Acremonium-like submerged conidiation inPaecilomyces nostocoides andP. lilacinus

Paecilomyces nostocoides, in which conidia of smaller or larger sizes appear in chains, was newly isolated from Japan. In addition to the typicalPaecilomyces-type conidiation, the Japanese isolate showed additionalAcremonium-like submerged conidiation in and/or on some agar media. The submerged conidiation was also observed in the ex-type strains, but not in the type specimens ofP. nostocoides. The same submerged conidiation was observed inP. lilacinus, the species most similar toP. nostocoides. The species definitions ofP. nostocoides andP. lilacinus were emended to include the submerged conidiation.Paecilomyces-type conidia were uninucleate in bothP. nostocoides andP. lilacinus. Paecilomyces nostocoides andP. lilacinus had the Q-10(H₂) ubiquinone system.     

Contribution of leaf surface fungi to the air spora

High concentrations of airborne fungal spores frequently occur from spring through fall in temperate areas of the world. Although it is generally assumed that fungi on leaf surfaces are contributors to the air spora, little data are available comparing the types of fungi found on leaf surfaces with those in the atmosphere. Air sampling was carried out with a Burkard Spore Trap located on the roof of a building on the University of Tulsa campus using standard methods. Leaf samples were aseptically collected from Ulmus americana and Quercus palustris trees on campus, placed in sterile plastic bags, and brought to the lab. For each leaf, 4 cm² areas of both upper and lower leaf surfaces were swabbed and plated on malt extract agar with streptomycin. Cultures were incubated at room temperature for 5–7 days and then examined microscopically. Results were expressed as colony forming units (CFU)/cm². Twenty-one fungal taxa were identified from the air samples. The most abundant taxa were Cladosporium, ascospores, basidiospores, and Alternaria; together these four spore types comprised over 90% of the yearly total. Yeasts were the most abundant fungi isolated from both leaf types. Among the mycelial fungi were Phoma species, followed by Cladosporium and Alternaria. Overall twenty genera of filamentous fungi were identified. Yeasts and Phoma are normally splash dispersed and were not identified in the Burkard air samples. However, 10 taxa isolated from leaf surfaces were registered in air samples. Crude estimates of the leaf surface area of each tree suggest that the total fungal load was approximately 5.04×10⁸ CFU for Ulmus and 2.71×10⁸ CFU for Quercus. Of these levels, 19% were from fungi also detected in air samples. The data suggest that some leaf-surface fungi are major contributors to the air spora.     

Dynamics of the fungal symbionts in the gallery system and the mycangia of the ambrosia beetle,Xylosandrus mutilatus (Blandford) (Coleoptera: Scolytidae) in relation to its life history

The dynamics of the fungal symbionts in the gallery system and the mycangia of the ambrosia beetle,Xylosandrus mutilatus, were studied in relation to its life history using both isolation experiments and scanning electron microscopy (SEM). In the galleries,Ambrosiella sp. was predominant during the larval stages but its relative dominance gradually decreased during the development of the larvae. In contrast, yeasts (mainlyCandida sp.) andPaecilomyces sp. dominated continuously in the galleries after eclosion.Ambrosiella sp. was consistently stored in the mycangia in all adult stages, except in the teneral and overwintering adults when the other fungi were dominant. No fungal spores occurred in the mycangia of the adult beetles reared under aseptic conditions from the pupal stage, while onlyAmbrosiella sp. was stored in those reared from the teneral-adult stage. These results suggest that: (i) Xmutilatus is associated with at least three fungal species, among whichAmbrosiella sp. is the most essential food resource for development of the broods; (ii) immediately after eclosion, new female adults may take at least four associated fungal species, with no or incomplete selection, into their mycangia from the walls of the cradles; and (iii) conditions may well be produced in the mycangia of both matured and dispersing beetles whereby only the spores ofAmbrosiella sp. can proliferate.     

Nematicidal activity of <Emphasis Type="Italic">Paecilomyces</Emphasis> spp. and isolation of a novel active compound

Many species of Paecilomyces are entomogenous fungi and several are efficacious toward nematodes. To study the potential of Paecilomyces species in controlling nematodes, fungal extracts of 40 Paecilomyces spp. were evaluated for their nematicidal activity against Bursaphelenchus xylophilus and Panagrellus redivivus. The extracts of six Paecilomyces spp. exhibited the nematicidal activity against P. redivivus, and 11 species exhibited the nematicidal activity against B. xylophilus. The methanol extract of strain 1.01761 incubating on Czapek solid medium killed more than 95% P. redivivus in 24 h at 5 mg/ml, and the filtrate of strain 1.01788 cultured in Sabouraud’s broth medium resulted in 90% mortality of B. xylophilus in 24 h at 5 mg/ml. A novel nematicidal compound 4-(4′-carboxy-2′-ethyl-hydroxypentyl)-5,6-dihydro-6-methylcyclobuta[b]pyridine-3,6-dicarboxylic acid, was isolated from Paecilomyces sp. YMF1.01761. The LD₅₀ value of the compound within 24 h against P. redivivus was 50.86 mg/L, against Meloidogyne incognita was 47.1 mg/L, and against B. xylophilus was 167.7 mg/L.     

Development of a serial bioreactor system for direct ethanol production from starch using<Emphasis Type="Italic">Aspergillus niger</Emphasis> and<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Aspergillus niger hyphae were found to grow with unliquefied potato starch under aerobic conditions, but did not grow under anaerobic conditions. The raw culture ofA. niger catalyzed saccharification of potato starch to glucose, producing approximately 12 g glucose/L/day/ The extracellular enzyme activity was decreased in proportion to incubation time, and approximately 64% of initial activity was maintained after 3 days. At 50°C,A. niger hyphae growth stopped, while the extracellular enzyme activity peaked. On the basis of theA. niger growth property and enzyme activity, we designed a serial bioreactor system composed of four different reactors. Fungal hyphae were cultivated in reactor I at 30°C, uniquefied starch was saccharified to glycose by a fungal hyphae culture in reactors II and III at 50°C, and glucose was fermented to ethanol bySaccharomyces cerevisiae in reactor IV. The total glucose produced by fungal hyphae in reactor I and saccharification in reactor II was about 42 g/L/day. Ethanol production in reactor IV was approximately 22 g/L/day, which corresponds to about 79% of the theoretical maximum produced from 55 g starch/L/day.     

Growth-promoting factors for yeast cells ofParacoccidioides brasiliensis

Low-density seedings of yeast cells ofParacoccidioides brasiliensis give poor growth (as assessed by plating efficiency test) on conventional mycological agar media, and therefore growth-promoting factors for this fungus were sought. Water-extracts of yeast cells of sixP. brasiliensis isolates were all considerably effective in promoting the growth of low-density seedings ofP. brasiliensis isolates Pb-18 and Hachisuga, but had little effect on isolate Bt-4. Horse serum, at a concentration range of 2–4%, moderately or considerably promoted the growth of theseP. brasiliensis isolates. Combinations of the fungus cell extracts with horse serum were highly effective in promoting the growth of all of the fungal isolates. The fungus cell extracts showed siderophore (microbial iron carrier) activity. An iron-chelator, ethylenediaminetetraacetic acid, at a concentration of 100 μM also highly promoted the growth of the fungal isolates in the presence of horse serum, and ferric ion added to culture medium was considerably effective in the growth promotion. These results suggest that deficient utilization of external iron by the fungus cell is one of the growth-limiting processes for low-density seedings of yeast cells ofP. brasiliensis on conventional mycological agar media.     

The role of meteorological factors in determining the annual variation ofAlternaria andCladosporium spores in the atmosphere of Palencia, 1990–1992

A study was made of the daily content ofAlternaria andCladosporium spores in the atmosphere in Palencia city (Spain) for three consecutive years 1990–1992.Alternaria andCladosporium represented 55% of the total identified spores, presenting an annual distributional pattern of which the maximum values were reached in summer. Multiple regression analyses showed a positive correlation between minimum temperature andCladosporium spore concentrations, while forAlternaria there was a positive correlation with maximum temperature and a negative correlation with precipitation. Duncan's multiple range test among means ofAlternaria data indicated that winds coming from a northeasterly direction were associated with significantly higher concentrations the spores.     

Conidial surface ultrastructure of human-pathogenic and saprobicCladosporium species

Freeze-fracturing of outer wall layers ofCladosporium conidia revealed two types of ultrastructure, coinciding with taxonomic characteristics. The outer conidial layers were essentially smooth in the human pathogenic species,C. bantianum, C. carrionii, andC. trichoides. In contrast, mosaic arrays of rodlets on conidia were observed with freeze-fracturing in the saprobic species,C. cladosporioides, C. coralloides, C. herbarum, C. sphaerospermum, andC. variabile. Conidia ofC. elatum were an exception among the saprobic species as they had smooth surfaces. The present study supports the suggestion that the human pathogenicCladosporium species should be transferred to another genus.     

Differential responses of three fungal species to environmental factors and their role in the mycorrhization of<Emphasis Type="Italic"> Pinus radiata</Emphasis> D. Don

Three ectomycorrhizal (ECM) isolates of Rhizopogon luteolus, R. roseolus and Scleroderma citrinum were found to differ markedly in their in vitro tolerance to adverse conditions limiting fungal growth, i.e. water availability, pH and heavy metal pollution. S. citrinum was the most sensitive, R. luteolus intermediate and R. roseolus the most tolerant species. Pinus radiata D. Don seedlings were inoculated in the laboratory and in a containerised seedling nursery with spore suspensions of the three ECM species. Colonisation percentage was considerably lower under nursery conditions, probably due to competition by native fungi. The effects of nursery ECM inoculation on seedling growth depended on the fungal species. Only R. roseolus-colonised plants showed a significantly higher shoot growth than non-mycorrhizal plants. All three fungi induced significantly higher root dry weights relative to control plants. Despite the low mycorrhizal colonisation, mycorrhization with all three species improved the physiological status of nursery-grown seedlings, e.g. enhanced root enzyme activity, shoot nutrient and pigment content, net photosynthesis rate and water use efficiency. Of the three fungal species, R. roseolus was the most effective; this species was also the most adaptable and showed the greatest range of tolerance to adverse environmental conditions in pure culture. It is, therefore, proposed as a promising fungal species for ECM inoculation of P. radiata in the nursery.     

Survey of aflatoxins and ochratoxin A in stored tubers ofCyperus esculentus L.

The mold incidence, moisture contents, pH and levels of mycotoxins (aflatoxins B₁, G₁ and ochratoxin A) on/of/in rootstock snack (tubers ofCyperus esculentus L.) samples were monitored during a 150-day storage period. Whereas the mold incidence, moisture and mycotoxin levels increased with storage time, the pH declined during the same period. Altogether, 12 fungal species, mostly toxigenic, includingAspergillus flavus, A. parasiticus andA. ochraceus were isolated. At collection period only 3 of the 9 snack samples analysed contained trace amounts of aflatoxins. By 120th day, all the 9 samples were contaminated and the average levels were 454 and 80 ppb for aflatoxin B₁ and aflatoxin G₁ respectively on the 150th day. Ochratoxin A was not detected before 120th day and then only at low levels, occuring in a maximum of four samples and ranging between 10 and 80 ppb.     

A simple polymerase chain reaction/restriction fragment length polymorphism assay capable of identifying medically relevant filamentous fungi

Because of the accumulating evidence that suggests that numerous unhealthy conditions in the indoor environment are the result of abnormal growth of the filamentous fungi (mold) in and on building surfaces, it is necessary to accurately reflect the organisms responsible for these maladies and to identify them in precise and timely manner. To this end, we have developed a method that is cost effective, easy to perform, and accurate. We performed a simple polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis on multiple members of species known to negatively influence the indoor environment. The genera analyzed were Stachybotrys, Penicillium, Aspergillus, and Cladosporium. Each organism underwent PCR with universal primers that amplified ribosomal sequences generating products from 550 to 600 bp followed by enzymatic digestion with EcoRI, HaeIII, MspI, and HinfI. Our results show that using this combination of restriction enzymes enables the identification of these fungal organisms at the species level.     

Chemotaxonomic discrimination among the fungal genera Tolypocladium, beauveria and Paecilomyces

Fungi of the genera Beauveria and Paecilomyces were found to produce cyclotetradepsipeptides, beauverolides. Production of beauverolides was not detected at the genus Tolypocladium. Analysis of beauverolides therefore provides a very simple chemotaxonomic test which seems to be suitable for fast discrimination between the genera Beauveria vs Tolypocladium and complementing morphological examination. A GC-MS study of β-hydroxy acid distribution in the beauverolide hydrolyzates revealed that all strains prdouce γ-methyl-β-hydroxy acids only. Their occurrence thus cannot be used as a taxonomic marker of different species within the genera Beauveria and Paecilomyces.     

Cultural characteristics and extraction of the fungal pigment phleichrome from the phytopathogenic fungus<Emphasis Type="Italic">Cladosporium phlei</Emphasis>

The cultural characteristics of the fungusCladosporium phlei were assessed in order to develop an improved method for the production of the fungal pigment, phleichrome, which is an intermediate in the production of a photodynamic therapeutic agent. The growth ofC. phlei, as measured by the hyphal growth rate and increase in biomass, varies significantly depending on the culture media utilized (V8 juice-based medium proved optimal for both growth rate and biomass increase). How-ever, even on a V8 juice plate, the growth ofC. phlei occurred slowly and in a limited fashion, in that the colony covered only 75% of the agar surface after more than 4 weeks of cultivation at 20°C. Supplementations of glucose, fructose, galactose, and sucrose increased both hyphal expansion and mass production, whereas supplementations of other carbon sources, including glycerol and sorbitol, exerted no detectable effects. The effect of inorganic nitrogen supplementation was negligible, whereas organic nitrogen evidenced significant effects, with enhanced growth with malt extract and growth inhibition with yeast extract and tryptone. Sporulation was enhanced under conditions of continuous light, and a minimum of 10³ spores per mL of liquid media was found to be necessary for the optimal mass increase. A simple extraction procedure was established in order to isolate the deep red pigment which was subsequently identified as phleichrome via NMR analysis. WhenC. phlei was cultured on V8 medium containing 5% glucose and 2% malt extract, the quantity of mycelial mass was estimated as 20.6 g (dry weight) per liter of culture. The expected phleichrome yields from the mycelia and culture filtrates were estimated to be 43 and 2 mg/L, repectively. There was an equal contribution of the reported research by the first two authors.     

Daily and seasonal variations ofAlternaria andCladosporium airborne spores in León (North-West, Spain)

The spores ofAlternaria andCladosporium are present throughout the year in the atmosphere of León (NW Spain), although they show an important seasonal variation. To understand the relationship between the number of spores and climatic factors,Alternaria andCladosporium spores counts for January 1994 to December 1995 were examined by means of correlation analyses. The results of weekly samples of both years showed that the spores concentration of two taxa are significantly and positively correlated with maximum and minimum temperature and sunshine hours and negatively with relative humidity. The statistical analysis of daily samples generally showed the same results. In the hourly distribution of spore concentrations we can see a similar behaviour ofAlternaria andCladosporium, with most spores collected in the 12–14 h period.     

Interactions ofCandida albicans with bacteria and salivary molecules in oral biofilms

The yeastCandida albicans coaggregates with a variety of streptococcal species, an interaction that may promote oral colonization by yeast cells.C. albicans andCandida tropicalis are the yeasts most frequently isolated from the human oral cavity and our data demonstrate that both these species bind toStreptococcus gordonii NCTC 7869 while two otherCandida species (Candida krusei andCandida kefyr) do not. Adherence ofC. albicans was greatest when the yeast had been grown at 30° C to mid-exponential growth phase. For 21 strains ofC. albicans there was a positive correlation between the ability to adhere toS. gordonii and adherence to experimental salivary pellicle. Whole saliva either stimulated or slightly inhibited adherence ofC. albicans toS. gordonii depending on the streptococcal growth conditions. The results suggest that the major salivary adhesins and coaggregation adhesins ofC. albicans are co-expressed.     

Cladosporium sphaerospermum as a new plant growth-promoting endophyte from the roots of Glycine max (L.) Merr.

Endophytic fungi are plant symbionts that produce a variety of beneficial metabolites for plant growth and protection against herbivory and pathogens. Fourteen fungal samples were isolated from the roots of soybean cultivar Daemangkong and screened on waito-c rice for their plant growth-promoting capacity. Twelve of the fungal isolates promoted plant growth, while two inhibited it. The fungal isolate DK-1-1 induced maximum plant growth in both waito-c rice and soybean. The plant growth promotion capacity of DK-1-1 was higher than the wild type Gibberella fujikuroi. Gibberellin (GA) analysis of culture filtrate of DK-1-1 showed the presence of higher amounts of bioactive GA₃, GA₄, and GA₇ (6.62, 2.1 and 1.26 ng/mL, respectively) along with physiologically inactive GA₅, GA₁₅, GA₁₉, and GA₂₄. Phylogenetic analysis of 18S rDNA sequence identified the fungal isolate as a new strain of Cladosporium sphaerospermum. Gibberellin production and plant growth-promoting ability of genus Cladosporium are reported for the first time in the present study. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Variation within isolates ofTyphula incarnata from localities differing in winter climate

Isolates ofTyphula incarnata, a snow mold fungus, were collected from four localities with different winter climates. Their ecological traits such as mycelial growth rate, sclerotium size, carpogenic germination of sclerotia, and aggressiveness were compared between populations in order to reveal infraspecific differentiation associated with climatic differences. Population variability was evident only in sclerotium germination: isolates from more snowy localities germinated faster than those from less snowy localities.T. incarnata is regarded as a versatile pathogen with no specialized forms in contrast withT. ishikariensis. The germination rate of sclerotia is considered very critical in the life history ofT. incarnata.     

Comparative structural study ofQuercus serrata andQ. acutissima formed byPisolithus tinctorius andHebeloma cylindrosporum

Ectomycorrhizas were synthesized in pots and growth pouches betweenQuercus serrata, Q. acutissima, and two ectomycorrhizal fungi,Pisolithus tinctorius andHebeloma cylindrosporum. Root morphology and the structure of the mantle and Hartig net were compared using light, fluorescence, scanning and transmission electron microscopy.P. tinctorius initially colonized root cap cells, and eventually produced a highly branched lateral root system with a complete mantle, whereasH. cylindrosporum promoted root elongation with few hyphae on the root apex surface indicating that interaction between roots differs with fungal species. Hartig net structure and hyphal inclusions varied between all the combinations tested. There were structural differences between mycorrhizas ofH. cylindrosporum/Q. acutissima grown in soil and growth pouches, which indicate that the growth pouch environment can induce artefacts in roots. Fruit bodies ofH. cylindrosporum developed in pots withQ. acutissima. AlthoughP. tinctorius has been used to inoculate oak seedlings in the nursery, results of this study indicate thatH. cylindrosporum may also be an effective ectomycorrhizal fungus forQ. serrata andQ. acutissima.     

Isolation of scopoletin from leaves of Hevea brasiliensis and the effect of scopoletin on pathogens of H. brasiliensis

Scopoletin (7-hydroxy-6-methoxy coumarin) which inhibited the conidial germinationof Corynespora cassiicola was isolated from the uninfected mature leaves ofHevea brasiliensis. Scopoletin was not detected in uninfected immature rubber leaves. The immature leaves produced scopoletin after being infected with C. cassiicola. The concentration of scopoletin in infected leaves was higher than in uninfected mature leaves. Scopoletin also inhibited the conidial germination of other fungal pathogens of H. brasiliensis. However, no correlation was observed between scopoletin accumulation and clonal resistance. This revised version was published online in June 2006 with corrections to the Cover Date.     

Biological species ofArmillaria and their mycoparasitic associations withRhodophyllus abortivus in Hokkaido

Specimens of basidiomes and/or rhizomorphs ofArmillaria mellea complex and basidiomes ofRhodophyllus abortivus, developing on the same decaying stumps or stems of forest trees, were collected in three forests in Hokkaido. Normal basidiomes ofR. abortivus were found near to, but free from, the rhizomorphs and/or basidiomes ofArmillaria, while abnormal basidiomes, as carpophoroid forms, were developed on the rhizomorphs ofArmillaria. Of three mycoparasiticArmillaria isolates found withR. abortivus, one was identified asA. gallica and two asA. jezoensis. The isolates ofR. abortivus showed excellent mycelial growth and rhizomorph formation on PDA. However, on MDA, RMDA and BMDA, they showed poor aerial mycelia growth and no rhizomorphs. In the contrapositional cultures, the growth ofA. gallica was completely inhibited byR. abortivus on PDA but only slightly inhibited on MDA and RMDA. On the other hand, mutual inhibition at a distance was observed on BMDA. The mycelial growth and rhizomorph formation inA. jezoensis were severely inhibited by the colony ofR. abortivus on PDA, but only slightly inhibited on MDA. On RMDA and BMDA, the colonies of twoArmillaria species andR. abortivus showed mutual inhibition at a distance and apparent rhizomorph formation by bothArmillaria species.