Conidial Wall Research Articles

Phylogeny of Stemphylium spp. based on ITS and glyceraldehyde-3-phosphate dehydrogenase gene sequences

The phylogenetic relationships among 44 isolates representing 16 species of Stemphylium were inferred from ITS and glyceraldehyde-3-phosphate dehydrogenase (gpd) sequence data. The results generally agree with morphological species concepts. There was strong support for monophyly of the genus Stemphylium. Analysis of the gpd fragment in particular was useful for establishing well-supported relationships among the species and isolates of Stemphylium. Species of Stemphylium that appear to have lost the ability to produce a sexual state are scattered among the species with the ability to reproduce sexually (Pleospora spp.). Species that are pathogenic to alfalfa are resolved into two groups. Stemphylium botryosum and two isolates with morphological characters similar to S. globuliferum had identical sequences at both loci. These two loci in S. vesicarium, S. alfalfae and S. herbarum are nearly identical but differ from S. botryosum. The separation of S. vesicarium, S. herbarum and S. alfalfae into separate species by morphometric evidence was not supported by the molecular data. Morphological and developmental characters such as size and shape of conidia, conidiophores, and ascospores, and size and time of maturation of pseudothecia are useful for diagnosing species. However, other morphological characters such as septum development and small variations in conidial wall ornamentation are not as useful.

Comparative morphological studies on tomato powdery mildew (Oidium neolycopersici)

Fourteen isolates of tomato powdery mildew (Oidium neolycopersici) and one isolate of the following species: Podosphaera fusca (= Sphaerotheca fusca), Erysiphe orontii (cucumber powdery mildews), Erysiphe cichoracearum (lettuce powdery mildew) and Erysiphe aquilegiae var. ranunculi (Ranunculus lingua powdery mildew) were used for comparative morphological studies. Basic characteristics of the anamorphs, including outer conidial wall patterns, were compared using light and scanning electron microscopy (SEM). In main morphological features, O. neolycopersici was strongly differentiated from E. cichoracearum, E. orontii and P. fusca. However, based on morphological features (e.g. germination type; appressorium shape; morphology of conidiophores) O. neolycopersici was close to E. aquilegiae var. ranunculi (both belong to Oidium subgen. Pseudoidium) and it probably could be placed to Erysiphe sect. Erysiphe (= Erysiphe s. str.)

Characterisation of the ‘C’ morphotype of the pine pathogen Sphaeropsis sapinea

Sphaeropsis sapinea is an opportunistic pathogen of Pinus spp. and other conifers. Infection occurs when host trees are predisposed by adverse environmental conditions or mechanical damage. Initially two morphotypes, A and B, were described for S. sapinea and these were defined based on cultural characteristics, texture of conidial walls and virulence. The existence of these two morphotypes has, furthermore, been confirmed through the use of RAPDs and DNA sequencing of the ITS region of the rRNA operon. A third RAPD group, including isolates from Indonesia and Mexico, has recently been reported and was designated as a C morphotype based on differences in conidial size. The objective of this study was to characterise isolates of the C morphotype of S. sapinea based on cultural characteristics, conidial morphology, growth rate and pathogenicity. Cultural and conidial characteristics of isolates belonging to the A and C morphotypes were more similar to each other than to those of the B morphotype. The growth rates of isolates belonging to the three different morphotypes were not significantly different. Artificial inoculation of both Granny Smith apples and Pinus patula seedlings revealed that isolates of the C morphotype were considerably more virulent than those of the A and B morphotypes. The discovery of the more virulent C morphotype of S. sapinea could have serious implications for management of this pathogen, as well as quarantine practises in countries that import pine seeds.

Interaction between Laccaria laccata and Trichoderma virens in co-culture and in the rhizosphere of Pinus sylvestris grown in vitro.

Interactions between the ectomycorrhizal fungus Laccaria laccata and the soil fungus Trichoderma virens in co-culture and in the rhizosphere of Pinus sylvestris seedlings growing in vitro were investigated by light and scanning electron microscopy. The growth of T. virens was inhibited in co-culture. Shortened, more branched and sometimes deformed or injured hyphae of T. virens were observed in the zone of inhibition. Two-month-old mycorrhizae of P. sylvestris/ L. laccata were inoculated with a conidial suspension of T. virens and examined at intervals of 7-24 h and 2, 3, and 6 days post-inoculation (p.i.). On non-mycorrhizal roots, conidia germination was high and long hyphae formed 3 days p.i. On mycorrhizal roots, short germ tubes were observed only sporadically. At 3 days p.i., the mantle hyphae of L. laccata grew towards the conidia and coiled around them. Extremely dense coils of hyphae were found around clusters of conidia. Deformation of conidia, breaks in conidial walls and their partial degradation were observed 6 days p.i.

Identification of species in Aspergillus section Flavi based on sequencing of the mitochondrial cytochrome b gene

The partial sequences of the mitochondrial (mt) cytochrome b gene (402 bp) were determined for species of Aspergillus section Flavi. On the basis of identities of DNA sequences, 77 strains were divided into seven DNA types, from D-1 to D-7. The type strains of A. sojae, A. parasiticus, A. flavus and A. oryzae together, A. tamarii, and A. nomius were placed in DNA types D-1, D-2, D-4, D-5 and D-7, respectively. These species could be differentiated from each other. Furthermore, two other DNA types, D-3 and D-6 were found. DNA type D-3 was closely related to A. parasiticus (D-2) and included one strain that deposited as A. flavus var. flavus and produced aflatoxins B and G. DNA types D-6 included one strain named A. flavus and closely related to A. tamarii. The observations of conidial wall texture by SEM (Scanning Electron Microscopy) supported the relationships derived from the cytochrome b gene. The production of aflatoxins was also examined. Using the DNA sequence of cytochrome b gene, several strains were reidentified. The derived amino acids sequences were all the same in the studied strains. The mt cytochrome b gene is useful and reliable in distinguishing and identifying the species in Aspergillus section Flavi.

Morphological variation and cultural characteristics of Coniothyrium leucospermi associated with leaf spots of Proteaceae

During an examination ofConiothyrium collections occurring on Proteaceae one species,C. leucospermi, was repeatedly encountered. However, it was not always possible to identify this species from host material alone, whereas cultural characteristics were found to be instrumental in its identification. Conidium wall ornamentation, which has earlier been accepted as crucial in species delimitation is shown to be variable on host material, making cultural comparisons essential. Using standard culture and incubation conditions,C. leucospermi is demonstrated to have a wide host range in the Proteaceae. In addition, microcyclic conidiation involving yeast-like budding from germinating conidia and hyphae in culture is newly reported for this species.

Differences in sialic acid density in pathogenic and non-pathogenic Aspergillus species.

ASPERGILLUS: fumigatus is a ubiquitous soil fungus that causes invasive lung disease in the immunocompromised host. The structure of the conidial wall has not been well characterized although it is thought that adhesins present on the surface are involved in attachment of the conidia to host lung cells and proteins, which is a prerequisite for the establishment of infection. Negatively charged carbohydrates on the conidial surface have been previously identified as the molecules responsible for attachment of conidia to extracellular matrix proteins. The aim of this research was to identify carbohydrates on the conidial surface that contribute to its negative charge. Direct chemical analysis and indirect binding assays have demonstrated that A. fumigatus possesses sialic acids on the conidial surface. Pre-treatment of A. fumigatus conidia with sialidase decreased binding of a sialic acid-specific lectin, Limax flavus agglutinin (LFA), to the conidial surface and decreased adhesion of conidia to the positively charged polymer poly L-lysine. Two other sialic acid-specific lectins, Maackia amurensis agglutinin and Sambucus nigra agglutinin, exhibited negligible binding to A. fumigatus conidia indicating that 2,3-alpha- and 2,6-alpha-linked sialic acids are not the major structures found on the conidial surface. Mild acid hydrolysis and purification of conidial wall carbohydrates yielded a product that had the same R(F) as the Neu5Ac standard when analysed by high-performance thin-layer chromatography. A density of 6.7 x 10(5) sialic acid residues per conidium was estimated using a colorimetric assay. Conidia grown on a minimal medium lacking sialic acid also reacted with LFA, indicating that sialic acid biosynthesis occurs de novo. Sialic acid biosynthesis was shown to be regulated by nutrient composition: the density of sialic acids on the surface of conidia grown in minimal media was lower than that observed when conidia were grown on rich, complex media. It has previously been shown that pathogenic Aspergillus species adhere to basal lamina proteins to a greater extent than non-pathogenic Aspergillus species. To determine whether the expression of sialic acid on the conidial surface was correlated with adhesion to basal lamina, conidia from other non-pathogenic Aspergillus species were tested for their reactivity towards LFA. Flow cytometric analysis demonstrated that A. fumigatus had a significantly greater sialic acid density than three non-pathogenic Aspergillus species. Sialic acids on the conidial wall may be involved in adhesion to fibronectin, a component of the basal lamina, as binding of A. fumigatus conidia to fibronectin was strongly inhibited in the presence of a sialylated glycoprotein.

Leaf Spot Disease of Broad Bean Caused by Alternaria tenuissima in Japan.

In April 1999, a leaf spot of broad bean (Vicia faba L.) was observed in commercial fields in Shimane prefecture of Western Japan. Lesions were concentric and brown in color. Older leaves were particularly affected. In later stages of the disease, plants defoliated as leaves blighted from margin to the center. Isolation was made from infected leaf tissue. The isolated fungus produced conidia on V8 medium (2) either in dark or under continuous irradiation of near ultraviolet radiation (NUV) from BLB fluorescent lamps. Conidial chains were unbranched or rarely formed a few lateral branches with a few conidia. The conidia of the fungus grown under continuous NUV were dark and smoothly tapered into the apical beak, and each conidium had a conspicuously thickened primary septum with a constriction of the conidial wall and often a darker median transverse septum. The conidia measured 21.2 to 45.5 μm (mean = 32.9 μm) × 7.3 to 17.7 μm (mean = 11.4 μm ) on V8 medium. Conidia produced on leaves and stem collected from field were similar in size and appearance. The fungus was identified as Alternaria tenuissima based on its cultural and morphological characteristics (2). An isolate was also sent to CABI Bioscience Identification Services (Egham, UK), which also identified the fungus as A. tenuissima. A conidial suspension (107 spores/ml) was prepared and used to inoculate detached leaves and intact plants of broad bean. Intact plants were inoculated by spaying with spore suspension and covered with polyethylene bags for maintaining high humidity. Detached leaves in moist petri dishes were inoculated with drops of spore suspension. Symptom developed on both detached and intact leaves 3 to 4 days after inoculation. Reisolating the pathogen from infected leaves completed Koch's postulates. In June 2000, the leaf spot was observed in all 15 fields surveyed in other areas of Shimane prefecture. In some fields, plants were defoliated and stems and pods were also infected. Isolates of A. tenuissima also were obtained from those fields. This pathogen has been isolated from other hosts in Japan (1). This is the first report of A. tenuissima on broad bean in Japan.

The rapid and accurate determination of germ tube emergence site by Blumeria graminis conidia

The development of appressoria by germinating Blumeria graminis conidia depends on its germ tubes making contact with the host surface. Low angle, low temperature scanning electron microscopy showed that 80% or more of first-formed germ tubes of f. spp. hordei, tritici and avenae conidia emerged from close to the host leaf surface, and so made contact with it allowing them to become functional primary germ tubes. Light microscopy of f. sp.hordei confirmed this result and, in addition, showed that germ tubes frequently emerged close to, and contacted, various hydrophobic and hydrophilic artificial substrata. Geometric models of conidium-substratum interfaces were developed and a “surface point method” was derived to predict the frequency with which contact would result if germ tube emergence was a random phenomenon. However, observed contact frequencies were far higher (c. three to eight times) than predicted. Thus, the germ tube emergence site was determined as a response to substratum contact. In part, this appeared to be a non-specific response. Nevertheless, germ tube contact frequencies were greater on the curved surface of leaf epidermal cells than on planar surface, suggesting that specific recognition of leaf surface characteristics may promote directional emergence. The area of contact required to stimulate directional germ tube emergence was very small: contact with a microneedle tip or with a spiders’ suspension thread was sufficient for many conidia. Similarly, on leaves, the only contact is between the tips of a limited number of conidial wall projections and the edges of epicuticular leaf wax plates. Micromanipulation to roll conidia so that their original site of contact with a leaf was rotated away from it, led to the majority of first-formed germ tubes growing away from the leaf, i.e. emerging close to the site of original contact. The experiments indicated that the site of germ tube emergence is determined within 1 min of deposition. This implicated the release of conidial extracellular materials in recognition of the conidium-leaf surface contact site.

Biosynthesis and functions of melanin in Sporothrix schenckii.

Sporothrix schenckii is a human pathogen that causes sporotrichosis, an important cutaneous mycosis with a worldwide distribution. It produces dark-brown conidia, which infect the host. We found that S. schenckii synthesizes melanin via the 1,8-dihydroxynaphthalene pentaketide pathway. Melanin biosynthesis in the wild type was inhibited by tricyclazole, and colonies of the fungus were reddish brown instead of black on tricyclazole-amended medium. Two melanin-deficient mutant strains were analyzed in this study: an albino that produced normal-appearing melanin on scytalone-amended medium and a reddish brown mutant that accumulated and extruded melanin metabolites into its medium. Scytalone and flaviolin obtained from cultures of the reddish brown mutant were identified by thin-layer chromatography, high-performance liquid chromatography, and UV spectra. Transmission electron microscopy showed an electron-dense granular material believed to be melanin in wild-type conidial cell walls, and this was absent in conidial walls of the albino mutant unless the albino was grown on a scytalone-amended medium. Melanized cells of wild-type S. schenckii and the albino grown on scytalone-amended medium were less susceptible to killing by chemically generated oxygen- and nitrogen-derived radicals and by UV light than were conidia of the mutant strains. Melanized conidia of the wild type and the scytalone-treated albino were also more resistant to phagocytosis and killing by human monocytes and murine macrophages than were unmelanized conidia of the two mutants. These results demonstrate that melanin protects S. schenckii against certain oxidative antimicrobial compounds and against attack by macrophages.

Ultrastructure of conidiogenesis and mature conidia in the plant pathogenic Entomosporium mespili

Acervuli of E. mespili developed subcuticularly on both surfaces of infected Photinia leaves. Acervuli developed as single or aggregated, confluent structures. Conidium initials arose from hyphae which developed between the cuticle and epidermis. As conidia developed the cuticle was displaced and eventually ruptured. Development of the first conidium from a conidiogenous cell was holoblastic. Subsequent conidia developed in an enteroblastic, annellidic fashion. Three to four conidia arose from a single conidiogenous cell. A mature conidium consisted of four to six cells including an apical cell and a basal cell which developed first. These were separated by a septum with a plugged pore. A similar septum separated the basal cell from the conidiogenous cell. A long, slender, enucleate appendage then arose from the tip of the apical cell. During this time the basal cell gave rise to two to four small lateral cells, separated by septa from the basal cell. An appendage then developed from each lateral cell. Each cell of a mature conidium contained a single nucleus and a complement of cytoplasmic organelles and inclusions, many of which were more prominent in freeze substituted conidia than in chemically fixed conidia. The conidium wall labelled for chitin using wheat germ agglutin-gold conjugate. Its outer surface was coated with fibrillar material.

Comparison of cultural and morphological characters and ITS sequences in anamorphs of Botryosphaeria and related taxa

Anamorphs of 22 strains of botryosphaeriaceous fungi including Fusicoccum spp., Diplodia spp., Sphaeropsis spp., and Lasiodiplodia theobromae (teleomorph = Botryosphaeria rhodina) were compared utilizing conidial characters, cultural morphology, growth rates and nucleotide sequences of the nuclear rDNA internal transcribed spacers ITS 1 and ITS 2. The ITS data shared consensus with morphological and cultural characters in separating 7 of the 22 strains. The 7 strains were placed into one of three groups that corresponded with the morphological species L. theobromae, S. sapinea, and F. luteum. Only partial consensus was achieved with the remaining 15 strains as ITS sometimes grouped together strains with uniform morphological and cultural characters and other times not. The ten Fusicoccum strains, identified as anamorphs of B. dothidea or B. ribis, were divided into two ITS groups and up to five groups based on traditional characters. The two putative B. ribis strains were consistently grouped together and appear to represent a stable variant of B. dothidea. Two morphologically indistinguishable Sphaeropsis sp. strains, identified as anamorphs of B. obtusa, were separated into two ITS groups, one containing all S. sapinea strains and the other containing Diplodia mutila (teleomorph = B. sievensit) and D. quercina. Conidial color, wall texture, septa number, size and shape as well as mycelial growth under different temperatures were consistent characters within some presumed morphological species, and within some ITS groups, but none of the characters were universally informative. Moreover, conidial pleomorphy and plasticity of some morphological and cultural characters posed problems for differentiating strains using traditional characters, particularly with Sphaeropsis and Diplodia form-genera. The possibility that the ITS region is not informative at the species level for some of the taxa evaluated is considered.

Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1.

The fungicidal properties of the synthetic peptide D4E1 were studied with nongerminated and germinating conidia of Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Fusarium moniliforme, and Fusarium oxysporum. The minimal lethal concentrations (MLC) needed to kill 100% of germinating conidia of A. fumigatus, A. flavus, and A. niger were 12.5, 12.5, and 25 microM, respectively. The MLC value for nongerminated and germinating conidia of both Fusarium spp. was 3.0 microM. Except for A. fumigatus, D4E1 was inactive against the nongerminated conidia of the Aspergillus spp. Physicochemical studies showed D4E1 complexed with ergosterol, a sterol present in conidial walls. Cholesterol, present in nongerminated conidia of F. moniliforme, had a greater affinity for D4E1 than did ergosterol. D4E1 was more resistant to fungal and plant protease degradation than the natural peptide, cecropin A. These in vitro results suggest D4E1 is a candidate for transgenic expression in plants to enhance host resistance to fungal infection.

Fungicidal and binding properties of the natural peptides cecropin B and dermaseptin

In vitro fungicidal properties of cecropin B and dermaseptin were explored using non-germinating and germinating conidia from Aspergillus flavus, A. fumigatus, A. niger, Fusarium n2oniliforme and F oxysporum. Cecropin B produced LD50 values for germinating A. flavus, A. fumigatus and A. niger conidia of 30, 0.5 and 2.0 microM, respectively, while dermaseptin gave LD50 values of 4.0, 0.05 and 2.0 microM, respectively. Cecropin B produced an LD50 value of 0.2 microM for non-germinating F. moniliforme and F. oxysporum conidia, while dermaseptin did not reduce either as much as 50% at any level tested. LD50 levels for CB were 0.2 and 0.1 microM, respectively, for germinating F. moniliforme and F. oxysporum conidia. Dermaseptin was less effective, giving LD50 values for germinating F. moniliforme and F. oxysporum conidia of 0.3 and 0.8 microM, respectively. Neither peptide reduced conidial viabilities of non-germinating Aspergillus spp. Physicochemical studies indicated cecropin B and dermaseptin bound to ergosterol and cholesterol, conidial wall constituents, but not to chitin or beta-1,3-glucan.

Sialic acid-dependent recognition of laminin and fibrinogen by Aspergillus fumigatus conidia.

In an attempt to define the molecular basis of the adherence of Aspergillus fumigatus conidia to the host tissues, a step which might be mediated by the recognition of basement membrane laminin or fibrinogen, we analyzed the binding of these glycoproteins by flow cytometry and a microtiter plate adherence assay. Flow cytometry revealed that the binding of fluorescein isothiocyanate-labeled laminin to conidia was saturable and specific. Moreover, the ability of conidia to bind laminin increased with their maturation. Competition experiments showed a cross-reactivity between laminin and fibrinogen binding and a lack of interactions with glycosaminoglycans. In addition, the binding of laminin was not inhibited by the different adhesive synthetic peptides tested. Furthermore, the microtiter plate assay of adherence to chymotrypsin degradation products of laminin or fibrinogen purified by gel filtration suggested a unique binding site common to sequential degradation fragments or the presence of multiple binding sites on the two ligands. Therefore, the role of carbohydrates in the recognition process was investigated. Among the carbohydrates tested, constitutive of the conidial wall or of the oligosaccharide side chains of laminin and fibrinogen, only N-acetylneuraminic acid and sialyllactose inhibited the binding of these glycoproteins to conidia. In conclusion, these results strengthen the idea that the laminin and fibrinogen receptors in A. fumigatus are identical and suggest an interaction mediated by a sialic acid-specific lectin of the conidial wall.

Phylloplane bacteria antagonistic to the sunflower pathogen Alternaria helianthi

Thirty-eight of the 61 isolates of bacteria obtained from diseased and healthy sunflower leaves inhibited the germination of conidia and growth of germ-tubes of Alternaria helianthi in vitro. Inhibition included reduced conidial germination, germ-tube swelling causing vesicle formation, excessive germ-tube branching, lysis of germ-tubes, absence of sporulation and a reduced rate of hyphal growth. Some bacteria appeared to be endoparasitic, persisting inside the lumina of conidia and causing erosion of the conidium wall which resulted in the destruction of conidial cells. Bacteria attached themselves to conidia, hyphae and conidiophores and, in the field, are probably dispersed with the fungus. Five bacterial isolates that showed a high level of inhibition were identified as members of the genus Bacillus and comprised three species, B. subtilis, B. cereus and B. mycoides. The inhibitory substance produced by an isolate of B. subtilis was heat stable and therefore probably not an extracellular enzyme. Inhibition caused by the 38 isolates was not due to acid production by the bacteria. The size of lesions formed by A. helianthi was reduced when an isolate of B. mycoides was applied to sunflower plants with the conidial suspension. B. mycoides had no effect on infection by A. helianthi when applied to sunflower leaves 24 h prior to inoculation with conidia of A. helianthi.

Characterization of the mucilage sheaths of Lemonniera aquatica by lectin-gold labelling

A pre-embedding lectin-gold labelling method was used to characterize the carbohydrate components in the mucilage ofLemonniera aquatica. A specific tissue processing protocol was developed, namely: a) primary fixation in 2% paraformaldehyde and 0.2% glutaraldehyde in PIPES buffer (pH 7.2) for 30 min; b) secondary fixation in 2% glutaraldehyde in the same buffer system for 1 h; c) post-fixation in 1% aqueous OsO4 for 1h; d) embedment in Mollenhaur's resin. The three gold conjugated lectins used were: concanavalin A, wheat germ agglutinin andLimax flavus agglutinin, allowing detection of their complementary saccharides, namely α-d-mannose/α-d-glucose,N-acetyl-d-glucosamine (GluNAc), andN-acetylneuraminic acid (NANA), respectively.N-Acetyl-d-glucosamine and NANA residues were the major components of germ tube mucilage with only a small amount of α-d-manose/α-d-glucose. However, NANA was restricted to the mucilage in the region of germ tube emergence from the conidial arm. The abundance of GluNAc and NANA residues on hyphae and appressoria was less than that on the germ tube. Conversely, α-d-mannose/α-d-glucose was more abundant in the appressorial mucilage. Variability of mucilage composition was found to exist between different structures of the germinated conidium and also between different regions of the same structure. Further, the conidial cell wall ofL. aquatica is not chitinous, and lacks NANA and α-d-mannose/α-d-gluocse.

The Aspergillus parasiticus polyketide synthase gene pksA, a homolog of Aspergillus nidulans wA, is required for aflatoxin B1 biosynthesis.

Aflatoxins comprise a group of polyketide-derived carcinogenic mycotoxins produced by Aspergillus parasiticus and Aspergillus flavus. By transformation with a disruption construct, pXX, we disrupted the aflatoxin pathway in A. parasiticus SRRC 2043, resulting in the inability of this strain to produce aflatoxin intermediates as well as a major yellow pigment in the transformants. The disruption was attributed to a single-crossover, homologous integration event between pXX and the recipient A. parasiticus genome at a specific locus, designated pksA. Sequence analysis suggest that pksA is a homolog of the Aspergillus nidulans wA gene, a polyketide synthase gene involved in conidial wall pigment biosynthesis. The conserved beta-ketoacyl synthase, acyltransferase and acyl carrier-protein domains were present in the deduced amino acid sequence of the pksA product. No beta-ketoacyl reductase and enoyl reductase domains were found, suggesting that pksA does not encode catalytic activities for processing beta-carbon similar to those required for long chain fatty acid synthesis. The pksA gene is located in the aflatoxin pathway gene cluster and is linked to the nor-1 gene, an aflatoxin pathway gene required for converting norsolorinic acid to averantin. These two genes are divergently transcribed from a 1.5 kb intergenic region. We propose that pksA is a polyketide synthase gene required for the early steps of aflatoxin biosynthesis.

The Effects of Tricyclazole, Pyroquilon, Phthalide, and Related Fungicides on the Production of Conidial Wall Pigments by Penicillium and Aspergillus Species

Nine compounds that inhibit 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis in Pyricularia oryzae were used to study their effect on pigment production in conidial walls of 11 Penicillium and 5 Aspergillus species. Conidial pigmentation in all of the fungi except A. flavus and A. parasiticus was inhibited by tricyclazole and, where tested, by most of the other compounds that inhibit melanin synthesis in Py. oryzae. The stronger inhibitors of DHN-melanin synthesis (tricyclazole, chlobenthiazone, and pyroquilon) were the strongest inhibitors of pigment synthesis by the Penicillum and Aspergillus species, and four of the other compounds (phthalide, PCBA, PP-389, and MQ) had a similar order of effect on pigment synthesis in the Penicillium and Aspergillus species as previously reported for melanin synthesis in Py. oryzae. Two of the weakest melanin inhibitors, coumarin and TQ, did not inhibit pigment synthesis. Flaviolin, a shunt product from the DHN-melanin pathway, was isolated from tricyclazole-treated cultures of 10 of 14 of the fungal isolates when they were grown on a special alkaline medium. Conidial wall pigmentation was inhibited by tricyclazole in the alkaline medium, and the effects of tricyclazole on conidial wall pigmentation and flaviolin accumulation occurred concurrently. These findings indicate that some of the Aspergillus isolates and all of the Penicillium isolates used in this study contain a reductase enzyme(s) that is involved in the biosynthesis of green pigments and that is inhibited by compounds that prevent DHN-melanin biosynthesis in a number of brown to black fungi. The accumulation of flaviolin in cultures containing tricyclazole suggests that pentaketide metabolites are used in the synthesis of these pigments.

DewA encodes a fungal hydrophobin component of the Aspergillus spore wall

An anonymous cDNA clone, pCAN4, was shown previously to correspond to an mRNA that accumulates preferentially during asexual sporulation of the filamentous fungus Aspergillus nidulans. The peptide encoded by pCAN4 is a fungal hydrophobin, a group of small, hydrophobic cell wall proteins. When the CAN4 gene was disrupted, conidia and conidiophores appeared to be normal, but sporulating colonies wetted more rapidly with detergent solutions than did the wild type. We renamed CAN4 dewA for the detergent wettable phenotype and mapped it to chromosome V, 24 map units from cysC. The A. nidulans rodA gene also encodes a sporulation-specific fungal hydrophobin. Spores of a dewA- rodA- double mutant were less hydrophobic than those of either mutant alone, showing that dewA and rodA contribute independently to spore-wall hydrophobicity. Immunolocalization of DewA by epitope tagging demonstrated that DewA is present in the spore wall, but not in the walls of germ tubes, hyphae or cells of the spore-producing conidiophore. We conclude that dewA encodes a new fungal hydrophobin component of the conidial wall.