Several independent molecular phylogenetic analyses have indicated that the genus Neolecta has a unique position within the Ascomycota. It is the only taxon outside the core-group of filamentous, ascoma-forming ascomycetes that also has the ability to form ascomata. Light and electron microscope studies indicate that hymenial structure and development in Neolecta spp. are unique. Ascogenous hyphae in N. vitellina branched repeatedly and successively to produce asci. Non-ascogenous hyphae were multinucleate, often with nuclei in pairs. Nuclear pairing was particularily prominent in the ascogenous hyphae. A basal septum delimited the dikaryotic ascus. Ascosporogenesis was initiated by nuclear fusion followed by a meiotic and mitotic division to form eight nuclei. The ascus apex was thin with an annular subapical thickening. Ascospores were forcibly released through a ‘split’ in the ascus apex. Woronin bodies were frequently associated with hyphal septa. Attempts to culture N. vitellina and to obtain molecular information from the type species, N. flavovirescens, were unsuccessful. However, N. flavovirescens showed several microscopic characters that indicated close relationships with the two other species in the genus, N. vitellina and N. irregularis. The position of Neolecta spp. within the Ascomycota is discussed.

Individual tall fescue (Festuca arundinacea) plants infected with both Neotyphodium coenophialum and N. lolii endophytes, and individual perennial ryegrass (Lolium perenne) plants infected with both N. lolii and Neotyphodium LpTG-2, were obtained following inoculation of naturally infected seedlings with the second endophyte. Differences in the ability of the endophytes to produce conidia, together with colony characteristics, enabled the endophytes in plants to be identified following incubation of excised leaf tissue on potato dextrose agar. Most tillers of dually infected plants were infected with just a single endophyte, but tillers infected with two endophytes were identified in three tall fescue plants. In these tillers one endophyte was always present at a much higher concentration than the other. Over time the incidence of dually infected tillers decreased and no tillers with both N. coenophialum and N. lolii were present 5 months after the associations were established. No evidence was obtained of exchange of nuclei between different endophytes present in dually infected tillers giving rise to heterokaryons or interspecific hybrids.

Most species of Ceratocystis sensu stricto are virulent pathogens of a wide variety of plants including forest and fruit trees, sweet potato, pineapple and sugar cane. Confusion exists regarding the taxonomy of the species in this genus. The aim of this study was to develop a rapid and reliable PCR-based RFLP identification method and to consider phylogenetic relationships among the better-known species of Ceratocystis. A 1·6 kb fragment within the ribosomal DNA operon was directly amplified from living fungal tissue, without extracting DNA. The amplified fragment included part of the small (SSU) and large (LSU) sub-unit rRNA genes, the 5·8S rRNA gene and the internal transcribed spacers (ITS) 1 and 2. The PCR fragments were digested with eighteen restriction enzymes. Four of these (Alu I, Dra I, Hae III and Rsa I) produced RFLPs that separated the species of Ceratocystis. The amplification products from the best-known species were sequenced, and the delimitation of taxa based on this phylogenetic analysis generally agreed with results of previous studies using isozymes and rDNA sequence analysis. This study provides an extended understanding of the relationships among species of Ceratocystis and will form a sound foundation for further taxonomic studies of the group.

That the symbiotic fungus of leaf-cutting ants only occasionally produces the sexual phase makes their identification confusing. This has occurred so rarely, either in laboratory nests, or in unbalanced field nests, that the possibility of contamination of the fungal garden by other fungi cannot be disregarded. In this paper we describe the formation of several basidiomata in a healthy and free-living nest of the leaf-cutting ant Acromyrmex hispidus fallax, the cultivation in vitro of the sterile mycelia (isolated from the fungal garden) with their typical inflated tips, and the similarity of both forms confirmed by RAPD analysis of their genomic DNA. The fungus was identified as Leucoagaricus gongylophorus.

In order to investigate the barotolerance of marine fungi and to elucidate their ecological role in the deep sea, high-pressure equipment was built and tested. Cultures of the marine yeasts Debaryomyces hansenii, Rhodotorula rubra and Rhodosporidium sphaerocarpum were inoculated into gas-permeable plastic foil bags and incubated in pressure vessels filled with hydraulic fluid that serves also as an oxygen reservoir. The equipment was used at temperatures from 7° to 34°C and pressures from 0·1 to 80·0 MPa. Comparison of five types of plastic foil showed effects on total cell number in batch culture. An exchange of the hydraulic fluid increased yield by replenishing oxygen, the growth-limiting factor. There were no detectable growth differences between buffered (TRIS, HEPES, imidazole, MES) and unbuffered media and thus unbuffered medium was used. Yeasts were best cultivated in an unbuffered sea-water medium (glucose–peptone–yeast extract) sealed in bags made from polyethylene foil. The fluorocarbon liquid FC-77 provided the best oxygen reservoir owing to its high oxygen and carbon dioxide solubility. An exchange of the hydraulic fluid is not necessary if sensitive growth determination methods are used. All marine yeasts cultivated under simulated deep sea conditions were able to grow at least up to a pressure of 20 MPa, with Rhodotorula rubra and Rhodosporidium sphaerocarpum growing at 40 MPa, corresponding to 4000 m depth. Thus, marine yeasts are able to grow under simulated deep sea conditions and may participate in the degradation of organic matter in the deep sea.

Genetic variation in Alternaria brassicae, A. brassicicola, and A. raphani collected from geographically diverse regions of the world was studied with RAPD and RFLP markers. Twenty 10-mer primers of arbitrary nucleotide sequences were tested for amplification of genomic DNA of A. brassicae using PCR. Of these, five primers amplified the genomic DNA from 20 A. brassicae isolates and produced reproducible RAPD profiles. UPGMA analysis of RAPD data showed that isolates collected from geographically distinct regions could be broadly classified into four groups. Intra-regional variation between isolates was less apparent. Variation was, however, higher in A. brassicicola, as based on RAPD analysis. Two isolates (from Canada and France) of A. raphani also showed variability with different RAPD profiles generated by all five primers tested. Five polymorphic, distinct RAPD products were used as hybridization probes for RFLP analysis to detect inter- and intra-specific variation. Variation among A. brassicae, A. brassicicola, and A. raphani was evident. Non-radioactive probes were also used to hybridize with Southern blots of A. brassicae, A. brassicicola, Leptosphaeria maculans, Rhynchosporium secalis and Brassica juncea for the selection of A. brassicae-specific probe(s). Probe AbP3 specifically hybridized with restriction digests of A. brassicae but not with those of A. brassicicola or other tested species. This probe should, therefore, be useful for distinguishing between two important pathogens of crucifers, i.e. A. brassicae and A. brassicicola both in culture and infected tissues.

Strains of Ophiostoma ulmi, O. novo-ulmi subsp. americana, O. novo-ulmi subsp. novo-ulmi and O. himal-ulmi were examined for optional introns/insertions within the following mitochondrial genes: small subunit RNA gene (rns), large ribosomal subunit gene (rnl) and the cytochrome oxidase subunit I gene (coxI). Insertions were noted in the rns and coxI genes in strains of O. ulmi, the less aggressive species, but absent in strains of the more aggressive O. novo-ulmi subsp. americana. Strains of all species examined had a group I intron present in the U11 region of the mitochondrial-rnl gene. In all but two strains of O. novo-ulmi subsp. americana, this rnl-U11 intron was about 1.5 kb in length whereas a 2.6 kb version of this element was present in all strains representing O. ulmi, O. novo-ulmi subsp. novo-ulmi, and Ophiostoma himal-ulmi. Irrespective of size, this intron based on RNA folds is a class IA1 group I intron and it encodes a putative ORF for the rps3 ribosomal protein. The size variation of the rnl-U11 intron was examined in detail for two strains of O. novo-ulmi subsp. americana and sequence data suggests the presence of a complex ORF within the 2.6 kb version of this intron; here a homing endonuclease-like gene has been inserted in frame and fused to the carboxyl-terminus of the putative rps3 coding region. The mitochondrial optional introns/insertions in combination with nuclear markers might be useful in distinguishing among the various species and subspecies of the O. ulmi s. lat. complex.

Entomophaga maimaiga is a naturally occurring fungal pathogen specific to larvae of the gypsy moth, Lymantria dispar. E. maimaiga is thought to be native to Asia where its epizootics can suppress gypsy moth outbreaks. However, in the USA this beneficial fungal pathogen was not observed until 1989, although an isolate of E. maimaiga from Tokyo was released in Massachusetts to control gypsy moths as early as in 1910–1911, and another isolate from Ishikawa Prefecture in Japan was later released in 1985 and 1986 in New York and Virginia. Our objectives were to: (1) test the hypothesis that E. maimaiga populations in the USA have reduced genetic variability due to founder effects compared to the putative ancestral populations in Asia; (2) track the origin of the North American populations of this fungus; and (3) assess whether genetic differences among E. maimaiga isolates are correlated to morphological differences. We compared genetic diversity among 30 E. maimaiga isolates originating from seven states in the USA, five prefectures in Japan, one province of China and one region of far eastern Russia by AFLPs. Among 14 USA isolates, only ten polymorphic AFLP loci were found, whereas 56 polymorphic loci were found among 16 Asian isolates; 29 loci were polymorphic among 12 isolates from Japan alone. Average gene diversity (h¯) for the polymorphic loci was 0.223±0.005 for Asia (including Japan), 0.131±0.006 for Japan only, and 0.041±0.004 for the USA. Thus, native populations from Asia were more diverse than the USA populations. These results are consistent with the expectation of a population founded from a source population by a small number of individuals. Distance and parsimony analyses of AFLP data showed that the isolates from the USA formed one distinct clade that was most closely related to Japanese isolates collected outside the Tokyo area. No morphological variation of E. maimaiga from different geographical locations was detected.

The entomopathogenic hyphomycete Paecilomyces fumosoroseus is a promising candidate for biocontrol of economically important agricultural pests. Assessment of genetic relatedness of this species appears to be essential to gain insight into the monitoring of such biocontrol products. Intraspecific variation within the internal transcribed spacer region of the ribosomal RNA gene (rDNA-ITS) was studied in 48 isolates of P. fumosoroseus. These strains were isolated from different geographical and biological origins, more particularly from the silverleaf whitefly, Bemisia tabaci-argentifolii (Homoptera: Aleyrodidae), a major insect pest in field and greenhouse crops. PCR amplification of the ITS1-5.8S-ITS2 rDNA region followed by restriction analysis of the PCR products underlined the overall highly variable nature of this region. Digestion with the six endonucleases AluI, HaeIII, Hin6I, HpaII, NdeII, and SmaI allowed the separation of the isolates into three distinct groups. The group 1, representing 25 isolates, is composed of strains isolated only from the host B. tabaci-argentifolii. By contrast, the group 3 is more diffuse as it included strains from various insect host and geographical origins. Phylogenetic analysis of rDNA-ITS sequence data strongly supported the conclusions of the PCR-RFLP analysis and recognized three monophyletic groups within the P. fumosoroseus complex. The high level of polymorphism within the P. fumosoroseus species is discussed in the light of their biological origin.

Phytophthora quercina, a new species associated with oak decline in Europe, has been assigned to Waterhouse's Group I of Phytophthora. The level of intraspecific variation and evidence of affinities to other Group I species and another, as yet unidentified, species from oak, were examined at the molecular level using four random ten-mer primers to amplify total DNA (RAPDs). Sequences and restriction fragment length polymorphisms of a 900 bp PCR product consisting of the ITS1 and ITS2 regions, and the 5.8S subunit of ribosomal DNA were also examined to estimate relatedness to a broader range of Phytophthora species. The RAPD banding patterns of ten isolates of P. quercina from eight sites in Germany, Hungary and Italy were almost identical and distinct from all the other species tested. Their ITS restriction fragment patterns were also identical, as were the ITS sequences of four selected isolates. Phylogenetic analysis of the ITS sequence data confirmed its unique position in this section of the genus which comprises P. quercina, another five Group I species, P. infestans (Group IV) and P. nicotianae (Group II). Isolates of P. quercina formed a distinct branch at the base of this clade showing no close affinities with any other species. Such data support morphological, physiological and pathological evidence that P. quercina is distinct, although it has some affinity with the other Group I species. The results support earlier reports that Waterhouse's groupings of morphospecies do not fully correspond to phylogenetic relationships indicated by molecular studies. The unidentified Phytophthora sp. 2 from oak was closely related to P. ilicis (Group IV); both were distinct on molecular criteria from all other species in the study.

From the alkali-extractable material obtained from cell walls of 44 species (49 strains) of Penicillium a water-soluble fraction (F1S) was isolated. The main component (F1S-S) of fraction F1S was purified by gel permeation chromatography through Sepharose CL6B, analysed chemically and its structure determined by 1H- and 13C-NMR. Galactose was the predominant neutral sugar in this fraction. Six different polysaccharidic structures were found and the species investigated were arranged into six groups according to these structures. These results illustrate the complexity of the cell wall components of Penicillium and add to existing information on the relatedness of species of the genus with Eupenicillium and Talaromyces. The results show that there are several types of cell wall in Penicillium.

Twenty-one Aspergillus strains representing different A. awamori, A. phoenicis and A. foetidus isolates were studied in order to explore the potential of a fast RFLP analysis to identify fungal strains. The patterns were compared with those characteristic for A. niger, A. tubingensis, A. carbonarius, A. japonicus and A. aculeatus represented by A. niger CBS 120.49, A. tubingensis NW 756, A. carbonarius CBS 111.26, A japonicus CBS 114.51 and A. aculeatus CBS 101.43 and also with those of the type strains of A. heteromorphus CBS 117.55 and A. ellipticus CBS 707.79.

Sma I digested chromosomal DNA revealed characteristic rDNA patterns after ethidium bromide staining which were used in combination with hybridization patterns of Pst I/Sal I double digested chromosomal DNA with well-defined probes. This allowed clear distinction of eight separate species within the Aspergillus sect. Nigri group. The probes used were a 0·9 kb fragment of the 28S rDNA from Agaricus bisporus, an internal fragment of the pkiA gene from A. nidulans, and the pelA gene from A. niger. All the strains examined including those indicated as A. awamori and A. phoenicis were shown to belong either to A. niger, A. tubingensis or a group representing isolates of A. foetidus varieties, amongst which A. foetidus var. acidus CBS 564.65 and A. foetidus var. pallidus CBS 565.65.

The production of fumonisins, fusaproliferin and beauvericin by Gibberella fujikuroi different mating populations isolated from maize in Argentina was evaluated. From 203 strains of Fusarium verticillioides (G. fujikuroi mating population A), 193 were fumonisin producers. Among members of mating population A, female fertile strains produced 20% more toxin than female sterile ones. Among 78 Fusarium proliferatum strains (G. fujikuroi mating population D) 65 produced fumonisins. The percentage of strains that were high, intermediate and low level toxin producers varied according to the species evaluated and the area from which the strains were isolated. Fusarium subglutinans (G. fujikuroi mating population E) strains produced low levels or were no fumonisin producers. Strains from both G. fujikuroi mating populations D and E were able to produce fusaproliferin and beauvericin. Among the members of F. subglutinans (G. fujikuroi mating population E) the fusaproliferin production was more constant. Co-production of fumonisin, fusaproliferin and beauvericin among the strains belonging to G. fujikuroi D and E was also observed. The co-production of fumonisin, beauvericin and fusaproliferin in maize need to be considered, since from the toxicological point of view interactions between these toxins could occur. The toxigenic ability of the strains evaluated prompt us that is necessary to determine the natural occurrence of fusaproliferin and beauvericin in Argentinean maize.

Previous studies of morphological variation in the homobasidiomycete Hyphoderma setigerum have lead to suspicions of a species complex. This study explores variation in DNA sequences from the nuclear ribosomal ITS region of 45 specimens from America, Asia, and Europe in a phylogeographic context. Based on molecular analysis, morphological studies, and crossing tests, nine preliminary taxa are shown to exist inside the species complex, and the two previously described segregate species H. subsetigerum and H. nudicephalum are confirmed. The molecular analysis shows evidence of allopatric differentiation over intercontinental distances. Only one of the nine well-supported clades has a geographic distribution spanning more than one continent, probably indicating the importance of vicariance in the evolution of this species complex. The basionym of H. setigerum, Thelephora setigera, is neotypified to fix the application of that name.

The antifungal activity of flavone and flavanone was tested in three different concentrations, 0·5, 2 and 8 × 10−4 M, against 34 fungi. In general, flavone was the substance with the higher activity, and could at 2 × 10−4 M completely inhibit several fungi. No linear relationships between the concentration and the antifungal activity could be observed for some fungi. Chaetomium globosum was the only fungus which was totally inhibited by flavanone at 8 × 10−4 M. Several fungi did, however, show a higher inhibition rate at 2 × 10−4 M.

The extracellular subtilisin-like protease activity of several fungal pathogens of nematodes, insects and plants was recorded after batch cultivation. Nematophagous and entomophagous isolates of Verticillium, Paecilomyces, Beauveria and Metarhizium produced Suc-(Ala)2-Pro-Phe-pNA hydrolysing enzymes that differed in serological properties based on Western blot analysis with polyclonal antibodies. The antibodies used were raised against the main subtilisin-like proteases from one species from each of the four genera. From each strain of V. chlamydosporium tested, between one and four isoforms with different pI values were separated by isoelectric focusing and visualized by enzymoblots. The occurrence of multiple isoforms with unique N-terminal sequences in single strains might suggest the presence of subtilisin gene families. Using as a probe a fragment of the Pr1 gene from M. anisopliae, RFLPs were observed that confirmed qualitative differences between subtilisin-like genes within and between species. In contrast to these pathogens of invertebrates, plant-pathogenic species of Verticillium did not produce subtilisin-like proteases, nor were homologous genes detected. Such activity, however, and the corresponding genes, were detected in species weakly pathogenic to plants, or saprotrophs.

21 strains with all typical morphological characteristics of eight Verticillium species (Phyllachorales) were studied in this work, together with representatives from four Hypocreales species (11 strains), that were previously classified as members of the genus. The PCR products from two nuclear genes, i.e. the ITS1-5.8S-ITS2 region and RNA polymerase II largest subunit gene (rpb1), together with four mitochondrial genes, i.e. the small ribosomal rRNA subunit (rns), the two NADH dehydrogenase subunit genes (nad1 and nad3), and the cytochrome oxidase subunit III gene (cox3) were sequenced and analyzed. Similarly, antibodies raised against one strain of each of the species examined (V. nubilum and V. theobromae excluded) were used against the proteins of all other strains. The number and relative area of precipitates formed after crossed electrophoreses served to estimate the degree of immunochemical relatedness. Combined molecular and immunochemical data clarified the phylogenetic relationships of all true Verticillium species and provided a convincing insight into the evolutionary relation of the sect. Nigrescentia with members of the sect. Verticillium and sect. Prostrata that until recently were included in Verticillium.

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.

Four group I introns, designated Bb1, Bb2, Bb3 and Bb4, were identified in the entomopathogenic hyphomycete Beauveria bassiana. Sequence analyses of these introns verified that they were invariably inserted at specific target sequences after conserved positions Ec2563, Ec2449, Ec2066 and Ec1921 of the large nuclear subunit (LSU) rDNA 3′-end. Secondary structure modelling confirmed that Bb1 and Bb3 belonged to subgroup IE while Bb2 and Bb4 belonged to subgroup IC1. Intron presence, distribution and size-variation were studied in a population of 125 B. bassiana strains using site-specific primers. Nucleotide sequences and secondary structures were compared and showed considerable variations usually at P1, P6 and P9 helices, but concomitantly, high homology between members of the same site-specific group. Intron distribution studies revealed that few (7.2%) strains were intron-less, most contained one (28%), two (48%) or three (16%) introns, while only one strain contained all four introns. Bb4-like introns (Ec1921) were the most abundant (86.4%), whereas the other three introns were evenly represented (ca 30%) in the B. bassiana population. Analysis of intron genotype distribution indicated a tenuous dependence upon geographic origin or insect host species. Phylogenetic analysis of all B. bassiana LSU introns and their close relatives from other entomopathogenic fungi showed a strong correlation between specific insertion sites and intron subgroups, fully supported by corresponding clades, suggesting common ancestry of the site specific LSU introns.

The susceptibility of mustard beetle, Phaedon cochleariae, larvae to the insect pathogenic fungus Metarhizium anisopliae, was influenced by the topography of the host plant, the dosage and the larval instar. At high doses (>109 conidia ml−1) more insects were likely to acquire conidia. Most of the inoculum adhered to the mid-ventral region of the abdomen. Inoculum applied to the leaf became diluted during leaf expansion and this decreased beetle mortality. Leaf expansion was slower at 10°C than at alternating temperatures of 23/10° or when constant at 23°. Slightly more larvae acquired conidia when fed on oilseed rape than Chinese cabbage or turnip. Most conidia adhered to the legs and the mid-ventral region of the abdomen. Mortality was usually less on oilseed rape than Chinese cabbage and turnip. These observations suggest that fungistatic compounds of oilseed rape were interfering with the infection process.