred in only one out of seven years. The two major functional groups of saprotrophic and mycorrhizal species behaved similarly over the years, as in the Swiss study.

Annual variation was high for species richness alone and for richness combined with abundances. The log transformed species richness of plots correlated well with parameters that take the abundances or yearly frequencies of species into account. If other mushroom assemblages show similar regularities, it may be possible to omit abundance counts in future surveys. The species composition of the plots varied strongly over the years. Many species did not reach their maximum abundance in the richest year. Species showed their maximum abundances in different plots in the same year more often than expected by chance. Thus the presence and abundance of species depended on factor(s) other than the general productivity of a year, and years showed (a) different aspect(s) than productivity alone. Long-term surveys are important for understanding the structure of mushroom assemblages and their biodiversity.

The effect of simulated rain on the persistence of oil and water formulations of conidia of the entomogenous fungus Metarhizium anisopliae when applied to oilseed rape foliage was investigated, using third instar larvae of the mustard beetle (Phaedon cochleariae) as the target host. Rain significantly (P < 0.01) reduced the susceptibility of the beetle larvae to M. anisopliae but the amount of inoculum removed was influenced by the formulation. Larvae exposed to plants treated with conidia formulated in aqueous Tween, Shellsol T, or sunflower oil/Shellsol T resulted in 55, 82.5 and 72.5%, mortality, respectively. The mortality for these respective formulations was reduced by 42, 57 and 51% if the plants were exposed for 1 h to simulated rain. Laboratory and field studies showed that more inoculum collected beneath plants sprayed with conidia formulated in Shellsol T or aqueous Tween than in the more viscous sunflower/Shellsol T mixture. Mortality studies on leaves taken from field plots suggested that conidia on leaf surfaces could be replenished by repeated application. The number of conidia isolated from field plots was greater where inoculum was applied bi-weekly than once weekly.

The effects of growth media, temperature, pH and light on the development of four isolates of Coniothyrium minitans (CONIO and CH8 (colony type 3), G4 (colony type 4) and G9 (colony type 5)) were examined. Conidial germination, pycnidial production and hyphal extension rate were initially studied on seven different agar-based growth media at 18–20 °C. Potato-dextrose agar (PDA) and malt extract agar (MEA) consistently gave the greatest conidial germination, pycnidial production and hyphal extension rate for all four isolates. Growth and development on molasses-yeast agar was equivalent to that on PDA and MEA except that hyphal extension rate was slower. Subsequently, the effects of temperature, pH and light on the development of C. minitans were investigated on PDA only. The temperature range of conidial germination and pycnidial production of the four isolates was between 10–25° with the optimum at approximately 20°. Hyphal extension occurred over a greater temperature range, between 4 and 25°, with a maximum extension rate of approximately 3·5 mm d−1 for all isolates occurring between 20–25°. Conidial germination, pycnidial production and hyphal extension occurred over a pH range between 3–8 with optimum values for all growth assessments occurring between pH 4·5 and 5·6. Increasing light period from continuous dark, to 12 h light/12 h dark or continuous light had no effect on conidial germination or extension growth, but significantly increased pycnidial production.

Isolates G4 and G9, previously characterized by sparse production of pycnidia in comparison with CONIO and CH8, consistently exhibited a reduced production of pycnidia on all media, at all temperatures and pH ranges, and all light regimes tested. This demonstrates the stability of this character among these isolates of C. minitans. The significance of these results for improving production of inoculum of this biocontrol agent and in the identification and classification of isolates of C. minitans is discussed.

Botrytis cinerea (anamorph of Botryotinia fuckeliana) is a filamentous ascomycete that causes grey mould especially on grapevine. Based on the presence or the absence of two transposable elements (Boty and Flipper) two sibling sympatric populations named transposa and vacuma have been described. Among the vacuma population, some strains (designated HydR1) were found to be resistant to fenhexamid (a sterol C-4 demethylase inhibitor) and to show an increased sensitivity to 14α-demethylase inhibitors (DMIs). In order to assess whether or not mutations at the target gene level (CYP51) could underlie increased sensitivity to DMIs in HydR1 strains, we cloned the CYP51 gene and determined its DNA sequence in various B. cinerea strains. The gene was highly polymorphic, with mutations detected at 58 positions in the 35 strains analysed. The polymorphisms did not discriminate between transposa and vacuma strains, but did distinguish between HydR1 and non-HydR1 ones. Two expressed mutations were present in all HydR1 strains, namely phenylalanine to leucine at position 15 of the inferred protein, and serine to asparagine at position 105. These data, combined with the existence of morphological differences and somatic incompatibility between HydR1 and non-HydR1 strains, suggest that these two groups comprise distinct genetic entities.

The current circumscription of Lasiosphaeria includes taxa with a wide variety of ascomatal walls, ascomatal wall vestitures, and ascospore morphologies and a broad range of putative anamorphs. Despite the complexity of morphological characters in the genus, species within Lasiosphaeria can be arranged into four groups based on ascospore morphology. Taxa which possessed ascospores in each of the four groups were used in phylogenetic analyses of partial nuclear large subunit (LSU) rDNA sequences to test the monophyly of the genus and determine relationships among its species. Lasiosphaeria was found to be highly polyphyletic in that species segregated into seven well-supported monophyletic clades dispersed among several orders. Three new genera, Echinosphaeria, Hilberina, and Immersiella, are erected for three of these clades while the genus Lasiosphaeris is reintroduced for a fourth clade. These data support Ruzenia as a previously established genus and the transfer of Lasiosphaeria raciborskii to Chaetosphaeria. The circumscription of Lasiosphaeria has been considerably narrowed to better reflect a natural classification. These taxonomic changes are additionally supported by a combination of morphological characters which are discussed in relation to the phylogenetic trees.

Progress in understanding the biology of arbuscular mycorrhizal fungi is hampered by the limited number of species that can be successfully propagated and studied in vitro. We report the establishment of monoxenic cultures of Glomus etunicatum in association with excised Ri T-DNA transformed carrot roots. The fungus can be propagated in vitro using monoxenically formed resting spores and/or colonized root fragments. Modified White's medium buffered with 10 mM MES (pH 6) or MOPSO (pH 6·5) was most optimal for the host root growth as well as for G. etunicatum spore germination and mycorrhiza formation. The number of resting spores formed in vitro correlated positively with the length of roots occupied by arbuscular mycorrhizal structures, including arbuscules and vesicles. Spores first appeared in dual cultures within two weeks of root inoculation. Sporulation was asynchronous and continued until root senescence. Under applied culture conditions, spores achieved mature appearance within 5–7 d after their initiation. Approximately 6% of monoxenic spores were aborted at different stages of their development. Although G. etunicatum spores formed in vitro exhibited general morphological and anatomical similarity to soil-borne inoculum, they were significantly smaller and had thicker spore walls than their soil-borne counterparts. Caution should, therefore, be exercised in utilizing the in vitro system as a model of growth and development of glomalean fungi in soil.

Partial sequences of the mtLSU rDNA were obtained from the arbuscular mycorrhizal (AM) fungi Glomus proliferum (isolate DAOM 226389) and G. intraradices (isolates JJ291 and BEG75). The exon sequences of the two species showed regions of strong divergence. There was no evidence of intra-isolate sequence heterogeneity as it is found in variable regions of nuclear ribosomal genes of Glomeromycota. In G. intraradices JJ291, two introns were found in the partial LSU sequence. One of the introns contained an ORF for a putative site-specific homing endonuclease of the LAGLIDADG family. In G. intraradices BEG75, one of the introns was missing and the other had a DNA sequence distinct from JJ291. G. proliferum had no introns in the region sequenced. A PCR primer was designed to amplify the fragment of the mtLSU of a different, distinguishable G. intraradices genotype from colonized roots of a field sample. These mitochondrial gene sequences are the first reported from the phylum Glomeromycota. Our findings indicate that the intra-individual sequence heterogeneity of the Glomeromycota may be a peculiar feature of the nuclear genes. Therefore, mtLSU and its introns have the potential to be highly sensitive genetic markers for these fungi in the future.

The Amphisphaeriaceae (sensu lato) presently includes 36 genera and 23 synonyms and is a heterogeneous assemblage of ascomycetes. In this paper molecular and morphological data and teleomorph–anamorph connections are examined and confirm that the Amphisphaeriaceae (sensu stricto) should be confined to ten teleomorphic genera and their Pestalotia-like anamorphs. These include Amphisphaeria, Broomella, Discostroma, Ellurema, Griphosphaerioma and Neobroomella which are described and illustrated; and Blogiascospora, Lepteutypa, Paracainiella and Pestalosphaeria which are discussed.

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.

Release and dispersal of basidiospores of Amanita muscaria var. alba and their potential to infiltrate a nearby residence were investigated. Basidiospore release mainly occurred in the first three days following the expansion of the caps. The concentrations of released basidiospores near basidiomata were 77137, 75062, and 41738 spores m−3 in the first three days, respectively, with the highest concentration at 281738 spores m−3 air. After three days, the concentration dropped by 95%. At the second location, airborne basidiospore concentrations dropped 96–99% after three days with the concentrations of 940, 575, and 1359 spores m−3 in the first three days, respectively. The diurnal pattern showed a relatively extended night peak. Relative humidity and dew were positively correlated with basidiospore release and short distance dispersal. Rain and rain rate were positively correlated with basidiospore release, but not correlated with short distance dispersal. The basidiospore release period of Amanita muscaria var. alba was short, but within such a period it released a large amount of basidiospores. However, only less than 5% of basidiospores released were dispersed to the second location 5.2 m away and 2.7 m above the basidiomata. Only <0.1% of basidiospores dispersed from the basidiomata were found inside a nearby residence. Amanita muscaria var. alba showed a low potential of infiltrating the residence.

Growth of A. ochraceus at 30°C was significantly influenced by changes in both solute (NaCl, glycerol) and matric potential (PEG 8000). Optimum growth rates were at approx. −10·0 MPa with the non-ionic solute, and between −3·5 (=0·975 aw) and 14·0 MPa (=0·90 aw) with the ionic solute in a sucrose-based minimal salts medium. The optimum matric potential for growth was at −7·0 MPa (=0·95 aw). Growth rates were influenced by whether colonies were grown directly on the agar medium or on cellophane overlays. Sclerotia were profusely formed on media >−1·5 MPa (=0·99 aw), only initials at −3·5 MPa, and none under optimum conditions for growth (−7·0 to −10·0 MPa). The contents of low and high mol. wt polyols (glycerol, erythritol, arabitol and mannitol) and sugars (glucose and trehalose) were quantified in whole colonies and conidia grown under different osmotic and matric potential conditions for the first time. There was a higher total amount of polyols (μg mg−1D.W..) in conidia than in whole colonies. In glycerol-amended osmotic media maximum total polyols were present at −4·5 (=0·965 aw) and −17·0 MPa (=0·88 aw) in mycelial colonies and conidia respectively, with glycerol and mannitol being predominant in both, with small amounts of erythritol also present in conidia. By contrast, in matrically-modified media, the high mol. wt mannitol was the major component (>75% of the total) with small amounts of arabitol and the low mol. wt erythritol and glycerol, regardless of matric potential. For sugars, trehalose was predominant in conidia (>90%) from osmotically-modified media with low levels of glucose present in both conidia and mycelial colonies at all potentials tested. By contrast, in matric media trehalose was the predominant sugar in both conidia and whole colonies. This study suggests that xerophilic fungi may use different mechanisms for overcoming osmotic and matric stress.

Although Eutypa lata is the main agent of Eutypa dieback of grapevine, another species of Eutypa has been isolated from diseased grapevines in Northern California. Stromata of this recently discovered Eutypa were also collected from Acer macrophyllum, A. negundo, and Fraxinus latifolia in the vicinity of vineyards, and appeared commonly on Umbellularia californica in some mixed-evergreen forests of Napa and Sonoma counties. This second species of Eutypa was distinguished from E. lata because of the sulcate ostiole of the perithecium and smaller ascospores. A morphological comparison with type specimens revealed identical features between the Californian isolates and E. leptoplaca sensu Rappaz (1987). This identification was confirmed through phylogenetic analyses of Eutypa spp. based on the complete sequence of the internal transcribed spacer (ITS) of the rDNA and partial sequence of the β-tubulin gene. These analyses also separated collections of E. maura, E. sparsa, E. lejoplaca, E. tetragona, E. leptoplaca and E. lata, confirming the previously proposed species concepts. The pathogenicity of E. leptoplaca on grapevine was established using isolates collected from Vitis vinifera, U. californica, and A. macrophyllum. The importance of E. leptoplaca in relation to Eutypa dieback and its role as a necrotrophic pathogen are discussed.

This study investigated the morphology and infectivity of fine endophyte in a mediterranean environment. Five morphological groups of fine endophyte differing in up to 11 characters were present within two pot cultures of fine endophyte from south- western Australia. Occurrence and abundance of a particular type of fine endophyte were not affected by the application of calcium carbonate. It appeared that there may be more than one fungus included among the two sources of fine endophyte, however it was not possible to separate the fungi. A second experiment demonstrated that infective propagules of fine endophyte can be very small (<38 μm) and fresh roots acted as a potential source of infective propagules. There was no infectivity of fine endophyte in the soil at the end of the growing season but it reoccurred in similar quantity in the next growing season.

Pycnidial fungi belonging to the genus Ampelomyces are the most common natural antagonists of powdery mildews worldwide. During a study of the interactions between apple powdery mildew (Podosphaera leucotricha) and Ampelomyces mycoparasites, 52 new Ampelomyces isolates were obtained from P. leucotricha and, in addition, 13 new isolates from other species of the Erysiphaceae in four European countries. Their genetic diversity was screened using single-stranded conformation polymorphism (SSCP) analysis of the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA). For comparison, 24 isolates obtained from genetic resource collections or other sources were included in this study. Based on the ITS-SSCP patterns, the isolates were placed in eight groups. The isolates belonged to two types based on their growth in culture. The faster-growing and the slower-growing isolates were included in different SSCP groups. A phylogenetic analysis of the ITS sequences of representatives of these groups confirmed the results obtained with the SSCP method, and showed that the faster-growing isolates do not belong to Ampelomyces as suggested by earlier studies. All the isolates from P. leucotricha fell into a distinct SSCP group of genetically homogeneous isolates. This suggests that Ampelomyces mycoparasites which occur in apple powdery mildew are slightly different from the other Ampelomyces groups which contain mycoparasites from various powdery mildew species. This may be because the main growth period of Ampelomyces mycoparasites in apple powdery mildew is isolated in time from that of Ampelomyces isolates that occur in other species of the Erysiphaceae. P. leucotricha starts its life-cycle early in the season, usually in March–April, while most powdery mildews are active in the same environments only late in the year.