A molecular survey of ectomycorrhizal hyphae in a California Quercus–Pinus woodland

Ectomycorrhizal (ECM) hyphal communities have not been well characterized. Furthermore, there have been few studies where the ECM hyphal community is compared to fungi detected as sporocarps or ECM-colonized root tips. We investigated fungi present as hyphae in a well-studied California Quercus–Pinus woodland. Hyphal species present were compared to those found as sporocarps and ECM root tips at the same site. Hyphae were extracted from root-restrictive nylon mesh in-growth bags buried in the soil near mature Quercus douglasii, Quercus wislizeni, and Pinus sabiniana. Taxa were identified using PCR, cloning, and DNA sequencing of internal transcribed spacer and 28s rDNA. Among the 33 species detected, rhizomorph-forming ECM fungi dominated the hyphal community, especially species of Thelephoraceae and Boletales. Most fungi in soils near Quercus spp. and P. sabiniana were ECM basidiomycetes, but we detected two ECM ascomycetes and three non-mycorrhizal fungi. Many ECM species present as hyphae were also previously detected at this site as sporocarps (18%) or on ECM root tips (58%). However, the hyphal community was mostly dominated by different taxa than either the sporocarp or ECM root communities.     

Vacuolar Reticulum in Oomycete Hyphal Tips: An Additional Component of the CaRegulatory System?

Cultures ofAchlyasp.,Phytophthora cinnamomi, Saprolegnia diclina, S. ferax,andS. parasitica,treated with 6-carboxyfluorescein diacetate solution, accumulate 6-carboxyfluorescein in a reticulate system of fine tubules. The network shows longitudinal polarity within the hyphae, tubules being finest toward the hyphal tips. In more mature subapical regions the network is connected with large vacuoles that also accumulate 6-carboxyfluorescein. A morphologically similar system has also been identified in freeze-substituted hyphae ofS. ferax.The network is considered to be vacuolar, but differs from the tubular vacuole system of true fungi in that tubules are less motile, more frequently branched, and do not alternate with clusters of spherical vacuoles. The appearance of the network resembles patterns of calcium-sensitive dye staining and it is suggested that the vacuolar reticulum in the tip region of oomycete hyphae may act as a Ca²⁺sink. The tubular reticulum in oomycetes is very fragile and can be shown with 6-carboxyfluorescein in only those hyphal tips with a motility and organelle distribution characteristic of growing hyphae with normal morphology. Diverse abnormal hyphae show a range of other fluorochrome localizations. These include large irregular compartments filled with fluorochrome, and fluorescent cytoplasm with organelles and vacuoles standing out in negative contrast. These localizations in abnormal hyphae are correlated with other structural changes indicative of damage. Special care is required in experiments with oomycetes to avoid such artefacts of localization.     

A Wiskott–Aldrich syndrome protein is involved in endocytosis in Aspergillus nidulans

Endocytosis is vital for hyphal tip growth in filamentous fungi and is involved in the tip localization of various membrane proteins. To investigate the function of a Wiskott–Aldrich syndrome protein (WASP) in endocytosis of filamentous fungi, we identified a WASP ortholog-encoding gene, wspA, in Aspergillus nidulans and characterized it. The wspA product, WspA, localized to the tips of germ tubes during germination and actin rings in the subapical regions of mature hyphae. wspA is essential for the growth and functioned in the polarity establishment and maintenance during germination of conidia. We also investigated its function in endocytosis and revealed that endocytosis of SynA, a synaptobrevin ortholog that is known to be endocytosed at the subapical regions of hyphal tips in A. nidulans, did not occur when wspA expression was repressed. These results suggest that WspA plays roles in endocytosis at hyphal tips and polarity establishment during germination.     

Karyology and hyphal characters as taxonomic criteria in Ascomycetous black yeasts and related fungi

Mycelial development of seventy-three strains of black yeasts and related fungi were studied, and numbers of nuclei per hyphal cell were counted. Two main patterns were apparent in expanding hyphae, viz. (1) uninucleate expanding hyphal cells, septum formation strictly following mitosis, and (2) multinucleate, branched, aseptate hyphal tips, septa being formed in a later stage, leading to oligo- or uninucleate mature cells. Characteristic genera in the two groups areExophiala andAureobasidium, respectively. InZasmidium and in someRamichloridium species all mycelial cells are oligonucleate. The character is indicative for relationships at the family level in black yeasts.     

GROWTH AND DIFFERENTIATION OF NEUROSPORA HYPHAE

Zalokar , Marko . (Yale U., New Haven, Conn.) Growth and differentiation of Neurospora hyphae. Amer. Jour. Bot. 46(8): 602–610. Illus. 1959.—Neurospora hyphae grow only at their tips. New protoplasm is formed throughout the hyphae and transported to the tips by active cytoplasmic streaming. This enables the fungus to grow at a prodigious rate. The use of radioactive precursors demonstrated that the rate of protein and ribonucleic acid formation does not decrease substantially in hyphal regions distant from the growing point. Apical growth of hyphae results in a morphological differentiation into young regions filled with cytoplasm and dense with nuclei and old regions, vacuolated and laden with fat droplets. Cytochemical methods were used to study the biochemical differentiation of hyphae and attention was focused on the growing region. It was found that hyphal tips, i.e., the first 50–100 μ of the growing end of the hypha, differed substantially from the rest of the young hyphae. Hyphal tips were slightly richer in proteins and significantly richer in protein-bound sulfhydryl groups and ribonucleic acid. Glycogen was absent at the tips. Peroxidase increased at the tips, while cytochrome oxidase and succinic dehydrogenase decreased. Alkaline phosphatase exhibited strong activity in a short region behind the extreme tip. β-galactosidase, in a few cases, was more active at the tips than in the rest of the hyphae. The results of cytological and cytochemical investigations are discussed in connection with the problem of cellular differentiation.     

Dynamics of mitochondria during the <Emphasis Type="Italic">Neurospora crassa</Emphasis> tip growth

Tip growth of the mycelial fungus N. crassa vegetative hyphae is realized owing to the combined activities of tens of the cells and diverse intracellular structures, such as microvesicles, microtubules, microfilaments, mitochondria, etc. Using a vital mitochondrial probe Mitotracker Red (10 μM, 10 min) we have found that the same mitochondria can move hundreds of microns along the hyphae within several hours. Analysis of the mitochondria distribution along 100 μm of the tips in intact hyphae as well as in the isolated apical fragments has shown that the congregation of mitochondria in the growing tips can correlate with the rate of elongation. These data together with the earlier electrophysiological estimations of the membrane potential gradients along the hyphal tips suggest that the electrical gradients along the hyphal apical part can be involved in the regulation of the energy supply of the tip growth.     

Apical assemblies of intermediate filament‐like protein FilP are highly dynamic and affect polar growth determinant DivIVA in Streptomyces venezuelae

Streptomyces spp. grow as branching hyphae, building the cell wall in restricted zones at hyphal tips. The organization of this mode of polar growth involves three coiled‐coil proteins: DivIVA and Scy, which form apical protein complexes referred to as polarisomes; and the intermediate filament‐like protein FilP, which influences cell shape and interacts with both Scy and DivIVA. Here, we use live cell imaging of Streptomyces venezuelae to clarify the subcellular localization and dynamics of FilP and its effect on hyphal morphology. By monitoring a FilP‐mCherry fusion protein, we show that FilP accumulates in gradient‐like zones behind the hyphal tips. The apical gradient pattern of FilP localization is dependent on hyphal tip extension and immediately dissipates upon growth arrest. Fluorescence recovery after photobleaching experiments show that FilP gradients are dynamic and subject to subunit exchange during vegetative growth. Further, the localization of FilP at hyphal tips is not directly dependent on scy, even though the strongly perturbed morphology of most scy mutant hyphae is associated with mislocalization of FilP. Finally, we find that filP has an effect on the size and position of the foci of key polar growth determinant DivIVA. This effect likely contributes to the phenotype of filP mutants.     

The role of the motile tubular vacuole system in mycorrhizal fungi

Mycorrhizal fungi, to be effective for the plant, must be able to transfer mineral nutrient elements from sites of uptake at hyphal tips across various distances to the exchange region in the mycorrhiza. Vacuoles are likely to be important in this transport, since they contain elements of nutritional significance in abundance. In tip cells of hyphae of most fungi –- known to include three ectomycorrhizal basidiomycetes, an ericoid mycobiont, and two arbuscular mycorrhizal fungi –- the vacuoles form a motile tubular reticulum. The vacuoles are most active in hyphal tips, but non-motile vacuoles at a distance from the tip can be induced to become motile by environmental changes. Neither the tubular vacuolar reticulum nor its contents are properly preserved by conventional fixation and embedding. Vacuolar tubules are readily shown in vivo with fluorescent tracers, throughout the extramatrical mycelium and in outer hyphae of the sheath in eucalypt mycorrhizas synthesised with Pisolithus sp., but they have proved harder to label in field-collected ectomycorrhizas and ericoid mycorrhizas. Freeze-substitution does preserve the structure of vacuoles and vacuolar tubules, and careful anhydrous techniques allow them to be microanalysed, indicating high content of K and P in vacuoles of hyphal tips, and also in sheath and Hartig net of ectomycorrhizas. Vacuoles contain polyphosphate in diffuse, non-granular form. Polyphosphate is present right up to the tip region of hyphae as well as in sheath and Hartig net: thus important mineral nutrient elements are present at both ends of the long hyphal transport pathway. Exactly what happens in between, however, remains to be elucidated.     

Cytoplasmic vesicles in germinating spores ofGilbertella persicaria

Summary Germ tube apices ofGilbertella persicaria contain cytoplasmic vesicles, similar to the secretory vesicles found at the tips of vegetative hyphae. The vesicles are present at all stages of development, from the time of germ tube initiation to the establishment of branched hyphae. In contrast to the abundant vesicles at tips of established hyphae, the germ tubes have only a few apical vesicles in a layer next to the plasma membrane. When germinated spores are treated by washing and centrifuging prior to fixation, the cytoplasm is often disrupted near the apex, and the clusters of apical vesicles disappear. The findings indicate the delicate nature of hyphal tips and the necessity of avoiding prefixation stresses in order to preserve the apical apparatus of growing hyphae.     

Study on the hyphal responses of Aspergillus fumigatus

The hyphal responses of an A. fumigatus isolate to a trizolederivative-fluconazole (FCZ) were studied with a Bio-Cell Tracer system. The numerical data were recorded as the original growth rate (Pre-GR), the time needed for FCZ reaching to its target in hypha (τ_on), the growth rate under the FCZ effect (Exp-GR) and the growth rate after FCZ was removed (Post-GR). Based on above numerical data, the inhibitory rates in the exposure and post exposure periods were calculated as the Exp-I% and Post-I% values. It was found there were variable inhibitory rate values (I%) in individual hyphae corresponding to different FCZ concentrations. It was shown by correlation analysis of the numerical data that the Pre-GR values were negatively correlated with the τ_on values and positively correlated with both the Exp-I% and Post-I% values. Additionally, the τ_on values are negatively correlated with the Exp-I% and Post-I% values. Those results suggested that the hyphal growth rate and the susceptibility of the FCZ target be the important factors to determine the hyphal responses to the FCZ effect. Serial morphological alternations were captured while the hyphal growth curves were changing under the FCZ effects. Of the morphological data, the interesting alternations were visualized when the hyphae were affected by 16 μg/ml FCZ. As shifting of the hyphal growth curves, the hyphae were repeatedly seen as swollen tips and germination from the swollen sites. It is indicated that the hyphal tips are the most sensitive parts of this mycelia fungus to the FCZ affects. Additionally, because the hyphal regrowth was observed as germination from the swollen tips before FCZ was removed, an adaptation phenomenon could be proposed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Wall structure of the Neurospora hyphal apex: immunofluorescent localization of wall surface antigens.

Antisera have been raised in rabbits against three wall fractions from Neurospora crassa. Fractions were separated according to Mahadevan & Tatum (1965), i.e. fraction I, glucan-peptide-galactosamine complex; fraction III, laminarin-like glucan; and fraction IV, chitin. Distinct patterns of immunofluorescent staining were obtained using an indirect staining method. Hyphae stained with antiserum to fraction I showed maximum fluorescence in the apical and/or subapical regions: in both cases, fluorescence showed a sharp decrease with distance behing the subapical region. Hyphae stained with antiserum to fraction III showed faintly fluorescent tips with fluorescence increasing with distance from the tip. Hyphae stained with antiserum to fraction IV showed faint fluorescence, equivalent to levels of autofluorescence, except at the sites of hyphal fractures. Antisera were also raised against whole walls from 24 and 120 h cultures. Hyphae stained with antisera against whole walls which had previously been absorbed to remove antibodies to fractions I, III, and IV showed preferential staining of apices. The uncharacterized tip antigen(s) thus revealed was also demonstrated on immunodiffusion plates. This pattern of immunofluorescence was compared to the fluorescence of apices after staining with an optical brightener. Enzymic dissection procedures did not generally give reliable results with apices from 24 h cultures. Untreated apices appeared amorphous, while a drastic chemical treatment revealed randomly oriented microfibrils which were shown to be alpha-chitin. The apical hyphal walls were significantly thinner than those from more mature hyphal regions.     

Penetration of Hyphae of Sclerotinia sclerotiorum by Coniothyrium minitans Without the Formation of Appressoria

In a study using scanning electron microscopy (SEM), the mode of hyperparasitism of Coniothyrium minitans on its host Sclerotinia sclerotiorum was investigated. The SEM micrographs confirm previous reports, from light microscopic studies, that hyphal tips of C. minitans invade the host hypha by direct penetration, without developing appressoria, and that indentation of the host cell wall at the point of penetration is often evident. There is no functional distinction between amain branch and a side branch hypha of the hyperparasite and tips of either type of hyphae are capable of invading host hyphae by direct penetration.     

An F-actin-depleted zone is present at the hyphal tip of invasive hyphae of Neurospora crassa

The distribution of filamentous actin (F-actin) in invasive and noninvasive hyphae of the ascomycete Neurospora crassa was investigated. Eighty six percent of noninvasive hyphae had F-actin in the tip region compared to only 9% of invasive hyphae. The remaining 91% of the invasive hyphae had no obvious tip high concentration of F-actin staining; instead they had an F-actin-depleted zone in this region, although some F-actin, possibly associated with the Spitzenk?rper, remained at the tip. The size of the F-actin-depleted zone in invasive hyphae increased with an increase in agar concentration. The membrane stain FM 4-64 reveals a slightly larger accumulation of vesicles at the tips of invasive hyphae relative to noninvasive hyphae, although this difference is unlikely to be sufficient to account for the exclusion of F-actin from the depleted zone. Antibodies raised against the actin filament-severing protein cofilin from both yeast and human cells localize to the tips of invasive hyphae. The human cofilin antibody shows a more random distribution in noninvasive hyphae locating primarily at the hyphal periphery but with some diffuse cytoplasmic staining. This antibody also identifies a single band at 21 kDa in immunoblots of whole hyphal fractions. These data suggest that a protein with epitopic similarity to cofilin may function in F-actin dynamics that underlie invasive growth. The F-actin-depleted zone may play a role in the regulation of tip yielding to turgor pressure, thus increasing the protrusive force necessary for invasive growth.     

Cytology and ultrastructure of interactions between <Emphasis Type="Italic">Ustilago esculenta</Emphasis> and <Emphasis Type="Italic">Zizania latifolia</Emphasis>

Ustilago esculenta is a biotrophic smut fungus that parasitizes Zizania latifolia, an edible aquatic vegetable of the southern China region. Infection results in swelling of the upper parts of the Z. latifolia culm which are called jiaobai and have a unique flavor and delicacy and are popular among Chinese. The infection process of Z. latifolia by U. esculenta was investigated with light and electron microscopy. Distribution of hyphae was uneven in plants; hyphae were mainly present in the swollen upper parts (jiaobai), the nodal regions of mature culms and old rhizomes and buds or shoots. Hyphae were rare in the internodes of mature culms and were fewer in the internodes of old rhizomes. All new buds produced on the nodes of culms and rhizomes were infected by hyphae in November before and in March after overwintering. The hyphae grew into the buds from the parent nodes via intervascular tissues only or via parenchyma tissues and vascular bundles. Hyphae extended within and between the host cells and frequently formed hyphal aggregations or clusters, not only in the mature tissues but also in developing tissues. The typical interface between the fungal hyphal wall and invaginated host plasma membrane comprised a sheath. The sheath surrounding a hyphae comprised an outer electron-opaque matrix and an inner electron-dense layer. The electron-opaque matrix layers were thicker in jiaobai tissues, ranging from 0.28 to 0.85 μm. The electron-dense hyphal coatings were more conspicuous in the young buds or shoots and mature culms than in the jiaobai. The intercellular hyphae caused large cavity formation between the cells or rupture of host cell walls, for gaining entry into host cells. The broken host cell wall fused with the electron-opaque matrix of the hyphal sheath as an interactive interface. The teliospore wall and wall ornamentation development was the same in postmature jiaobai tissues with sporadic sori and in the huijiao (jiaobai tissues containing the massive sori), but a sheath enveloping the teliospore was more transparent in the process of teliospore development in the jiaobai than in the huijiao.     

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.     

甲霜灵对大豆疫霉野生菌株及突变菌株生长发育影响的细胞学研究

本文利用电子显微镜技术研究了内吸性杀菌剂甲霜灵(Metalaxyl)对大豆疫霉Phytophthoras ojae野生菌株和突变菌株的形态学及超微结构的影响。结果表明:不同浓度甲霜灵处理后可导致野生菌株和突变菌株发生一系列不同的变化。低浓度(1μg/mL)处理后,野生菌株在培养基上的生长即可受到抑制,菌丝呈现不规则的肿胀、过度分枝;菌丝细胞壁不规则加厚,菌丝细胞内液泡增加,脂肪粒累积,细胞器排列紊乱,原生质最终坏死。随浓度的升高,野生菌株立即停止生长,菌丝干瘪坏死。而突变菌株只在高浓度(10μg/mL)甲霜灵处理后顶端菌丝出现少量较小的分枝,菌丝细胞壁无增厚现象,但细胞内脂肪粒大量积累,明显高于敏感性菌株;突变菌株在高浓度甲霜灵压力下仍继续生长。     

Investigation of the infection process of macrophomina phaseolina on the surface of soybean roots using scanning electron microscopy

Sclerotial germination, hyphal growth pattern, and appressorial formation of the charcoal rot fungus, Macrophomina phaseolina (Tassi) Goid. on the surface of soybean roots, Glycine max (L.) Merrill was observed using scanning electron microscopy. Sclerotial germination is followed by a hyphal growth pattern which does not appear to be associated with any specific structural feature on the corrugated root surface. Appressoria are produced on the root surface at the tips of both primary hyphae and side branches within three days after inoculation.     

Demonstration of dihydro-orotate dehydrogenase in Neurospora crassa hyphae with a cytochemical procedure

Synopsis Dihydro-orotate dehydrogenase activity was demonstrated in the hyphae of the fungusNeurospora crassa by a cytochemical technique. The activity was slightly stronger in the hyphal tips thus demonstrating the more intense metabolic activity in these parts of the mycelium. Several control reactions showed that the staining reaction was specific.     

The cellular structure of the Hartig net: coenocytic and transfer cell-like organization

The organization of the Hartig net in the mature state was studied in mycorrhizas of Amanita muscaria (Pers. ex. Fries) Hook. Picea abies (L.) Karst. grown in vitro. The tips of the fan-like branched hyphae contain dense cytoplasm with a large number of mitochondria and rER frequently stretched in the direction of the hyphal growth, indicating that active transfer of solutes between host and fungus is localized here. Lack of septation of the hyphae and their intimate juxtaposition, leaving no space in between, result in a coenocytic, transfer cell-like structure of the Hartig net. The multinucleate status of the cells is shown. The advantage of this organization for nutrient exchange between host and fungus and for nutrient transport within the hyphal system is discussed.     

The SH3/PH domain protein AgBoi1/2 collaborates with the Rho-type GTPase AgRho3 to prevent nonpolar growth at hyphal tips of Ashbya gossypii

Unlike most other cells, hyphae of filamentous fungi permanently elongate and lack nonpolar growth phases. We identified AgBoi1/2p in the filamentous ascomycete Ashbya gossypii as a component required to prevent nonpolar growth at hyphal tips. Strains lacking AgBoi1/2p frequently show spherical enlargement at hyphal tips with concomitant depolarization of actin patches and loss of tip-located actin cables. These enlarged tips can repolarize and resume hyphal tip extension in the previous polarity axis. AgBoi1/2p permanently localizes to hyphal tips and transiently to sites of septation. Only the tip localization is important for sustained elongation of hyphae. In a yeast two-hybrid experiment, we identified the Rho-type GTPase AgRho3p as an interactor of AgBoi1/2p. AgRho3p is also required to prevent nonpolar growth at hyphal tips, and strains deleted for both AgBOI1/2 and AgRHO3 phenocopied the respective single-deletion strains, demonstrating that AgBoi1/2p and AgRho3p function in a common pathway. Monitoring the polarisome of growing hyphae using AgSpa2p fused to the green fluorescent protein as a marker, we found that polarisome disassembly precedes the onset of nonpolar growth in strains lacking AgBoi1/2p or AgRho3p. AgRho3p locked in its GTP-bound form interacts with the Rho-binding domain of the polarisome-associated formin AgBni1p, implying that AgRho3p has the capacity to directly activate formin-driven actin cable nucleation. We conclude that AgBoi1/2p and AgRho3p support polarisome-mediated actin cable formation at hyphal tips, thereby ensuring permanent polar tip growth.