Competitive interactions among bark beetle associated fungi are potentially influenced by abiotic factors. Water potential, in particular, undergoes marked changes over the course of beetle colonization of tree hosts. To investigate the impact of water potential on competition among three southern pine beetle associated fungi, Ophiostoma minus, Entomocorticium sp. A and Ceratocystiopsis ranaculosus, we utilized artificial media with water potentials of 0, −5, −10, and −20 MPa. Growth of all three fungi, when grown alone, decreased on media with lower water potentials. Growth rates of all three fungi were likewise reduced in competition experiments. At −5 to −10 MPa, C. ranaculosus (a fungus with beneficial effects toward southern pine beetle) was nearly equal in competitive ability to O. minus (a fungus with antagonistic effects towards southern pine beetle). This was not true on control media, nor at other water potentials tested. The range of water potentials used in our assays was similar to the range of water potentials we measured in loblolly pines within a southern pine beetle infestation. This study indicates that water potential may alter the outcome of competitive interactions among bark beetle-associated fungi in ways that favour bark beetle success.

Visual markers detectable by histochemical staining have been developed for analysing the time course and tissue specificity of maize infections by Fusarium moniliforme. Three F. moniliforme strains, RRC 374, MRC 826 and RRC PAT, were transformed with a plasmid, pHPG, containing the gusA reporter gene which codes for β-glucuronidase (GUS) and the hph gene for hygromycin resistance as the selectable marker. Introduction of plasmid DNA into germinating conidia yielded 1·2×10−7 transformants per conidium; expression of both gusA and hph was however, transient. Stable transformants were obtained using protoplasts as the recipient, but transformation frequency was reduced. Southern blot and PCR analyses confirmed incorporation of pHPG into the genome of all three F. moniliforme strains with gusA properly inserted in MRC 826 and RRC PAT, but apparently disrupted in RRC 374. The growth pattern for transformed F. moniliforme isolates and the parental wild types followed a sigmoid curve on minimal and enriched media. Hygromycin totally inhibited growth for wild type isolates, but not of transformants. Transformed isolates maintained the ability to infect the maize plant. Thus, this study is the first report of F. moniliforme transformed with a visibly detectable reporter gene to use for analysing this endophyte-host interaction of world-wide importance to animal and human health.

The dimorphic Mucor circinelloides requires an anaerobic atmosphere and the presence of 30% CO2 to grow as a multipolar budding yeast, otherwise hyphal growth predominates. Establishing other means to control the morphology would be a distinct advantage in the development of a fermentation process for this organism for the production of heterologous proteins. Thus, conditions suppressing polarised growth while at the same time abolishing the CO2 requirement were investigated in submerged cultivations. It was found that supplementing cultures with mixtures of ergosterol and Tween 80 resulted in yeast-like growth under 100% N2. Their impact on growth and morphological development was assessed at a range of concentrations. Maximum biomass levels and the specific growth rate decreased at elevated levels of ergosterol and Tween 80. Possible effects of carbon dioxide and the added fatty acid/sterol mixture on supporting yeast growth by influencing the fluidity of the plasma membrane or affecting polarised growth are discussed.

The fungal pathogen Entomophaga maimaiga, infecting gypsy moth larvae (Lymantria dispar), produces two types of spores; relatively short-lived conidia are produced externally on cadavers and are actively ejected to cause infection during the same season, while azygospores, produced within cadavers, are dormant after formation and survive unfavourable conditions. This is the first record providing experimental proof of differential reproduction resulting from infections initiated by these two types of spores. Cadavers of larvae that had died from infections initiated by azygospores produced only conidia, regardless of host instar or relative humidity after host death. This is in stark contrast to infections initiated by conidia, which could yield either conidia only, azygospores only, or both spore types, with reproductive outcome primarily influenced by host age. Production of only conidia from infections initiated by azygospores increases opportunities for secondary transmission during the eight week field season; if infections initiated by azygospores produced azygospores, chances for amplification of infections with subsequent epizootic development would be decreased.

The synthesis of plant growth hormone auxin, indole-3-acetic acid (IAA), is not only confined to flowering plants. Yeasts and other fungi are also known to produce this hormone and in many cases at higher levels than plants. The main concern of this study was to determine the physical and chemical conditions necessary for optimal biosynthesis of this hormone by Lentinus sajor-caju. Glucose was determined to be superior to sucrose as carbon and energy source. The synthesis of IAA in a nitrogen-free medium or in a medium with low external phosphate was substantially reduced. Light exposed and non-agitated cultures grown in dark had also reduced levels of IAA compared to agitated cultures grown in dark. The highest (0.18 mg ml−1) IAA level was determined in cultures grown in glucose containing medium (pH 7.5) on a rotary shaker (150 rpm) at 30 °C in dark. The biological activity of IAA obtained from the extra-cellular culture of Lentinus sajor-caju was determined using oat coleptile growth test.

Spores of Tetrachaetum elegans were isolated in early autumn from submerged leaves which were randomly collected at a single site from a small stream in southwestern France. In order to investigate potential substrate preferences, spores were sampled in four leaves from seven different plant genera. A total of 96 monosporic isolates were screened for genetic variation in RAPD patterns using nine different primers. Fifty-two fragments were identified and used for calculation of similarity coefficients. A large number of fragments were shared across all isolates, suggesting that the population was genetically homogeneous. The combination of 11 polymorphic fragments contributed to the delineation of 13 RAPD types. Colonization of a single leaf by multiple genotypes was frequently encountered. Fungal genetic assemblages differed between plant genera. Variations in the frequency of occurrence of the various genotypes are discussed in terms of ecological strategies and in reference to previous work at the community level. We suggested two hypotheses: first, substrate preferences are found among aquatic hyphomycete genotypes, and secondly, sequential occupation by distinctive populations may result in different assortments of genotypes on leaf litters of different plants at any given time of sampling.

Sequences of nuclear ITS (nrDNA) regions were obtained from 22 species of Tyromyces s.l., a group of important wood-rotting, poroid holobasidiomycetes, belonging to Coriolaceae. Different reference collections of a species generally produced identical molecular sequences. Parsimony analysis of these sequences resulted in trees compatible with the currently accepted delimitation of genera, but the alignment of sequences has proven to be difficult owing to regions of uncertainty. Analyses based on inclusion and exclusion of ambiguous regions in the alignment demonstrate that closely related taxes are, however, always clustered together, especially at the generic level. It is considered that ITS sequences are reliable in distinguishing genera and species of Tyromyces s.l., but not able to provide sufficient resolution for relationships between some formerly recognized genera. The taxonomic implications of ITS variation are also discussed. Species of Oligoporus and Postia clustered in one clade, indicating a close relationship as shown from morphological research. Species of Skeletocutis are characterized by encrusted hyphal ends in the hymenophore, but the type species of Tyromyces, T. chioneus, is embedded within the Skeletocutis clade, indicating a strong link between these two genera. Species of Antrodia and of Spongipellis included in this study were not well supported as monophyletic groups, although they are clustered together within the trees.

Conidia of Metarhizium flavoviride were suspended in oil formulations and received fatty and other acids before germination. This was done by the acids being added either to the conidial suspensions before inoculation onto agar plates or directly into the agar before the conidia were applied. Certain acids of chain lengths C10 and below, including lactic, salicylic, caprylic and capric acids produced significant inhibition of germination when added to the oil formulations. When incorporated into the agar, all acids of C10 and below were inhibitory, with the exception of sebacic and succinic acids. Sebacic acid (a C10 saturated dicarboyxlic acid) and all the longer chain fatty acids sampled produced no inhibition of germination, either when added to the formulation or the agar. Tests were carried out on a sample of conidia which showed no germination; a few acids stimulated some germination. These included sebacic acid and longer chain fatty acids such as stearic and (inconsistently) lauric acids. Conidia from a long term storage experiment showed increased germination on the addition of stearic and/or lauric acids. The inhibitory effect of capric acid on germination was overcome by a 4–15 times higher concentration of stearic acid.

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.

Four isolates of the entomopathogenic fungus Erynia neoaphidis, from different hosts and locations in Switzerland, were distinguished by their DNA fragment banding pattern obtained by RAPD–PCR. The isolates differed in sporulation and germination rates, but not in virulence (overall mean LD50 16 conidia mm−2) against the aphid Acyrthosiphon pisum. The sporulation rate was 6 to 24 conidia mm−2 10 min−1. On nutrient agar, 24–73% of the conidia produced secondary conidia and 32–68% produced germ-tubes on polystyrene. Differentiation of primary into secondary conidia or germ-tubes is possibly dependent on the presence of free water on the contact surface; on a dry surface, conidia germinated mostly with a germ-tube, whereas on a wet surface the conidia produced a secondary conidium. Experimental support is provided for the claim that, in addition to LD50 values, features such as sporulation rate, spore germination, capacity to form secondary conidia and intraspecific variation should be considered in selecting isolates of E. neoaphidis for biocontrol agents.

The genetic relationships among 132 isolates of Sclerotium rolfsii (teleomorph Athelia rolfsii) collected during 1967–97 from 36 different host species over a wide geographic range representing 13 countries were investigated using mycelial compatibility groupings and RAPD analysis. A smaller group of 15 Sclerotium delphinii isolates from five host species and a limited geographic distribution was also studied. The development of aversion reactions following mycelial pairings of isolates in all possible combinations on potato dextrose agar was used to differentiate 71 mycelial compatibility groups (MCG) in S. rolfsii and five MCG in S. delphinii. Many MCG were unique single-member groups and these generally were found in widely separated geographic regions or countries. There was no clear relationship between host of origin and MCG, except for a majority of isolates of S. rolfsii from turfgrass that belonged to MCG 1. Within a specific geographic region, e.g. California, there usually were several different MCG present, some of which were recovered from the same host species. In addition, specific MCG of S. rolfsii were recovered from widely separated geographic regions as well as different host species, e.g. MCG 1 was recovered from turfgrass, carrot, tobacco, and tomato in California, Georgia, North Carolina, and Mexico, respectively. The extent of genetic diversity within and among MCG of S. rolfsii and S. delphinii was studied using RAPD analysis. Isolates from different MCG could be differentiated by their unique banding patterns using six primers. There were no discernible relationships among the various MCG using UPGMA analysis. Isolates within a particular MCG were also genetically diverse, but shared greater numbers of common bands and clustered together. Only a few members of some MCG in S. rolfsii and S. delphinii that had identical RAPD patterns were considered to be clonally derived. The extent of genetic diversity among isolates of S. delphinii was lower than that observed in S. rolfsii.

Oxalate decarboxylase activity was present in liquid culture medium and mycelium of Agaricus bisporus. Enzyme activity in the mycelium peaked at two-weekly intervals after primary growth phase and into secondary metabolism, with activity peaks in the medium occurring 7 d later than in the mycelium. The enzyme was partially purified and had two isozymes with pIs 3.0 and 3.4. Characterization of the protein by SDS–PAGE and Western blotting against a polyclonal antibody raised to oxalate decarboxylase from Collybia velutipes showed a major protein band of 64 kDa. Oxalate decarboxylase was also detected in the fruit body up to the cup stage of development. The pH optimum for the enzyme was 3.6 and temperature optimum was 35 °C.

Several Trichoderma virens (syn. Gliocladium virens) strains have good biocontrol activity against Rhizoctonia solani on cotton but lack some of the commercially desirable characteristics found in other Trichoderma species. In an attempt to combine these desirable characteristics, we used a highly effective biocontrol strain of T. virens in protoplast fusions with a strain of T. koningii, which had good storage qualities, but little biocontrol efficacy. All fusants were morphologically similar to one of the parental species. However, when compared to the morphologically similar T. koningii parent, two fusants showed significantly better biocontrol activity against R. solani on cotton. In addition, one T. virens-like fusant gave significantly less control than the T. virens parent. Fusants also differed from the morphologically similar parent in the production of secondary metabolites. One fusant was obtained which maintained biocontrol activity during storage for up to a year.

Entomophthorales pathogenic to insects and mites often cause epizootics in their host populations, but some have been difficult to culture in vitro and, therefore, to develop as biopesticides. Grace's insect cell culture medium supplemented with lactalbumin hydrolysate and yeastolate has allowed growth of several species which until recently were referred to as obligate parasites. The research reported here was designed to evaluate the effects of the salts, vitamins and amino acids used to prepare the insect cell culture medium on in vitro growth of Batkoa sp. and Furia sp., pathogens of the spittlebug pests of pasture and sugar-cane in Brazil, and Neozygites floridana, a pathogen of several mite species. Also, several sources of carbon and nitrogen were examined. Batkoa sp., Furia sp. and N. floridana were similar concerning their growth patterns in a basic medium with added salts, vitamins and amino acids, as well as with a combination of all three compoments. The addition of salts to the basic medium of sugars plus lactalbumen hydrolysate and yeastolate caused a significant increase in biomass production of the three fungal species. The addition of vitamins and amino acids had less effect. Batkoa sp., Furia sp. and N. floridana are similar in growth patterns in media with various sources of carbon, but different in media with different sources of nitrogen. The production of the three fungal species is significantly higher in medium containing 2.66% glucose than in medium with 2.66% sucrose. The addition of 0.1% monossacarides to media containing 2.66% sucrose did not significantly increase biomass production.

Diplodia pinea (syn. Sphaeropsis sapinea) is a well-known latent pathogen of Pinus spp. with a worldwide distribution. As such, this fungus is native where pines are endemic in the northern hemisphere and it has been introduced into all countries of the Southern Hemisphere where pines are exotic. The newly described D. scrobiculata (formerly known as the B morphotype of D. pinea) is thought to have a much more limited distribution. D. scrobiculata was first reported as an endophyte and weak pathogen of P. banksiana, where it was found to coexist with D. pinea. Diplodia scrobiculata is now known to have a much broader distribution in Northern America and Europe. In this study, seven Simple Sequence Repeat (SSR) markers were used to evaluate genetic diversity and gene flow between populations of D. scrobiculata. Results indicate a strong geographic isolation between populations of D. scrobiculata from different regions in North America, with unique alleles fixed in the different populations. The data fits the isolation by distance model indicating limited dispersal. Geographic isolation in combination with isolation by distance suggests prolonged reproductive isolation. Intensive collections of endophytes from native P. radiata in California have yielded only D. scrobiculata and not the significantly more pathogenic D. pinea. SSR analysis of three populations of D. scrobiculata from native P. radiata identified many shared alleles among the populations and moderate to high gene flow between them. The three Californian populations are distant and distinct from populations of D. scrobiculata from elsewhere. Under stress conditions, P. radiata is known to be very susceptible to D. pinea in plantations in the Southern Hemisphere. Native P. radiata is currently experiencing severe stress due to pitch canker caused by Fusarium circinatum. Such stress would provide ideal conditions for an associated outbreak of D. pinea. Thus, it is critical to prevent the movement of D. pinea into the last remaining native stands of P. radiata.

The steroidal glycoalkaloid α-tomatine of tomato plants has been reported to protect Lycopersicon species against fungal attack. Two isolates from Fusarium solani were found to produce an extracellular enzyme inducible by α-tomatine. TLC showed that the enzyme catalyzes the hydrolysis of the glycoalkaloid into β-lycotetraose and tomatidine. The enzymatic activity was concentrated against polyethylene glycol 35000, and the enzyme was partially purified by preparative isoelectric focusing, preparative gel electrophoresis and ion-exchange chromatography. The enzyme was found to be a monomer of about 32 kDa by both SDS-PAGE and gel filtration. This molecular mass differs from that of the tomatinase of Fusarium oxysporum (50 kDa). Moreover, polyclonal antibody anti- tomatinase of F. oxysporum f. sp. lycopersici did not recognize the tomatinase from F. solani, suggesting that this tomatinase may be a novel enzyme.

A total of 479 samples of nests or feeding sites of 38 species of Australian termite were plated onto a medium selective for isolation of Metarhizium spp. Sixty-seven samples were positive for Metarhizium spp. and a total of 97 isolates of M. anisopliae were obtained. Very few isolates were obtained directly from infected termites. Most isolates were obtained from nest-mound material from eastern Australia. Termite-associated material from the two common mound-building species of termite, Nasutitermes exitiosus and Coptotermes lacteus, provided 75 of the isolates. Similar material from 26 species of termites revealed no Metarhizium. A detailed study of two sites found that some of the Metarhizium isolates found in nest-mound material, including also some M. flavoviride and M. album, were of morphological types also present in adjacent soil. The DNA of isolates from mounds and the adjacent soil were compared using RAPDs and sequence analysis of the ITS region of the nuclear rDNA and the same types were found from both sources. The possible role of the fungus in termite ecology is discussed and it is thought most likely that Metarhizium is only opportunistically a pathogen of termites. Thus, isolates obtained from termite nest material are probably there because of the incorporation of soil into termite nests.

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.

Although Plasmopara viticola causes grape downy mildew in most, if not all, wine producing countries, many basic biological or chemical aspects are still unknown and thus the histopathological changes during development of this oomycete pathogen were studied. The fluorochromes Aniline blue and Uvitex 2B were successfully used in whole leaf stainings to visualise intercellular hyphae and to investigate septal-development in sporangiophores. The occurrence and transfer of cytoplasm in sporangiophores was studied with the aid of Chlorazole Black E and Phloxin B. Application of Chlorazole Black E was the most reliable method to differentiate between cytoplasm and septa in sporangiophores due to a high contrast between the dark cytoplasm and the pale septa. In P. viticola, septa were found in the stem and branches of the sporangiophores, but not in the intercellular hyphae, which was in contrast to other oomycetes, such as P. tabacina, Pseudoperonospora cubensis and P. humuli, where septa were frequently found in the mycelium. In order to verify chemical composition of septa, sporangiophores were digested using chitinase and β-1,3-glucanase. After staining with Aniline blue and Uvitex 2B, the intense fluorescence of septa was conserved after the application of chitinase but not after β-1,3-glucanase, indicating that septa are mainly composed of β-1,3-glucans. Pretreatments of infected vine leaves with 2-deoxy-D-glucose (2-DOG) at low concentrations (1–5 mM) led to a disorganisation of the sporangiophore structure of P. viticola, whereas the production of septa was unaffected. Application of 2-DOG at higher concentrations resulted in a reduced length of intercellular hyphae (10 mM) or total inhibition (50 mM) of the intercellular mycelium. Thus, 2 DOG or its analogues interferes with morphogenesis in Plasmopara viticola and may have a deleterious effect on the spread of this downy mildew between plants.

A new pyrenomycete, Monosporascus ibericus sp. nov., isolated as an endophyte from roots and stems of three plant species growing on sand flats and salt marshes in the Ebro Delta (Spain), is described. The main characteristic of this fungus is the presence of a higher number of ascospores per ascus (up to six), compared to the other species of the genus: M. cannonballus, M. eutypoides, and M. monosporus. M. ibericus produces a cleistothecial ascomata with a tomentose peridium and lacks an anamorph. In addition to the morphological data, the comparative analysis of the ITS-region sequences of M. ibericus and the other Monosporascus spp., has supported the recognition of the new species. A phylogenetic study based on the sequences of the 18S rDNA did not allow us to assess clearly the taxonomic position of the genus Monosporascus, although the results indicated the genus might have affinities to the Xylariales rather than to the Sordariales.