Genetic variability among isolates of the ectomycorrhizal fungus genus Pisolithus collected in Brazil, USA and France was examined, based on nucleotide sequence of ITS from rDNA genes and restriction polymorphism of the mitochondrial DNA (mtDNA). Alignment of the ITS sequences showed 97.0% homology among isolates from Brazil, 97.8% homology among isolates from USA and France and 75.7–78.5% among the isolates from Brazil and USA/France. These sequences were aligned with ITS sequences of Australian, Brazilian and Kenyan Pisolithus isolates described in the literature. A consensus tree generated by sequence data of all Pisolithus isolates showed the presence of a number of species among the isolates. The mtDNA analysis was carried out by total DNA restriction analysis with different enzymes (Dra I, EcoR I, EcoR V, Hha I–Xba I, Hind III and Rsa I) and probing with purified mtDNA labelled with fluorescein d-UTP. The size of the mitochondrial genome of isolates Pt 90A, Pt 185R and PF cleaved with EcoR I was estimated to be, approx. 40.5, 47.2 and 47.6 kb, respectively. Cluster analysis based on the mtDNA restriction fragments grouped the isolates into two distinct groups, which coincide with their host specificity and geographical origins. PCR-RFLP analysis of mitochondrial rDNA (mt rDNA) from 12 Pisolithus isolates did not show any polymorphism. These data corroborate previous results obtained in our laboratory with RAPD-PCR and rDNA PCR-RFLP markers and support recent taxonomic studies where a number of Pisolithus species were described.

A polyclonal antibody was raised against microsomes prepared from Cunninghamella elegans and used to screen a C. elegans cDNA library. A cDNA clone from this screen was obtained, which corresponds to the C. elegans enolase gene. A single open reading frame (ORF) of 1311 bases was found, which encodes a protein of mol. wt 46882.9. The gene was found to start with an unusual initiation codon TCG, not a standard ATG codon. This was demonstrated first by ORF analysis of the determined sequence and was further confirmed by alignment of the protein with other published enolase sequences. The enolase gene was sub-cloned and over-expressed in Escherichia coli using the expression vector pQE30. Purified recombinant protein from this sub-clone showed the predicted molecular size in SDS–PAGE gel separations. Enolase enzyme activity was also detected that indicating the TCG initiation codon was correct. The C. elegans enolase was found to cluster with other fungal enolases in a phylogenetic analysis. Bacterial enolases and those of animals were separated in different clusters.

The nucleotide sequence of the internal transcribed spacer region of the ribosomal RNA gene (rDNA-ITS) was obtained for strains belonging to 10 special forms (f. spp.) of the phytopathogenic fungus Fusarium solani (teleomorph Haematonectria haematococca). Phylogenetic analysis of nucleotide sequences of the rDNA-ITS region showed that most clusters were composed of strains belonging to the same special form. F. solani f. sp. eumartii and f. sp. phaseoli were excluded from the cluster composed of all other special forms. In order to use the molecular data to identify each special form, several restriction enzymes specific for each special form were selected from the mapping data. The digestion pattern of the rDNA-ITS region with these restriction enzymes was able to distinguish F. solani f. spp. phaseoli, cucurbitae race 1, batatas, piperis, eumartii, and xanthoxyli. Although f. spp. mori, robiniae and pisi were not differentiated by restriction analysis, they were separated in the phylogenetic tree.

Filamentous fungi and yeasts control cytoplasmic osmotic pressure through ion accumulation and synthesis of compatible osmolytes including polyhydric alcohols (polyols), proline, and trehalose. Authoritative data on the osmotic effects of these compounds were obtained using vapour pressure deficit osmometry. All osmolytes tested were characterised by nonlinear relationships between concentration and osmotic pressure. At high concentrations larger polyols generated higher osmotic pressures than smaller ones, though differences between the osmotic effects of polyols with three, four, five and six carbon atoms were not pronounced at lower (physiological) concentrations. Proline shared a similar relationship between concentration and osmotic pressure with polyols with five carbon atoms, while at concentrations above 0.5 M trehalose generated higher osmotic pressures than any of the polyols tested. Mixtures of trehalose and glycerol boosted osmotic pressure in a synergistic rather than additive fashion. These data provide new clues to the adaptive significance of glycerol accumulation, and also suggest that complex patterns of osmolyte synthesis are not due to differences between the osmotic effects of these compounds.

Crinipellis perniciosa is the cause of witches' broom disease of cocoa, a serious problem in South America. The aim of the project was to develop a measure of fungal biomass in cocoa tissue infected with C. perniciosa using chitin as a marker. Axenic cultures of primary and secondary mycelium of C. perniciosa were exposed to strong hydrochloric acid to hydrolyse the chitin. The glucosamine so formed was converted to hexamitol by sodium borohydride and the acetylated and derivatized products were separated by gas chromatography and identified by mass spectrophotometry. As a proportion of the mycelial D.W., chitin was found to be 14% in primary phase mycelium and 17% in the secondary phase. An average figure was used to calculate the amount of fungal biomass from estimates of chitin in dried and fresh cocoa broom tissue infected with C. perniciosa. The green brooms, which represented an early stage of infection, contained 81 μg fungal biomass mg−1D.W., whereas brown brooms from a late stage in infection contained 161 μg fungal biomass mg−1D.W. of broom. In a detailed analysis of a single fresh broom most fungal biomass was found at the base (215 μg mg−1D.W.) but declined towards the shoot tip (10 μg mg−1D.W.). The assay was sufficiently sensitive to measure a minimum fungal biomass in cocoa of 40 ng mg−1D.W. of plant tissue. The method developed was specific to chitin, and significantly more sensitive than previously described methods.

To understand the evolutionary history of Erysiphaceae tribe Cystotheceae, phylogentic trees were constructed from the nucleotide sequences of the ribosomal DNA internal transcribed spacer of 28 taxa of the fungal ingroup and two outgroup taxa. The first split of the ingroup taxa occurred between a clade composed of the genus Cystotheca and a clade composed of the genera Podosphaera and Sphaerotheca. Podosphaera and Sphaerotheca did not separate into clades. Instead, Podosphaera species parasitic to Prunus and Sphaerotheca section Magnicellulatae grouped together, with the remaining Podosphaera species and Sphaerotheca section Sphaerotheca forming another subclade. Since the first splits were shared by Podosphaera species in both subclades, it was assumed the ancestral features of both subclades were Podosphaera-like and the genus Sphaerotheca derived from a Podosphaera-like ancestral taxon on at least two independent occasions. The results of this study suggest that ancestral fungi of the tribe Cystotheceae were originally arbor-parasitic, and transition from arbor-parasitism to herb-parasitism may have occurred at least twice independently, accompanied by a morphological change of appendages. The mycelioid appendage of the genus Sphaerotheca and other herb-parasitic genera seems to have evolved convergently at multiple times as an adaptation to herb-parasitism.

The genetic variation of Armillaria mellea sensu stricto was studied within and among non-heterothallic populations from Japan and Africa, and heterothallic populations from Europe and North America, using somatic incompatibility tests and RAPD analysis. Non-heterothallic isolates from Japan and Africa were divided into four somatic compatibility (SI) groups (A, B, C and D). SI group A contained half of all Japanese isolates and all African isolates. SI groups B and C each contained four Japanese isolates and D contained one isolate. All European and North American isolates were clearly separated from each other by somatic incompatibility tests. From the RAPD analysis, three distinct groups were separated (non-heterothallic, European and North American groups). The non-heterothallic group was divided into three subgroups (Ia, Ib and Ic). Subgroup Ia and Ib corresponded to SI groups A and B, however, Ic contained SI groups C and D. Subgroup Ia, in which 12 of 14 isolates had the same haplotype, showed a little variation. Non-heterothallic populations were less variable than heterothallic populations. The distribution of the isolates belonging to each SI group overlapped within Japan.

Of three hundred and sixty Trichoderma strains investigated, fourteen, identified as T. aureoviride, T. harzianum and T. viride, grew well at 5 °C on both minimal and yeast extract agar media. In dual culture tests at 10°, most strains antagonized the phytopathogens Rhizoctonia solani and Fusarium oxysporum f. sp. dianthi. T. aureoviride and T. viride were more effective against Pythium debaryanum than T. harzianum. The activities of extracellular chitinases (EC 3.2.1.30), β-glucosidases (EC 3.2.1.21) and proteases (EC 3.4.21.1; EC 3.4.21.4), which are thought to be involved in the mycoparasitic process, were also examined and results showed that these enzymes were highly active at 5° in the cold-tolerant strains.

Sequences of the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA of ectomycorrhizal fungi were analysed and a specific PCR primer pair for Tricholoma matsutake was designed. Using the primer pair, part of the ITS region of T. matsutake (400 bp) was amplified, but no amplified fragment was detected for other ectomycorrhizal fungi. PCR was performed on DNA extracted from mycorrhizas of T. matsutake and Shiro soil (extramatrical mycelium of T. matsutake and soil complexes) and the 400 bp fragments were also specifically amplified. This indicates that presence of T. matsutake at any of its life stages can be easily confirmed by PCR typing.

A molecular study of the mycorrhizal symbiosis between barley and Glomus intraradices used differential display PCR and a synchronous colonization method to identify genes that are differentially expressed in symbiosis. Several PCR products were consistently differentially amplified. PCR amplification of genomic DNA from either G. intraradices or barley as templates showed that three such products were encoded by G. intraradices. Sequence analysis of the deduced amino acid sequences of the fungal fragments, following extension by 3′-RACE, revealed similarities to proteins from higher eukaryotes. One (GINMYC1) shows similarity to TRIP15, a human protein that interacts in a hormone-dependent manner with the thyroid receptor. A second (GINMYC2) is similar to O-linked N-acetylglucosamine transferases from vertebrates, and the third (GINHB1) contains a putative leucine zipper and a homeodomain which indicates that it binds DNA and may act as a transcriptional regulator. Fragments of the expected sizes were amplified by RT–PCR from mRNA of mycorrhizal barley roots for all three fungal cDNAs, which indicates that the corresponding genes are expressed during intraradical growth of G. intraradices. The results provide a promising insight to fungal gene expression early in formation of this compatible and mutualistic symbiosis.

The β-1,3-glucanase system of the mycoparasitic T. harzianum, isolate T-Y, was found to be composed of at least five different enzymes. Their migration distance in acrylamide gels corresponded to peptides with molecular masses of 30–200 kDa, and they were named accordingly. The largest enzyme – Gβ-1,3–200, was the most abundant when T-Y was grown with no carbon source. Its secretion was almost eliminated when T-Y was grown on media containing a high concentration of carbon sources such as N- acetylglucosamine (GlcNAc) or malic acid. Several isolates of T. harzianum were found to have a β-1,3-glucanase secretion system, controlled by catabolite repression. Each isolate exhibited a different β-1,3-glucanase profile. Gβ-1,3–200 was isolated and purified: its molecular mass was approximately 75 kDa, and its activity was of the exo-type, specific to β-1,3-glucan linkages. Four short peptides resulting from proteolysis of this enzyme were sequenced, and their sequences were most homologous to LAM1.3, a previously isolated β-1,3-glucanase.

Ribosomal DNA sequences were assessed for their usefulness in distinguishing among Trichoderma isolates and for their robustness in resolving their phylogenetic relationships. DNA sequences from the D2 region of the 28S rRNA gene were determined for 50 Trichoderma isolates representing seven species. Eight distinct sequence types existed, and were mostly consistent with groupings based on morphology. Sequence variability within the D2 region alone was not sufficient to provide a reliable phylogeny. Sequences from the ITS1, 5.8S and ITS2 regions were subsequently determined for 18 of the isolates. Eight distinct ITS sequence types were detected among these 18 isolates. The ITS sequence types were generally consistent with morphology, ITS1 sequence data supported the identification of the Th3 T. harzianum group of Muthumeenakshi et al. (1994) as T. atroviride. The data also confirmed that the biocontrol strains of this study were different from those causing disease problems in the mushroom industry in Europe and North America. Results from the phylogenetic analysis stress the importance of testing the robustness of data used to predict phylogenies. Two ITS sequence data sets for the same group of isolates produced significantly different phylogenies. Congruence analysis detected that T. inhamatum (GJS90-90) was corrupting tree topologies and ‘first order pruning’ was performed by removing its sequence from the two ITS data sets. Subsequent differences in the topologies of pruned ITS1 and ITS2 trees were attributed to a lack of phylogenetic information in the ITS2 sequence region. Although ITS sequences successfully differentiated among morphologically distinct isolates within Trichoderma, it did not provide a sufficient phylogenetic signal to resolve all of their relationships.

Segregation and recombination in sexual progeny of Phaeosphaeria spp. were analysed using three PCR based techniques which targeted arbitrary DNA sequence (random amplified polymorphic DNA, RAPD–PCR), microsatellites (microsatellite primed PCR, MP–PCR) and dispersed repetitive DNA elements (rep-PCR). Polymorphic DNA markers were not detected in three sets of P. avenae f. sp. triticea ascospores isolated sequentially from single asci. It is hypothesised that ascospores of P. avenae f. sp. triticea were produced from a sexual process in which fusion of genetically identical nuclei took place. On the other hand, polymorphic DNA fragments were found in three sets of eight P. nodorum isolates recovered from ascospores sequentially liberated from single asci produced by in vitro mated ‘+’ and ‘−’ strains. Among 172 polymorphic markers identified in two sets of isolates (ascospores) of the fungus, 171 segregated in a ratio 1[ratio ]1. We have demonstrated for the first time that the inheritance of PCR based markers in P. nodorum progeny follows Mendelian genetics. Thereby, a molecular proof for bipolar heterothallism of the fungus was provided. In contrast, of 48 polymorphic markers detected in the third cross, only 26 segregated in an expected Mendelian frequency 4[ratio ]4 and the other 22 demonstrated either aberrant segregation pattern or possessed new DNA fragments amplified in PCR which were not detected previously in parental DNA profiles. The reasons underlying the high frequency PMS type markers identified almost exclusively in one cross have yet to be conclusively determined. If such recombinants were found in the progeny of in vitro mated P. nodorum isolates it is obvious that they also occur under natural conditions. Thus, the study provides a molecular evidence for generation of novel genotypes of the pathogen.

To elucidate relationships among Alternaria, Ulocladium, and Stemphylium species, nuclear internal transcribed spacer (ITS) and mitochondrial small subunit (SSU) ribosomal DNA (rDNA) sequences from 18 Alternaria, four Ulocladium and four Stemphylium spp. were determined and compared. Phylogenetic analysis of the ITS and SSU rDNA sequences, performed by the neighbour joining and maximum parsimony methods, revealed that the Stemphylium spp. were phylogenetically distinct from the Alternaria and Ulocladium spp. Most Alternaria spp. and the Ulocladium spp. were placed together in a large Alternaria/Ulocladium clade. Within this large clade, the Alternaria spp. clustered into several distinct species-clades, most of which correlated with species-groups previously established based upon morphological characteristics. The Ulocladium spp. were placed into two species-clades, each of which also included Alternaria spp. A. longissima was distantly related to the other Alternaria spp., as well as the Ulocladium and Stemphylium spp. Based upon ITS and nuclear 18S rDNA sequence identities, A. longissima was most closely related to Leptosphaeria.

The effects of varying carbon, nitrogen, phosphate and sulphate source on solubilization of natural gypsum by Aspergillus niger were assessed. The fungus was grown on Czapek Dox agar amended with 0.5% (w/v) gypsum. Solubilization activity was monitored by measuring the clear zone formed underneath and around the growing colonies. On different concentrations of glucose, nitrate, ammonium, urea, phosphate and sulphate, linear growth rate [Rg] was not significantly correlated with gypsum solubilization rate [Rs], but the solubilization ratio [Rs/Rg] was increased by increasing the concentration of glucose, nitrate, and urea. On ammonium nitrogen, the mycelial dry weight was negatively correlated with linear growth, and gypsum solubilization activity was markedly lower than that on nitrate or urea. Gypsum solubilization was strongly correlated with both biomass dry weight and oxalic acid production. The fungus was unable to grow on carbonate, as a carbon source, but the addition of sucrose alleviated the effect of alkalinity caused by sodium carbonate. The optimum C[ratio ]N ratio for gypsum solubilization was 50[ratio ]1, and the importance of phosphate, rather than sulphate, was also shown. Implication of critic acid in the solubilization process was also suggested. This work emphasizes the importance of organic acids, particularly oxalic acid, production by fungi in gypsum solubilization and revealed the optimum nutritional conditions for the solubilization process.

Apple fruits and grapevine leaves were inoculated with four wild-type strains of Botrytis cinerea. Two types of symptoms were observed on apple, firm or soft rot. Extracts from infected tissues were analysed for polygalacturonase (PG) and pectin methylesterase (PME) isozyme profiles. They showed that different specific PG isozymes occur in grapevine leaves and in soft rot on apple fruit. PG and PME profiles were also studied in genetic transformants altered in their colonization ability and/or the type of rot that they caused on apple fruit. No PG activity was found in apples infected by firm-rotting wild-type strains or transformants, whereas two neutral PG isozymes (pIs 7.3 and 7.6) were expressed in soft-rotting strains. No association was found between the production of these PG isozymes and the colonization level. The PME patterns were not correlated with the rotting type nor the colonization level. In vitro, firm-rotting strains and transformants showed no PG activity in glucose medium whereas soft-rotting ones produced several PG isozymes. In polygalacturonic acid cultures, all strains and transformants produced a single, common acidic isozyme. Unlike PG, no variation in the PME isozyme patterns in vitro could be correlated with the culture substrate on apple or the rotting type. The variation in pectinase profiles between in vitro and in planta contexts and the role of PGs in B. cinerea pathogenicity on apple fruit is discussed. We suggest that neutral PG isozymes are involved in the soft-rotting type on apple fruit.

An extracellular protease designated BBP (Beauveria bassiana protease) was purified from a B. bassiana isolate and subsequently characterized. It was produced in the late stages of growth in a gelatin medium, and was purified using a combination of ion-exchange chromatography and preparative gel electrophoresis. Inhibition by phenylmethylsulphonyl fluoride and chymostatin indicates that BBP is a serine type with chymotrypsin activity. Substrate specificity indicates that the protease has elastase activity as well. The protease was stable at 25 °C and had an alkaline pH optimum (7.5–9.5). Another protease was purified from the same isolate that produced BBP, and was identified as Pr1 based on its isoelectric point, amino terminal sequence, substrate specificity and cuticle degrading ability. A comparison between the two showed that BBP had a lower isoelectric point (pI 7.5) than Pr1 (pI [ges ] 10) and was 0.5 kDa smaller. The proteases also exhibited differences in substrate specificity with Pr1 being most active against Suc-Ala-Ala-Pro-Phe-pNA, and BBP being most active against MeOSuc-Ala-Ala-Pro-Met-pNA. The two proteases had equal cuticle-degrading activity and both were expressed in cuticle media, although the timing of their expression differed.

Phylogenetic relationships among Phytophthora species were investigated by sequence analysis of the internal transcribed spacer region I of the ribosomal DNA repeat unit. The extensive collection of isolates included taxa from all six morphological groups recognized by Waterhouse (1963) including molecular groups previously identified using isozymes and mtDNA restriction fragment length polymorphisms. Similar to previous studies, the inferred relationships indicated that molecular groups of P. cryptogea/drechsleri-like and P. megasperma-like taxa are polyphyletic. Morphological groups V and VI, which are differentiated by the presence of amphigynous or paragynous antheridia, are not monophyletic: species of the two groups are interspersed in the tree. Species with papillate and semi-papillate sporangia (groups I–IV) clustered together and this cluster was distinct from those of species with non-papillate sporangia. There was no congruence between the mode of antheridial attachment, sporangial caducity, or homo- or heterothallic habit and the molecular grouping of the species. Our study provides evidence that the antheridial position together with homo- or heterothallic habit does not reflect phylogenetic relationships within Phytophthora. Consequently, confirming studies done previously (Cooke & Duncan 1997), this study provides evidence that the morphological characters used in Phytophthora taxonomy are of limited value for deducing phylogenetic relationships, because they exhibit convergent evolution.

The geographical distribution and ecological habitats of the Pleurotus eryngii complex in Israel are described from a study of 60 genotypes from 10 populations. Both MEA and PDA media were suitable for growth of all genotypes at all temperatures tested (4–37 °C). There was a high correlation between a coefficient of growth and the mean colony diameter growth rate. All genotypes reached the maximum growth at 27°. Growth rate at 30° was greater than at 19°. We found highly significant (P < 0.0001) variability among growth rates of Israeli genotypes, indicating ecogeographical differences among populations. The growth rate of 12 European genotypes from the Ukraine and Slovakia was higher than most of the Israeli genotypes when grown at 27° (non-stress conditions), but Israeli genotypes tolerated 37° (stress conditions) better, and recovered faster than European populations when returned to 27°. We conclude that Israeli genotypes are better adapted to hot and dry climates than European genotypes. Based on multiple regression analyses, strong correlations were found between relative humidity and rainfall parameters, but not temperature, of the locations from which Israeli isolates were collected, and their growth rates. This indicates that, paradoxically, relative humidity and rainfall have a stronger effect than temperature on the adaptability of this complex to different environments.

Isolates from potato in an isolated garden in North Wales in 1995 consisted of essentially two distinct phenotypes. Phenotypes P1 and P2, of A1 and A2 mating type respectively, had distinctive multi-locus (RG57) and telomeric RFLP fingerprints. In addition, about 6% of isolates forming abundant oospores were self-fertile and showed a combination of both fingerprints. Among a total of 40 single-hyphal-tip and single-sporangial propagations from two self-fertile isolates, 16 were P2 whereas only one propagation was P1; four propagations were still self-fertile and still carried the molecular markers typical of both P1 and P2. Most of the remainder were A1 but otherwise carried both P1 and P2 molecular markers. Uninucleate zoospore lines from two of these were also of A1 mating type but had segregated molecular markers typical of P2; a zoospore line from a third segregated both A2 mating type and markers typical of P2. The original self-fertile isolates appeared to have heterokaryotic hyphae and sporangia with P1 and P2 nuclei and also nuclei of A1 mating type but P2 markers. Tetraploid nuclei within a heterokaryon, resulting from fusion of P1 with P2 nuclei, could have segregated to yield the latter.