Search

Aspergillus flavus contaminates agricultural products worldwide with carcinogenic aflatoxins that pose a serious health risk to humans and animals. The fungus survives adverse environmental conditions through production of sclerotia. When fertilized by a compatible conidium of an opposite mating type, a sclerotium transforms into a stroma within which ascocarps, asci, and ascospores are formed. However, the transition from a sclerotium to a stroma during sexual reproduction in A. flavus is not well understood. Early events during the interaction between sexually compatible strains of A. flavus were visualized using conidia of a green fluorescent protein (GFP)-labeled MAT1-1 strain and sclerotia of an mCherry-labeled MAT1-2 strain. Both conidia and sclerotia of transformed strains germinated to produce hyphae within 24 h of incubation. Hyphal growth of these two strains produced what appeared to be a network of interlocking hyphal strands that were observed at the base of the mCherry-labeled sclerotia (i.e., region in contact with agar surface) after 72 h of incubation. At 5 wk following incubation, intracellular green-fluorescent hyphal strands were observed within the stromatal matrix of the mCherry-labeled strain. Scanning electron microscopy of stromata from a high- and low-fertility cross and unmated sclerotia was used to visualize the formation and development of sexual structures within the stromatal and sclerotial matrices, starting at the time of crossing and thereafter every 2 wk until 8 wk of incubation. Morphological differences between sclerotia and stromata became apparent at 4 wk of incubation. Internal hyphae and croziers were detected inside multiple ascocarps that developed within the stromatal matrix of the high-fertility cross but were not detected in the matrix of the low-fertility cross or the unmated sclerotia. At 6 to 8 wk of incubation, hyphal tips produced numerous asci, each containing one to eight ascospores that emerged out of an ascus following the breakdown of the ascus wall. These observations broaden our knowledge of early events during sexual reproduction and suggest that hyphae from the conidium-producing strain may be involved in the early stages of sexual reproduction in A. flavus. When combined with omics data, these findings could be useful in further exploration of the molecular and biochemical mechanisms underlying sexual reproduction in A. flavus.

The maintenance of cell shape requires finely tuned and robust vesicle trafficking in order to provide sufficient plasma membrane materials. The hyphal cells of filamentous fungi are an extreme example of cell shape maintenance due to their ability to grow rapidly and respond to the environment while keeping a relatively consistent shape. We have previously shown that two phospholipid flippases, which regulate the asymmetry of specific phospholipids within the plasma membrane, are important for hyphal growth in Aspergillus nidulans. Here, we examine the rest of the phospholipid flippases encoded by A. nidulans by obtaining single and double deletions of all four family members, dnfA, dnfB, dnfC, and dnfD. We find that deleting dnfC does not impart a noticeable phenotype, by itself or with other deletions, but that dnfD, the homolog of the essential yeast gene neo1, is important for conidiation. dnfD deletion mutants form misshapen conidiophore vesicles that are defective in metulae formation. We localize DnfD to late Golgi equivalents, where it appears just before dissociation of this organelle. We propose that DnfD functions in a trafficking process that is specifically required for the morphological changes that take place during conidiation.

Characteristics of hyphal structure and growth can provide insights into the mechanisms of polarized growth and support investigations of fungal phylogeny. To assist with the resolution of evolutionary relationships of the zygomycetes, the authors used comparative bioimaging methods (light [LM] and transmission electron [TEM] microscopy) to describe selected subcellular characters of hyphal tips of Conidiobolus coronatus. Growing hyphae of C. coronatus contain Spitzenkörper (Spk). Spk are most commonly present in hyphae of Dikarya (Ascomycota and Basidiomycota) and are rarely reported in zygomycete hyphae, which possess an apical vesicle crescent (AVC). Such findings raise questions regarding the evolution of the Spk and its relationship with the AVC. Descriptions of additional subcellular characters (e.g., mitotic-phase spindle pole bodies, cytoplasmic behavior, organelle structure) are also presented.

We have examined the hyphal tip structure in four zygomycetous fungi: Mortierella verticillata (Mortierellales), Coemansia reversa (Kickxellales), Mucor indicus and Gilbertella persicaria (Mucorales) using both light and transmission electron microscopy. We have used cryofixation and freeze-substitution methods to preserve fungal hyphae for transmission electron microscopy, which yielded improved preservation of ultrastructural details. Our research has confirmed studies that described the accumulation of secretory vesicles as a crescent at the hyphal apex (i.e. the apical vesicle crescent [AVC]) and provided a more detailed understanding of the vesicle populations. In addition, we have been able to observe the behavior of the AVC during hyphal growth in M. indicus and G. persicaria.

The Rho-related family of GTPases are pivotal regulators of morphogenetic processes in diverse eukaryotic organisms. In the filamentous fungi two related members of this family, Cdc42 and Rac1, perform particularly important roles in the establishment and maintenance of hyphal polarity. The activity of these GTPases is tightly controlled by two sets of regulators: guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Despite the importance of Cdc42 and Rac1 in polarized hyphal growth, the morphogenetic functions of their cognate GEFs and GAPs have not been widely characterized in filamentous fungi outside the Saccharomycotina. Here we present a functional analysis of the Aspergillus nidulans homologs of the yeast GEF Cdc24 and the yeast GAP Rga1. We show that Cdc24 is required for the establishment of hyphal polarity and localizes to hyphal tips. We also show that Rga1 is necessary for the suppression of branching in developing conidiophores. During asexual development Rga1 appears to act primarily via Cdc42 and in doing so serves as a critical determinant of conidiophore architecture. Our results provide new insight into the roles of Cdc42 during development in A nidulans.

The cytoskeleton provides structure, shape and movement to various cells. Microtubules (MTs) are tubular structures made of α and β-tubulin heterodimers organized in 13 protofilaments, forming a hollow cylinder. A vast group of MT-associated proteins determines the function, behavior and interaction of the MTs with other cellular components. Among these proteins, molecular motors such as the dynein-dynactin complex and kinesin superfamily play roles in MT organization and organelle transport. This article focuses on the MT cytoskeleton and associated molecular motors in the filamentous fungus Neurospora crassa In addition to reviewing current available information for this fungus and contrasting it with knowledge of other fungal species, we present new experimental results that support the role of dynein, dynactin and conventional kinesin in MT organization, dynamics and transport of subcellular structures (nuclei and secretory vesicles). In wild type hyphae of N. crassa, cytoplasmic MTs are arranged longitudinally along hyphae and display a helical curvature. They interlace with one another to form a network throughout the cytoplasm. N. crassa dynein and dynactin mutants have a scant and disorganized MT cytoskeleton, an erratic and reduced Spitzenkörper (Spk) and distorted hyphal morphology. In contrast, hyphae of mutants with defective conventional kinesin exhibit only minor disruptions in MT and Spk organization. Although nuclear positioning is affected in all mutants, the MT-associated motor proteins are not major contributors to nuclear movement during hyphal growth. Cytoplasmic bulk flow is the vehicle for nuclear displacement in growing hyphal regions of N. crassa Motors are involved in nuclei saltatory movements in both retrograde or anterograde direction. In the dynein and kinesin mutants, micro and macrovesicles can reach the Spk, although growth is slightly impaired and the Spk displays an erratic path. Hyphal growth requires MTs, and their associated motors are required for their organization and dynamics and Spk integrity.

Light and chemical components of the host root exudate can induce hyphal growth and branching of arbuscular mycorrhizal fungi. Compounds that induce the same morphogenetic or biochemical response as light are referred to as photo-mimetic compounds (PCs). This is the first report of a synergistic response by Gigaspora gigantea, an arbuscular mycorrhizal fungus, to blue light and naturally occurring photomimetic compounds isolated from the exudate of host roots. The blue light treatment and exposure to photomimetic compounds were effective whether applied sequentially or simultaneously. The number of hyphal branches induced by blue light and photomimetic compounds together was greater than the sum of the branches generated by each separate treatment, and the synergism was greatest at the higher levels or orders of branches. The fact that blue light and PCs, individually, triggered the same hyphal branching response and when given together, they produced a synergistic response, indicated the activation of a second messenger in the induced-branching process. Delaying the application of PCs, after the initial light exposure, showed the second messenger was stable up to 3 h.

A new in vitro experimental system was developed to study the morphogenesis of discrete regions of a single extraradical mycelium of the arbuscular mycorrhizal (AM) fungus Glomus intraradices, growing simultaneously in six different agar-based media. The media were (i) unamended water agar (WA), (ii) WA+PO(4)(3-) (PO(4)(3-)), (iii) WA+NO(3)(-) (NO(3)(-)), (iv) WA+NH(4)(+) (NH(4)(+)), (v) WA+NH(4)(+)+MES (NH(4)(+)+MES) and (vi) minimal medium (M, complete nutrients). Each medium was amended with the pH indicator bromocresol purple. The extraradical mycelium of the fungus showed between-treatment differences in morphogenesis, architecture, formation of branched absorbing structures (BAS) and sporulation. Extraradical hyphae that developed in WA or PO(4)(3-) compartments exhibited an economic development pattern, in which runner hyphae radially extended the external colony. Extraradical hyphal growth in the NO(3)(-) compartments was characterized by increased formation of runner hyphae, BAS and spores and an alkalinization of the medium. In the two NH(4)(+)-amended media (NH(4)(+), NH(4)(+)+MES), sporulation was suppressed and considerable morphological changes were noted. These results show the plasticity of G. intraradices that lets it efficiently exploit an heterogeneous substrate.

Models of wall loosening in fungi and other walled eukaryotes require the action of proteins able to reduce the degree of linkage between components of the wall. In the oomycete Achlya ambisexualis, such a role has been proposed for a suite of endoglucanases that are secreted during branching and during the measurable wall softening associated with osmotic stress. We report here the isolation and characterization of one of these isoenzymes. The enzyme has a molecular weight of 32 kDa, a pH optimum of 6.75, a pI of 4.5, and a temperature optimum of 35 C. It is partially inhibited by sulfhydryl-binding reagents and completely inhibited by the tryptophan-binding reagent NBS. The enzyme has an endohydrolytic mode of action with substrate specificity towards glucans that contain β-(1,4) linkages, either alone (carboxymethyl cellulose) or as mixed linkage (1,4-1,3)-β-glucans (e.g., Avena glucan). It does not, however, degrade amorphous insoluble (phosphoric acid swollen) cellulose. Most significantly, the enzyme can also hydrolyze linkages in an Achlya cell wall fraction previously shown to consist of a mixed-linkage (1,4-1,3)-β-glucan. This property is consistent with the long-standing hypothesis that the branching-related endoglucanases of oomycetes play a role in cell wall loosening.

The accident at the Chernobyl Atomic Energy Station resulted in radiation contamination of large tracts of land and particularly the reactor building itself. Sustained exposure of microfungi to radiation appears to have resulted in formerly unknown adaptive features, such as directed growth of fungi to sources of ionizing radiation. We evaluate here spore germination and subsequent emergent hyphal growth of microfungi in the presence of pure gamma or mixed beta and gamma radiation of fungi isolated from a range of long term background radiation levels. Conidiospore suspensions were exposed to collimated beams of radiation and percent spore germination and length of emergent hyphae were measured. All fungal species isolated from background radiation showed inhibition or no response in germination when irradiated. Isolates from sites with elevated radiation showed a stimulation in spore germination (69% mixed radiation and 46% for gamma irradiation). Most isolates from low background radiation sites showed a significant reduced or no response to exposure to either source of radiation, whereas the stimulatory effect of experimental exposure to radiation appeared to increase in magnitude as prior exposure to radiation increased. We propose that the enhanced spore germination and hyphal growth seen in the exposure trials is induced by prior long term exposure to radiation and these factors could be important in controlling the decomposition of radionuclide-bearing resources in the environment.

1057 Abstract: An important function of the ectomycor- rhizal sheath may be the storage and delayed trans- location of nutrients to the host. In order to deter- mine whether there are such phases, we quantified seasonal change in phosphorus content in sheaths of Cenococcum geophilum-Pinus strobus (white pine) ec- tomycorrhizae. Our field site was a mixed deciduous forest in northern, lower Michigan. White pine nee- dle and root growth, and density of C. geophilum scle- rotia were determined weekly. Ectomycorrhizae were collected biweekly, as were buried meshes that served to index hyphal growth. Phosphorus content of sheaths of ectomycorrhizae was measured by energy- dispersive X-ray analysis of planed, frozen material. From May to Jun 1994, sheath P content declined 0.9%/d. From Jul to Oct sheath P content was low and varied little; C. geophilum hyphae grew, but nee- dles and roots did not. The following spring from Mar to May 1995, sheath P content increased 1.2%/ d. We suggest, on the basis of synchrony of changes

The antifungal activity of broth filtrate of Bacillus atrophaeus isolate #35 was studied on Ascosphaera apis (Ascosphaeraceae—Ascomycota), a pathogen of honeybee larvae. Inhibition of hyphal growth...

:::. :'''. : :' ' ::::':::::::::: :'':'''-''':'''' ". ...-' '..''..'' -:: . ..:::::::::::::: ::::::::: .::::::::::::::: :::::::::: : :::-/ , ::::::::::::::::::::::::::::::. ..... / § \:::::: ~~~~~~ ~~~~.:::: :::.. .. . ..: ......:::::::::::::::::\: : : : - : : :: : : : : : : :: : - : : : / , \::::-:::::::-:: -:::::::::::::... ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~~~.... :::::::::::::::: :::::::. .........: ::: ::: ::: ::... .. .. ::::::::: -:::: >, )

Magainin 2 from the African clawed frog is a member of a host defense peptide family with broad-spectrum antimicrobial activity related to its ef- fects on membrane integrity. This bioactive peptide was examined for its effects on the ultrastructure of the plant pathogens Thielaviopsis basicola, Rhizocton- ia solani, Fusarium oxysporum, Verticillium dahliae, and Pythium ultimum. Magainin 2 at 0.05 jig/jiL in potato dextrose broth in microtiter plate wells com- pletely inhibited all hyphal growth observable with a dissecting scope at 40 X, except for P. ultimum. These organisms were subsequently grown on potato dex- trose agar and the leading edge of each colony treat- ed with 1 pxg of magainin 2 in 10uL. Hyphal plugs from the treated area were taken at 12 and 24 h after treatment for transmission electron microscopic ex- amination. Observations on the ultrastructure of ma- gainin 2-treated hyphae revealed changes in the size and shape of the mitochondria, degradation of the mitochondrial and cytoplasmic matrices, reduction in the number of ribosomes and vacuolization of cy- toplasm after 12 h of treatment. The structural deg- radation of the cells was more severe after 24 h treat- ment. Magainin 2 at the concentration tested had no significant effect on the growth of P ultimum, per- haps due to the disparate metabolic chemistry of the Oomycetes compared to the other organisms. The low molecular weight and bioactivity of magainin 2 makes it a candidate for additional investigation into its use in bioengineered host resistance.

Two different measures of branching, the Interode Length Unit (ILU) and the Hyphal Growth Unit (HGU), were applied to Rhizoctonia solani Kiihn, AG4. The ILU (the mean length in 4um of 10 internodes) was simple to perform and yielded consistent results. Results using this measure and the HGU (the mean length of mycelium per hyphal tip) as a measure of branching were compared. Growth of R. solani on media containing different carbon sources or on different concentrations of sucrose or glucose all yielded ILU of ca. 200-225 ,tm; thus branching was not affected by these treatments. Internode lengths were also constant over 29 hours for entire colonies of R. solani. The hyphae were more highly branched (mean ILU = 134 ,m) when the fungus was grown with cellophane interposed between the mycelium and the culture medium. Hyphae aligned in the grooves of scored Parafilm were more highly branched (ILU = ca. 165 ,m) than hyphae on smooth Parafilm (ILU = ca. 300 ,um); these branching frequencies were not affected by the presence of a nutrient medium. The Internode Length Unit is a simple, repeatable, and descriptive unit for measurement of lateral branch production in Rhizoctonia solani.

Incubation of spores of Gigaspora margarita on agar medium containing activated charcoal resulted in an increased rate of hyphal growth. Circular patterns simulated by hyphal growth from groups of ...

The range of morphological and physiological variation of ten isolates of Radiomyces embreei and four of R. spectabilis was examined. Scanning electron microscopy revealed that a single R. embreei isolate (1186) produced significantly smaller sporangiospores than nine other R. embreei isolates grown under four different culture conditions. The effect of temperature on hyphal growth and sporangiospore germination revealed that R. spectabilis isolates grew from 15 to 40 C, and two of the four isolates grew at 45 C, whereas nine of the 10 R. embreei isolates grew from 15 to 45 C, and five of these nine grew at 50 C. The unusual R. embreei isolate (1186) grew only at temperatures between 15 and 35 C. Sporangiospore germination occurred in eight of 10 isolates of R. embreei at temperatures between 25 and 50 C, and nine of the 10 isolates germinated between 25 and 45 C, whereas germination did not occur with isolate 1186 at 40 C or above. Sporangiospore germination of R. embreei isolates usually was 90% or greater in as few as 4 to 6 h, while only 1 to 5% of 1186 sporangiospores germinated in up to 24 h. Slow sporangiospore germination also was seen with R. spectabilis isolates, with two reaching only I to 3% germination in 16 to 24 h, and one strain attaining 31% germination in 10 h. Spore germination of R. spectabilis isolates and R. embreei 1186 continued over the period of up to one wk. The genus Radiomyces contains two species, Radiomyces spectabilis Embree (1959) and Radiomyces embreei Benjamin (1960). These fungi are Zygomycetes, members of the Mucorales, and were placed in the family Radiomycetaceae by Ellis and Hesseltine (1974). Both species have been isolated from dung of rodents and lizards collected from the southwestern United States and Mexico, and R. embreei also has been isolated from soil in Arizona (Ranzoni, 1968). Recently, an isolate of R. spectabilis was obtained from India. Both species of Radiomyces are homothallic, produce smooth-walled zygospores (FIG. 1), and sporangiophores bearing either multispored sporangioles (R. spectabilis, FIG. 2), or singlespored sporangioles (R. embreei, FIG. 3). The ultrastructure of R. embreei sporangioles was demonstrated by Dykstra (1974), who reported that each sporangiospore is surrounded by a distinct outer sporangiole wall which fragments at the time of germination. Embree (1964) determined the rate of asexual spore germination and hyphal growth in relation to temperature with the type isolates of the genus. He reported both species to be thermotolerant, and capable of growth at 45 C or greater. Since the ability to

Three soft rot fungi were studied with respect to the ultrastructure of fine hyphae in wood cell walls. Inoculated birch sections showing different stages of cell wall attack were prepared for TEM using four fixation procedures. Comparisons were made between fine pen? etration, T-branch and proboscis hyphae. Fine hyphae of Phialophora hoffmannii have a cell wall; this was absent at hyphal apices of Humicola alopallonella and Monodictys putredinis. An electron-opaque region or halo found in the wood cell wall around all fine hyphae may be a pre-cellulolytic system secreted by these hyphae. Fine soft rot hyphae have a simple organelle composition, but T-branch and proboscis hyphae have membranous organelles thought to be derived from mature parent hyphae. The characteristic features of wood decay by soft rot fungi have been described previously and the progressive invasion and decay of wood cell walls by Phialopho? ra hoffmannii (Van Beyma) Schol-Schwarz and P. fastigiata (Lagerb. & Melin) Conant have been monitored using time-lapse cinemicrography (Hale and Eaton, 1985). After the colonization of wood surfaces by soft rot hyphae, the process of decay is initiated by the penetration of fine hyphae through wood cell walls. The penetration process shows many similarities to that occurring with some blue stain fungi. The process of blue stain penetration was thought to be primarily by mechanical pressure (Liese and Schmid, 1961, 1964) although Liese (1970) sug? gested that the penetration was achieved by localized enzymatic action at the hyphal tip with mechanical pressure pushing it through the cell wall. Decay of wood by soft rot fungi occurs either through erosion of the wood cell wall by enzymes released from hyphae on the lumen surface of the wall or by the excavation of cavities around hyphae in the S2 region ofthe cell wall. The establishment of fine hyphae within wood cell walls prior to cavity formation is considered here. Cavities are initiated by fine hyphae which grow in the wall and are then oriented parallel to the cellulose microfibrils. Krapivina (1960) showed that this initial process may happen either by a change in the direction of growth of the penetration hyphae to form L-bending hyphae (Levi, 1965; Nilsson, 1974a, b) or by branching, with each branch growing in the opposite direction, a process Corbett (1965) termed "T-shaped branching." Corbett and Levy (1963) only ob? served T-branches in the second S2 cell wall layer but T-branches have since been observed in the first S2 layer (Levy and Stevens, 1966). After a period of apical extension within the cell wall, hyphal growth ceases and a cavity is created by enzymatic activity along the length of the hyphae. During the formation of the cavity, the diam of the hypha increases. The emergence of a fine hypha, termed the proboscis hypha, is followed by a linear rate of growth within the cell wall, cessation of apical extension, and the formation of a cavity around the hypha, which also increases in diam. A series or chain of cavities is produced by repeated emergence of proboscis hyphae at

As pointed out in an earlier note on a " dry-rot " disease of Opuintia 2 the spots tend to run together so that the infected side is completely destroyed if the spots are not too far apart, and are rather inumerous (FIG. 1). The parasite must grow rather rapidly at first. WVhen plants like the one shown in the figure were brought to the laboratory for study it was noticed that the spots did not seem to increase very much more in size even after several weeks. Sections extending from healthy through to the diseased tissue disclosed the reason for this. After a certain period following infection the fungus becomes completely cut off from the healthy tissue on all sides by a well marked callus tissue consisting of three or four layers of cells (FIG. 2, a). Wolf3 described a similar reaction of the host against the advance of the parasite lHeIdcrsonia Opuntiae, the cause of " sun scald " of the prickly-pear, the mlain difference being that in the scald disease the suberized layers are laid down parallel to the hypodermis, while in our disease the callus cuts in obliquely from the epidermis, beginning just beyond the limits of hyphal growth, extending down under the diseased tissue and up obliquely again on the opposite side of the spot. Because of the callus, the disease does not usually extend completely through the segment. This differentiates it from the Splhacrella disease described by Wolf and others, a