Effect of the Antibiotics Radicicolin and Griseofulvin on the Fine Structure of Fungi

The cell walls of fungal hyphae laid down in the presence ofradicicolin and griseofulvin are thicker and stronger than innormal hyphae. In Aspergillus niger radicicolin and griseofulvininduced the formation of swollen and misshapen cells that differedfrom normal cells only in size and thickness of the cell wall.Electron micrographs revealed no abnormalities in orientationor thickness of the structural microfibrils in the walls ofthe swollen and misshapen cells. Hyphal septa were similar instructural detail to the rest of the cell wall and consequentlymay become thickened and malformed in the presence of theseantibiotics. The results support the suggestion that radicicolinand griseofulvin act, in the region of the cell wall boundedon the inner surface by the plasma membrane, to disrupt thenormal process of hyphal extension at the cell tip. The depositionof additional cell wall material on swollen hyphae is seen asa response on the part of the fungus to maintain the integrityof these wider malformed cells.     

Enviromentally Induced Changes in Clamp Connection Incidence in Hyphal Branching Systems of Coprinus disseminatus

The incidence of clamp connections among surface hyphae at thecolony margin of apparent dikaryons from four stocks of Coprinusdisseminatus has been investigated under several environmentalconditions. On 2 per cent malt agar, clamp connections are formed at allnodes of the main leader hyphae, but they are absent from theearlier-formed nodes of primary branch hyphae. Most primarybranches have begun to form clamp connections by their fifthcell division, and continue to do so subsequently. Onset ofclamp connection formation in primary branches is delayed whenthe concentration of malt in the medium is reduced. The occurrenceof clamp connections on main leader hyphae is reduced or preventedwhen nutrient supply in the medium is reduced, or when the mediumis ‘pre-staled’ by previous growth of C. disseminatus.Clamp-free main hyphal tips revert to formation of clamp connectionswhen the intact hyphal system is transferred to more favourableenvironmental conditions. Presence of clamp connections on bothmain and branch hyphae is associated with high hyphal diameter,and in some instances, also with high hyphal extension rate. The significance of these observations to mechanisms of growthof hyphal branching systems and their relevance to other speciesthat form clamp connections intermittently, are discussed.     

The In Vitro Action of Griseofulvin against Pathogenic Fungi of Plants

The action of griseofulvin in vitro against forty economicallyimportant fungal pathogens of plants has been examined. Thepathogens were assigned to one of four groups, according tothe severity of the morphological symptoms observed. It wasconcluded that griseofulvin has considerable potential valuefor the protection of plants against a wide range of fungaldiseases.     

Standardization of hyphal growth inhibition rate as a means of evaluating Microsporum spp. in vitro susceptibility to terbinafine, griseofulvin, and ciclopiroxolamine

Reference methods for antifungal susceptibility tests recommend the use of conidia as inoculum. However, some isolates produce few conidia, while the invasive form of filamentous fungi in general is hyphae making susceptibility tests infeaseble. These facts suggest that other than conidia broth dilution method is required for susceptibility tests. The aim of this study was to clarify if the hyphal growth inhibition rate could be used as a method of determining the antifungal susceptibility of genus Microsporum. For this reason, a method which traces hyphal tips automatically and measures their growth rate was standardized for Microsporum spp. Control growth curves and test growth curves obtained by real-time observation of the hyphae groups responses to different concentrations of terbinafine, griseofulvin, and ciclopiroxolamine were used to compare with minimum inhibitory concentrations (MICs) obtained by conidia broth microdilution method. A visible reduction in the growth inhibition rate was observed when hyphal activity was evaluated using the third or fourth serial two-fold dilution below the MIC determined by broth microdilution for terbinafine and ciclopiroxolamine. For griseofulvin, this reduction occurred after the fifth dilution below the MIC. This study highlights the importance of the inoculum type used to determine the in vitro susceptibility of Microsporum strains. We conclude that measurement of hyphal growth inhibition, despite being time consuming, could be a suitable method for evaluating antifungal susceptibility, particularly for fungi as Microsporum spp. that produce a small (or not at all) number of conidia.     

Ultrastructure of Wall Swellings of Germinating Sporangia of Phytophthora palmivora (Butl.) Butl.

Germ tubes of directly germinating sporangia of P. palmivoraincubated in yeast extract solution at 30 ?C usually developedinto prominent swellings from which hyphae later emerged. Thegerm tubes arose as an extension of a new germination wall formedinternal to the sporangial wall prior to germination. The germtube swellings contained typical hyphal organelles. The germtube swelling possessed a thicker wall than both hyphae growingout of it and germ tubes that did not form swellings.     

Internal Changes in Hyphae of Rhizopus sexualis (Smith) Callen and Mucor hiemalis Wehm. associated with Zygospore Formation

The distribution of nucleic acids, nuclei, mitochondria, andreserve foods in vegetative hyphae, zygophores, and developingzygospores of Rhizopus sexualis and Mucor hiemalis were examinedby differential staining. The extreme tips and growing zones of vegetative hyphae containeda high concentration of RNA and numerous mitochondria. Nucleiwere not present at the extreme tip but were numerous just behindit. In older parts of the hyphae the concentration of RNA waslow and both nuclei and mitochondria were fewer than in thezone of elongation. Glycogen and lipids were present in all parts of the livinghyphae except the extreme tips and were more highly concentratedin the older parts of the hyphae. Young zygophores showed a much lower RNA/DNA ratio than thatfound in the vegetative hyphal tips. Transfer of colonies from20? C to temperatures of less than 10? C, which is known toprevent zygospore initiation, caused some but not all recognizablezygophores of R. sexualis, but not those of M. hiemalis, torevert to the RNA/DNA ratio characteristic of vegetative hyphae.Some zygophores of Rhizopus and most of those of Mucor developedinto sporangiophores at low temperature, retaining the relativelylow RNA/DNA ratio throughout development. It is suggested thata reduction in the RNA/DNA ratio is an early step in the changefrom the vegetative state to the reproductive one. At firstthis step is reversible, but soon becomes irreversible by anadditional step, the nature of which is unknown. For some timeafter this the reproductive hyphae are capable of either producingasexual sporangia or of conjugating to produce zygospores. Onceconjugation has taken place development either ceases or continuesuntil the spore is fully mature, but it cannot under any circumstancesthen be reversed. The development and maturation of the zygospore involves a greatincrease in number of both nuclei and mitochondria and in theconcentration of glycogen and lipids.     

Scanning Electron Microscopy of the Infection of Beans by Rhizoctonia solani Propagules

Germination, hyphal spread, and formation of infection cushionsand lesions on bean hypocotyls by infective large propagulesof Rhizoctonia solani were studied with the scanning electronmicroscope. The surface of old, but not of young, hyphae appearedtypically wrinkled and shrunken. Infection cushions appearedas a network of several layers of crossed anastomosing hyphae.These features could not be observed under the light microscope.No differences in the surfaces of infective and non-infectivesmall propagules could be observed. Non-infective propagulesgerminating on the hypocotyls gave rise to spreading hyphaebut did not develop infection cushions.     

The Development and Behaviour of Mycelial Strands in Merulius lacrymans (Wulf.) Fr.: II. Hyphal Behaviour during Strand Formation

The system of hyphal branching by Merulius lacrymans was observedin mycelium which had grown from a wood food-base on to glassslides during incubation in sterile moist chambers. A hierarchyof branches and sub-branches arose from the region of clampconnexions, or nodes, of relatively wide main hyphae. Therewas evidence that the sequence of branches occurring at nodesin basipetal succession represented the time sequence of branchdevelopment at any one node. Later-formed branches at any nodewere smaller than earlier branches, but such earlier branchesusually became smaller towards the tip as growth continued.Mycelial strands were built up by growth and branching of thigmo-tropicallysensitive ‘tendril’ hyphae in association with thewide main hyphae. Tendril hyphae were characteristically narrow,thin-walled hyphae arising both as later-formed branches fromthe nodes of the main hyphae and as the narrowed tips of earlierbranches. Although this branching behaviour could be seen amongstaerial hyphae growing over agar media, hyphae growing in contactwith or within the agar behaved differently and did not formstrands.     

Growth of Hyphal Branching Systems in Coprinus disseminatus

Apical portions of hyphal branching systems of Coprinus disseminatuswere observed at the margins of colonies extending at a constantrate under one set of environmental conditions. Growth was consistentlymonopodial, the main hyphae continuing to extend in the colonymargin at a constant rate. Primary branch initiation occurredat regular intervals, closely associated with cell divisionof the parent hypha, and initiation of secondary branches tookplace in the older parts of the system. Extension rates of main,primary branch, and secondary branch hyphae were distinct, beingin the proportions 100: 66: 18. In the majority of systems successivebranches of the same category closely resembled one anotherand changes in extension rate of primary branches were smallfrom soon after initiation up to a length of 750µ. Ina minority of systems branch behaviour was less regular, somebranches increasing in extension rate whilst others decreased.Changes in older parts of the systems were not followed. young primary branch hyphae were consistently narrower thanmain hyphae. Although extension rate was correlated with hyphaldiameter for main and for branch hyphae separately, the extensionrate of equally wide hyphae was significantly higher for mainas compared with branch hyphae. Furthermore the wider, faster-extendingbranch hyphse temded tp arise from the wider, faster-extendingmain hyphae. The significance of these findings is discussed.     

Some Observations on the Water-Relations of the Hyphae of Neurospora crassa

The morphology of the colony and of individual cells of thehyphae of Neurospora crassa are described. The behaviour ofthe hyphal apices in solutions of differing molarity is describedand this behaviour is related to the behaviour of Fusarium oxysporum.Measurements of the water potential of the apical cells aremade and it is shown that this corresponds to the balancing-pointin this fungus and in F. oxysporum. Measurements are then madeof the water potential of cells at various points along thelength of the hyphae, and of the osmotic potential of thesecells, and turgor pressure is calculated. The lowest turgorpressure of 12.4 atmospheres is shown by the apical cells andthe highest of 17.5 atmospheres by the basal cells. The validity of the methods used is discussed.     

Contact‐induced apical asymmetry drives the thigmotropic responses of Candida albicans hyphae

Filamentous hyphae of the human pathogen, Candida albicans, invade mucosal layers and medical silicones. In vitro, hyphal tips reorient thigmotropically on contact with small obstacles. It is not known how surface topography is sensed but hyphae lacking the cortical marker, Rsr1/Bud1, are unresponsive. We show that, on surfaces, the morphology of hyphal tips and the position of internal polarity protein complexes are asymmetrically skewed towards the substratum and biased towards the softer of two surfaces. In nano‐fabricated chambers, the Spitzenkörper (Spk) responded to touch by translocating across the apex towards the point of contact, where its stable maintenance correlated with contour‐following growth. In the rsr1Δ mutant, the position of the Spk meandered and these responses were attenuated. Perpendicular collision caused lateral Spk oscillation within the tip until after establishment of a new growth axis, suggesting Spk position does not predict the direction of growth in C. albicans. Acute tip reorientation occurred only in cells where forward growth was countered by hyphal friction sufficient to generate a tip force of ～ 8.7 μN (1.2 MPa), more than that required to penetrate host cell membranes. These findings suggest mechanisms through which the organization of hyphal tip growth in C. albicans facilitates the probing, penetration and invasion of host tissue.     

ELECTRON MICROSCOPY OF CULTURED SPHERULES OF COCCIDIOIDES IMMITIS

Spherules of C. immitis have been grown in vitro in modified Roessler's medium under CO₂ tension and continuous cultures now maintained for over 18 months. Transformation of hyphae and development of the spherule form have been studied by thin section electron microscopy. Cells of organisms in the hyphal stage have thin (ca. 50 mµ), apparently structureless walls and a cytoplasmic membrane. Many nuclei, elongated mitochrondria with both transverse and longitudinal cristae, and lipid particles are present. The hyphal wall thickens and the cell transforms into spherules. A large central accumulation of electrontransparent polysaccharide appears in the spherule. The peripheral cytoplasm contains nuclei, each enclosed in a double-layered membrane, mitochondria, and small dense particles. Prior to cleavage the polysaccharide droplets are lost, while mitochrondria become small and spherical. Endospores are formed and liberated when the spherule wall breaks. These begin to grow and repeat the cleavage cycle.     

Nuclear and Non-nuclear Factors influencing Clamp Connection Formation in Coprinus disseminatus

Matings between sister single spore lines of Coprinus disseminatusshowed a cryptic tetrapolar pattern. The two groups of matingsthat resulted in formation of mycelia with clamp connections(apparent dikaryons) differed in rate of nuclear penetrationduring mating. In one group penetration occurred at rates comparablewith nuclear migration in other species and in the other groupit was often extensive but at a rate similar to, or less than,the dikaryon growth-rate. No differences were detected betweenthese two groups in stability, colony extension rate, frequencyof clamp connections, proportions of true clamp connectionsand pseudoclamps, or number of nuclei per hyphal tip cell. Cytological studies and the isolation of hyphal tips showedthat both groups of apparent dikaryons were heterokaryotic di-or trikaryons. The di- and trikaryotic conditions co-existedin the same mycelium, but adjacent cells of individual branchingsystems usually contained equal numbers of nuclei. Within apparentdikaryons the number and kinds of nuclei per cell were similarin hyphae with clamp connections and those with simple septa.Treatments that prevented clamp connection formation did notalter the nuclear status of most of the hyphae. Irregularities in nuclear distribution were infrequent and mostwere associated with pseudoclamps. Forty per cent of nodes withprobable pseudoclamps yielded homokaryotic branches, which wereof either constituent mating type. There was some indicationthat irregularities in nuclear distribution could also occurduring divisions associated with simple septa.     

Reduced15N-nitrogen Transport Through Arbuscular Mycorrhizal Hyphae to Triticum aestivum L. Supplied with Ammonium vs. Nitrate Nutrition

This study compared the influence of or nutrition on the contribution of extraradical hyphae of the arbuscular mycorrhizal fungusGlomus mosseae(Nicol. and Gerd.) Gerd. and Trappe (BEG 107)to or uptake byTriticum aestivum L. ‘Hano’(summer wheat) with sufficient or insufficient N supply in semi-hydroponicculture. Roots and root-distant hyphae were spatially separatedin compartmentalized pots. Although -fed plants supplied with sufficient N had higher N concentrationsthan their -fed counterparts, this did not favourably affect colonization rates, hyphal lengthdensities or¹⁵N amounts transported via hyphae to the plants.Ammonium supply did not result in higher P or reduced carbohydrateconcentrations in the plants, so these factors could not explainthe reduced hyphal lengths. It was concluded that the effectof supply on hyphal length may be related to the reduced root growth and/or a direct effectof on hyphal growth. Plant N deficiency reduced the percentage root length colonized, hyphallength, total¹⁵N uptake by hyphae and dry weight of both - and -fed mycorrhizal plants. This was more obvious for -fed plants because plant biomass and hyphal lengthsof -fed plants were relatively low in mycorrhizal plants irrespective of the N concentrationsupplied. Copyright 2001 Annals of Botany Company Ammonium, arbuscular mycorrhiza, Glomus mosseae, nitrate, nitrogen uptake,¹⁵N studies, Triticum aestivum, wheat     

Live-cell imaging of tubulin in the filamentous fungus Magnaporthe grisea treated with anti-microtubule and anti-microfilament agents

Summary. Microtubule dynamics were examined in live cells of the fungal plant pathogen Magnaporthe grisea transformed for constitutive expression of a fusion protein containing enhanced yellow-fluorescent protein and a Neurospora crassa benomyl-resistant allele of β-tubulin. Transformants retained their ability to differentiate appressoria and cause disease but remained sensitive to benomyl. Linear microtubule arrays and low-level cytoplasmic fluorescence were observed in vegetative hyphae, conidia, germ tubes, and developing appressoria. Fluorescence within nuclei was conspicuously absent during interphase but increased rapidly at the onset of mitosis. Treatment with either benomyl or griseofulvin resulted in the appearance of prominent brightly fluorescent aggregates, including a large aggregate near the apex, with the concomitant disappearance of most cytoplasmic microtubules. Electron microscope imaging of treated cells indicated that the aggregates lacked any obvious profiles of intact microtubules. During these treatments, hyphal tip cells continued to elongate in a nonlinear and aerial fashion at a much slower rate than untreated cells. With subsequent removal of griseofulvin, distal aggregates disappeared rapidly but the apical aggregates persisted longer. Treatment with latrunculin A caused hyphal tip swelling without apparent effect on linear microtubule arrays. Simultaneous treatment with griseofulvin and latrunculin A resulted in depolymerization of microtubules and a cessation of growth, but near-apical fluorescent aggregates were not observed. Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s00709-004-0081-3 Correspondence and reprints: Department of Biological Sciences, University of Delaware, Newark, DE 19716, U.S.A. Present address: Paradigm Genetics Inc., Research Triangle Park, North Carolina, U.S.A.     

A serological investigation of hyphal growth in Fusarium culmorum

Summary Apical growth of hyphae of Fusarium culmorum was demonstrated using an immunofluorescent labelling technique. An antiserum prepared against hyphal tips contained a series of antibodies, detected by immunodiffusion, not present in antisera against mature hyphae or conidia. Absorption of the tip antiserum with hyphae allowed a specific immunofluorescence reaction with hyphal tips only. The antiserum against mature hyphae gave non-fluorescent tips to the hyphae.     

Effects of latrunculin B on the actin cytoskeleton and hyphal growth in Phytophthora infestans

The actin cytoskeleton is conserved in all eukaryotes, but its functions vary among different organisms. In oomycetes, the function of the actin cytoskeleton has received relatively little attention. We have performed a bioinformatics study and show that oomycete actin genes fall within a distinct clade that is divergent from plant, fungal and vertebrate actin genes. To obtain a better understanding of the functions of the actin cytoskeleton in hyphal growth of oomycetes, we studied the actin organization in Phytophthora infestans hyphae and the consequences of treatment with the actin depolymerising drug latrunculin B (latB). This revealed that latB treatment causes a concentration dependent inhibition of colony expansion and aberrant hyphal growth. The most obvious aberrations observed upon treatment with 0.1 μM latB were increased hyphal branching and irregular tube diameters whereas at higher concentrations latB (0.5 and 1 μM) tips of expanding hyphae changed into balloon-like shapes. This aberrant growth correlated with changes in the organization of the actin cytoskeleton. In untreated hyphae, staining with fluorescently tagged phalloidin revealed two populations of actin filaments: long, axially oriented actin filament cables and cortical actin filament plaques. Two hyphal subtypes were recognized, one containing only plaques and the other containing both cables and plaques. In the latter, some hyphae had an apical zone without actin filament plaques. Upon latB treatment, the proportion of hyphae without actin filament cables increased and there were more hyphae with a short apical zone without actin filament plaques. In general, actin filament plaques were more resilient against actin depolymerisation than actin filament cables. Besides disturbing hyphal growth and actin organization, actin depolymerisation also affected the positioning of nuclei. In the presence of latB, the distance between nuclei and the hyphal tip decreased, suggesting that the actin cytoskeleton plays a role in preventing the movement of nuclei towards the hyphal tip.     

Anastomosis Formation and Nuclear and Protoplasmic Exchange in Arbuscular Mycorrhizal Fungi

We observed anastomosis between hyphae originating from the same spore and from different spores of the same isolate of the arbuscular mycorrhizal fungi Glomus mosseae, Glomus caledonium, and Glomus intraradices. The percentage of contacts leading to anastomosis ranged from 35 to 69% in hyphae from the same germling and from 34 to 90% in hyphae from different germlings. The number of anastomoses ranged from 0.6 to 1.3 per cm (length) of hyphae in mycelia originating from the same spore. No anastomoses were observed between hyphae from the same or different germlings of Gigaspora rosea and Scutellospora castanea; no interspecific or intergeneric hyphal fusions were observed. We monitored anastomosis formation with time-lapse and video-enhanced light microscopy. We observed complete fusion of hyphal walls and the migration of a mass of particles in both directions within the hyphal bridges. In hyphal bridges of G. caledonium, light-opaque particles moved at the speed of 1.8 ± 0.06 μm/s. We observed nuclear migration between hyphae of the same germling and between hyphae belonging to different germlings of the same isolate of three Glomus species. Our work suggests that genetic exchange may occur through intermingling of nuclei during anastomosis formation and opens the way to studies of vegetative compatibility in natural populations of arbuscular mycorrhizal fungi.     

Topoisomerase I (TopA) Is Recruited to ParB Complexes and Is Required for Proper Chromosome Organization during Streptomyces coelicolor Sporulation

Streptomyces species are bacteria that resemble filamentous fungi in their hyphal mode of growth and sporulation. In Streptomyces coelicolor, the conversion of multigenomic aerial hyphae into chains of unigenomic spores requires synchronized septation accompanied by segregation of tens of chromosomes into prespore compartments. The chromosome segregation is dependent on ParB protein, which assembles into an array of nucleoprotein complexes in the aerial hyphae. Here, we report that nucleoprotein ParB complexes are bound in vitro and in vivo by topoisomerase I, TopA, which is the only topoisomerase I homolog found in S. coelicolor. TopA cannot be eliminated, and its depletion inhibits growth and blocks sporulation. Surprisingly, sporulation in the TopA-depleted strain could be partially restored by deletion of parB. Furthermore, the formation of regularly spaced ParB complexes, which is a prerequisite for proper chromosome segregation and septation during the development of aerial hyphae, has been found to depend on TopA. We hypothesize that TopA is recruited to ParB complexes during sporulation, and its activity is required to resolve segregating chromosomes.