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.     

Ultrastructure at the Host-Parasite Interface of Phytophthora capsici in Roots and Stems of Capsicum annuum

The infecting hyphae of Phytophthora capsici grew intercellularly in infected tissues of roots and stems of pepper (Capsicum annuum). The vascular tissues were not markedly disorganized even when heavily infected. Intercellularly growing hyphae penetrated the host cells by forming haustorium-like bodies. The consistent features of ultrastructural changes in infected tissues of pepper roots and stems were degeneration of cell organelles and dissolution of host cell walls. The cytoplasm detached from the cell wall aggregated abundantly around some haustorium-like bodies or the penetration sites of fungal hyphae. The host cell walls were palely stained, thinned and swollen, possibly being biochemically altered by the action of fungal macerating enzymes. Electron-dense, wall-like material was apposed on the outer wall of xylem vessel contacted by fungal hyphae. The infecting hyphae were also surrounded by granular, dark-staining cytoplasm. Characteristics of host cell responses to the invading P. capsici were the deposition of papilla-like material on host cell walls next to hyphae and the encasement of haustorium-like bodies with wall appositions.     

马铃薯对晚疫病水平抗性的组织细胞学观察

以马铃薯晚疫病水平抗性品种LBr-12和感病品种费乌瑞它为材料,采用普通光学和电子显微镜技术,系统研究了马铃薯与晚疫病菌(致病疫霉)互作的组织细胞学反应特征。观察结果显示:(1)接种后,水平抗性材料LBr-12出现过敏反应,病菌被限制在侵染点的几个细胞中,菌丝产生较少的分支和吸器。(2)感病品种费乌瑞它被侵染细胞呈蔓延趋势,菌丝产生较多的分支和吸器。(3)电镜观察发现,抗病品种上病菌的胞间菌丝、吸器母细胞、吸器在细胞和亚细胞水平均发生了一系列异常变化,包括原生质的电子致密度加深、液泡增多变大、菌丝细胞壁不规则增厚、细胞器排列紊乱及解体、吸器母细胞及吸器形态异常、病菌最终畸形坏死,同时发现抗病品种受病菌侵染时可迅速产生结构防卫反应,形成的细胞壁沉积物使胞壁极度增厚或细胞膜上产生乳突状结构。     

Morphological and ultrastructural studies on protoplast production and reversion of the higher basidiomyceteAgaricus bisporus

Protoplasts have been isolated from young vegetative mycelia ofAgaricus bisporus by an enzyme mixture of novozym and chitinase. Protoplasts were released through ruptures in the wall, initially at the apices, but also later from older parts of the hyphae, indicating that they may lack the cell wall. The process of regeneration of these protoplasts has been investigated in liquid medium in which the protoplasts produced short chains of convoluted cells that finally produced a hypha. Electron microscopy has shown that at the start of regeneration two different kinds of fibrils were produced at the external surface of the protoplasts. Later, the thickness of the cell wall increased, and there was a deposit of amorphous material giving rise to a complete new wall.     

Trifluoromethanesulfonic acid-based proteomic analysis of cell wall and secreted proteins of the ascomycetous fungi Neurospora crassa and Candida albicans

Cell wall proteins from purified Candida albicans and Neurospora crassa cell walls were released using trifluoromethanesulfonic acid (TFMS) which cleaves the cell wall glucan/chitin matrix and deglycosylates the proteins. The cell wall proteins were then characterized by SDS–PAGE and identified by proteomic analysis. The analyses for C. albicans identified 15 cell wall proteins and six secreted proteins. For N. crassa, the analyses identified 26 cell wall proteins and nine secreted proteins. Most of the C. albicans cell wall proteins are found in the cell walls of both yeast and hyphae cells, but some cell type-specific cell wall proteins were observed. The analyses showed that the pattern of cell wall proteins present in N. crassa vegetative hyphae and conidia (asexual spores) are quite different. Almost all of the cell wall proteins identified in N. crassa have close homologs in the sequenced fungal genomes, suggesting that these proteins have important conserved functions within the cell wall.     

Morphological and Physiological Changes on Rhizopus stolonifer by Effect of Chitosan,Oligochitosan or Essential Oils

Effects of chitosan, oligochitosan and the essential oils of clove and cinnamon were evaluated on hyphal morphology, cell wall thickness, minimum medium pH changes and respiration of Rhizopus stolonifer. Changes in hyphal morphology were observed due to chitosan or oligochitosan treatment in this fungus. Mycelial branching, abnormal shapes and swelling were showed on hyphae of R. stolonifer treated with chitosan, whereas the development of hyphae was markedly inhibited by the effect of oligochitosan. Clove and cinnamon oils caused few morphological changes in the hyphae of R. stolonifer. Cell wall thickness was increased approximately 2‐ to 3‐fold by effect of chitosan, oligochitosan and the essential oil of clove. R. stolonifer grown in minimum medium generated a decrease in the medium's pH. However, the addition of chitosan or oligochitosan caused increases in pH of medium culture. The highest pH value (5.4) was observed in the presence of chitosan. The respiration of R. stolonifer was stimulated at low concentrations of chitosan, oligochitosan or essential oils. Significant changes in morphology and physiology of this fungus were demonstrated by the effect of all evaluated compounds. The most important changes were induced on cells of R. stolonifer treated with chitosan and oligochitosan.     

Ultrastructure of Ascodichaena rugosa on beech bark

Ascodichaena rugosa Butin is a corkinhabiting fungus, found frequently on the bark of Fagus sylvatica L. The hyphae of the fungus are distributed solely in the phellem cells, stopping their growth in the last-formed cork cell layer. The cell to cell invasion is effected by penetration hyphae, causing no extensive dissolution of the cork wall. Electron microscopical observations revealed fine structural details of the fruit bodies and of the intracellular hyphae. Of special interest were the finger-like hyaline hyphae in the last-formed layer of cork cells, which are interpreted as haustoria on the basis of the fine structure both of hyphae and host cells. This situation is considered as reflecting a parasitic relationship of Ascodichaena to beech bark. The activity of the fungus led also to the increased production of cork cells, perhaps related to the nutrient supply of the fungus.     

Melanin Production by a Filamentous Soil Fungus in Response to Copper and Localization of Copper Sulfide by Sulfide-Silver Staining

Gaeumannomyces graminis var. graminis, a filamentous soil ascomycete, exhibited enhanced cell wall melanin accumulation when exposed to as little as 0.01 mM CuSO(inf4) in minimal broth culture. Because its synthesis was inhibited by tricyclazole, the melanin produced in response to copper was dihydroxynaphthalene melanin. An additional hyphal cell wall layer was visualized by electron microscopy when hyphae were grown in the presence of copper and fixed by cryotechniques. This electron-dense layer was between the outer cell wall and the inner chitin layer and doubled the total wall thickness. In copper-grown cells that were also treated with tricyclazole, this electron-dense layer was absent. Atomic absorption spectroscopy demonstrated that up to 3.5 mg of Cu per g of fungal mycelium was adsorbed or taken up by hyphae grown in 0.06 mM CuSO(inf4). A method for silver enhancement was developed to determine the cellular location of CuS. CuS was present in cell walls and septa of copper-grown hyphae. Electron microscopy of silver-stained cells suggested that CuS was associated with the melanin layer of cell walls.     

The infection process ofFusarium oxysporum in cotton root tips

Summary Conidia ofFusarium oxysporum f. sp.vasinfectum started to germinate on the roots of cotton (Gossypium barbadense L.) 6 h after inoculation and formed a compact mycelium covering the root surface. 18 h later, penetration hyphae branched off and infected the root. The number of penetration hyphae increased with the number of conidia used for inoculation. The optimal temperature for penetration was between 28 and 30 °C. The highest numbers of penetration hyphae were found in the meristematic zone, 40 percent less in the elongation and root hair zones, and none in the lateral root zone. The fine structure of the infection process was studied in protodermal cells of the meristematic zone and in rhizodermal cells of the elongation zone. The penetration hyphae were well preserved after freeze substitution and showed a Golgi equivalent consisting of three populations of smooth cisternae. Plant reactions were found already during fungal growth on the root surface. In the meristematic zone, a thickening of the plant cell wall due to an apposition of dark and lightly staining material below the hyphae occurred. This wall apposition increased in size around the hypha invading the plant cell and led to the formation of a prominent wall apposition with finger-like projections into the host cytoplasm. In the elongation zone, the deposits around the penetration hypha appeared less thick and the dark inclusions were less pronounced. High pressure freezing of infected cells revealed, thatF. oxysporum penetrates and grows within the host cells without inducing damages such as plasmolysis, cell degeneration or even host necrosis. We suggest thatF. oxysporum has an endophytic or biotrophic phase during colonization of the root tips.Abbreviation Ph penetration hyphae     

Application of fluorescent probes to study structural changes in Aspergillus fumigatus exposed to amphotericin B, itraconazole, and voriconazole

The broad objective of this study was to document patterns of structural changes following antifungal treatment, and to determine any relationship with minimum inhibitory concentration (MIC) of an antifungal. Three clinical isolates of Aspergillus fumigatus, with high, intermediate, and low amphotericin B (AB), itraconazole (IZ), and voriconazole (VZ) MICs were studied in 24-well plates with cover slips. The fluorescent probes used were Calcofluor White (cell wall), propidium iodide (nucleus), and MitoTracker Green FM (mitochondria). Fluorescent microscopy as early as 3-h after exposure revealed that AB treated hyphae had intact cell wall with deformed mitochondria and nuclei while IZ and VZ treated hyphae revealed no intact cell wall, and deformation of mitochondria and nuclei. At 48 h, AB treated cells revealed rupture of hyphae and disintegration of mitochondria, and nuclei, IZ treated hyphae were swollen with disintegration of mitochondria, and nuclei while VZ treated hyphae showed rupture and disintegration of mitochondria and nuclei. The structural changes for the three strains studied were similar in fluorescent microscopy as long as the incubation time and their respective MICs were used. Thus, AB, IZ, and VZ induced gross organelle defects in A. fumigatus nuclei, mitochondria, and cell wall, which were consistent with respective MICs of antifungals used.     

Chalara elegal-Infected Tobacco Roots: Ultrastructural Observations on in vitro Host-Fungus Relationships

Evidence, based on ultrastructural observations of stages involved in root infection oi Nicotiana tabacum cv. Xanthi n.c. in vitro by the black root rot fungus Chalara elegans, indicates that host cells from various layers react differently when challenged by the pathogenic fungus. All the host responses observed were associated with host cell wall modifications. Host reaction to fungal invasion occurring in the epidermal cells was limited to a disorganization of the cytoplasm. In the hypodermal cell layer, fibrillar cell wall outgrowths and wall thickenings were the earliest and the most obvious host reactions. In parenchymal cells, the host reacted by depositing papilla-like wall appositions directly adjacent to the infecting hyphae; with secondary infection of these cells, a densely staining material was laid down, mainly around the distal region of the infecting hyphae. In all these tissues, infection also led to disorganization of the host cytoplasm. Colonization of the endodermis did not lead to any rapid lethal modifications in either the host or the fungus, and a biotrophic-like state seemed to occur at this stage of the infection. No hyphal infection occurred in the central cylinder.     

Characterization of the Sclerotinia sclerotiorum cell wall proteome

We used a proteomic analysis to identify cell wall proteins released from Sclerotinia sclerotiorum hyphal and sclerotial cell walls via a trifluoromethanesulfonic acid (TFMS) digestion. Cell walls from hyphae grown in Vogel's glucose medium (a synthetic medium lacking plant materials), from hyphae grown in potato dextrose broth and from sclerotia produced on potato dextrose agar were used in the analysis. Under the conditions used, TFMS digests the glycosidic linkages in the cell walls to release intact cell wall proteins. The analysis identified 24 glycosylphosphatidylinositol (GPI)‐anchored cell wall proteins and 30 non‐GPI‐anchored cell wall proteins. We found that the cell walls contained an array of cell wall biosynthetic enzymes similar to those found in the cell walls of other fungi. When comparing the proteins in hyphal cell walls grown in potato dextrose broth with those in hyphal cell walls grown in the absence of plant material, it was found that a core group of cell wall biosynthetic proteins and some proteins associated with pathogenicity (secreted cellulases, pectin lyases, glucosidases and proteases) were expressed in both types of hyphae. The hyphae grown in potato dextrose broth contained a number of additional proteins (laccases, oxalate decarboxylase, peroxidase, polysaccharide deacetylase and several proteins unique to Sclerotinia and Botrytis) that might facilitate growth on a plant host. A comparison of the proteins in the sclerotial cell wall with the proteins in the hyphal cell wall demonstrated that sclerotia formation is not marked by a major shift in the composition of cell wall protein. We found that the S. sclerotiorum cell walls contained 11 cell wall proteins that were encoded only in Sclerotinia and Botrytis genomes.     

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.     

Identification of VdASP F2-interacting protein as a regulator of microsclerotial formation in Verticillium dahliae

Verticillium dahliae, a notorious phytopathogenic fungus, causes vascular wilt diseases in many plant species. The melanized microsclerotia enable V. dahliae to survive for years in soil and are crucial for its disease cycle. In a previous study, we characterized the secretory protein VdASP F2 from V. dahliae and found that VdASP F2 deletion significantly affected the formation of microsclerotia under adverse environmental conditions. In this study, we clarified that VdASP F2 is localized to the cell wall. However, the underlying mechanism of VdASP F2 in microsclerotial formation remains unclear. Transmembrane ion channel protein VdTRP was identified as a candidate protein that interacts with VdASP F2 using pull-down assays followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, and interaction of VdASP F2 and VdTRP was confirmed by bimolecular fluorescence complementary and coimmunoprecipitation assays. The deletion mutant was analysed to reveal that VdTRP is required for microsclerotial production, but it is not essential for stress resistance, carbon utilization and pathogenicity of V. dahliae. RNA-seq revealed some differentially expressed genes related to melanin synthesis and microsclerotial formation were significantly downregulated in the VdTRP deletion mutants. Taken together, these results indicate that VdASP F2 regulates the formation of melanized microsclerotia by interacting with VdTRP.     

Endophyte differentiation inCasuarina actinorhizae

Summary This is an ultrastructural study of development of infected cells in nitrogen fixing root nodules ofCasuarina spp. While several aspects of development are similar to those found in many other actinorhizae, unusual aspects of development of the host cell and differentiation of the endophyte inCasuarina are correlated with unusual changes in the wall of the infected cell. Instead of vesicles the endophyte forms atypical hyphae in mature infected cells. These unusual hyphal forms are termed intracellular hyphae. Intracellular hyphae are nonseptate hyphae which originate and terminate within the same host cell, and have a varying diameter and a multidirectional growth and branching pattern. A laminate surface layer previously undescribed on hyphae ofFrankia is a feature common to mostCasuarina endophytic hyphae and is probably similar chemically to the laminae comprising the multilamellate envelope of endophytic vesicles in other actinorhizae.This paper is Florida Agricultural Experiment Station Journal Series No. 7350.     

Electron microscope studies on Tomentella

Summary Tomentella bombycina andT. fuscoferruginosa were examined by routine techniques of light and electron microscopy. Special attention was paid to the ultrastructure and inter-relationships of the generative subicular hyphae and subhymenial hyphae. The cell wall consisted of three layers in the generative subicular hyphae and only one layer in the subhymenial hyphae; this corresponded to the inner layer of the first and originated from it. Cells with two nuclei, normal mitochondria, large vacuoles, sparse endoplasmic reticulum and lomasomes were observed. Sometimes the nuclear membrane had unusually large pores and larger open gaps extending over 1/4 of the circumference. Septa with central dolipore-type pores were present. Clamp connections were not frequent. Finally, certain cells of the subhymenial hyphae appeared full of globular material, presumably lipids.     

Differential cytoplasm-plasma membrane-cell wall adhesion patterns and their relationships to hyphal tip growth and organelle motility

Summary Plasmolysis of hyphae of the oomycetesSaprolegnia ferax andAchlya ambisexualis and the ascomyceteNeurospora crassa produced abundant cytoplasmic strands between the retracted cytoplasm and punctate adhesions of the plasma membrane to the cell wall. These strands formed throughout the length of mature hyphae and are the first demonstration of Hechtian strands in hyphae. In contrast to similar strands in various plant cells, the strands inSaprolegnia lacked endoplasmic reticulum but contained F-actin, suggesting similarity between their adhesion sites and focal contacts in animal cells. However, strand adhesion to the wall was insensitive to RGD-containing peptides, suggesting that the trans-membrane adhesion molecules differ from animal integrins. The pattern of plasma membrane-cell wall adhesion varied in different zones along hyphae, with broad, irregular connections in the extreme apex, uniform and continuous connection in a transition zone, and small, punctate adhesions in the mature subapical zone, suggesting differential functions in these different regions. The apical adhesions are important in tip growth, as diverse inhibitors induced concomitant changes in hyphal growth and the adhesions in the apical and transition zones. Plasmolysis also induced cytoplasmic migrations throughout hyphae. Such migrations were dominated by the central cytoplasm, and produced distorted organelles which spanned central and peripheral cytoplasm, thus supporting the idea that the adhesions in mature zones of hyphae anchor the peripheral cytoplasm and facilitate cytoplasmic and organelle migrations.Abbreviations OM organic medium - RP rhodamine phalloidin - DIC differential interference contrast - PIPES piperazine-N,N-bis-2-ethanosulphonic acid     

Ultrastructural characterisation of the host–pathogen interface in white blister-infected Arabidopsis leaves

In this study transmission electron microscopy (TEM) was used to examine details of the host–pathogen interface in Arabidopsis thaliana cotyledons infected by Albugo candida, causal agent of white blister. After successful entry through stomatal pores, the pathogen developed a substomatal vesicle and subsequently produced intercellular hyphae. TEM observations revealed that coenocytic intercellular hyphae ramified and spread intercellularly throughout the host tissue forming several haustoria in host mesophyll cells. Intracellular haustoria were spherical and 4.5 μm in diameter. Each haustorium was connected to intercellular hyphae by a narrow, slender haustorium neck. The cytoplasm of the haustorium included the organelles characteristic of the pathogen. No obvious response was observed in host cells following formation of haustoria. Most of the mesophyll cells contained normal haustoria and the host cytoplasm displayed a high degree of structural integrity. Absence of host cell wall alteration and cell death in penetrated host cells suggest that the pathogen exerts considerable control over basic cellular processes and in this respect, response to this biotrophic Oomycete differs considerably from responses to other pathogens such as necrotrophs. Modification of the host plasma membrane (PM) along the cell wall and around the haustoria, was detected by applying the periodic acid-chromic acid-phosphotungstic acid (PACP) staining technique. After staining with PACP, the host PM was found to be intensely electron dense where it was adjacent to the host cell wall and the distal region of the haustorial neck. By contrast, the extrahaustorial membrane, where the host PM surrounded the haustorium, was consistently very lightly stained.     

Infection of Onion by the White Rot Pathogen, Sclerotium cepivorum

Infection of onion tissue by Sclerotium cepivorum occurred from germ tubes penetrating between adjacent epidermal cell walls or directly, via penetration pegs produced from slightly swollen hyphal tips or from beneath dome shaped infection cushions. After passing through the cuticle, the infection peg enlarged to form an infection hypha within the primary cell wall. Extensive degradation of the epidermal cell wall occurred, often at a distance of 2–3 cells from the advancing hyphae. As infection advanced, hyphae spread rapidly from the epidermis to the cortex growing between and within dead/dying host cells. Extensive host cell death resulted in localized collapse of the tissue around infection points. Complete colonization of the internal tissues of the root and stem base occurred within 5–7 days of inoculation.     

木耳菌丝老化过程的微形态学研究

分别从显微和超微结构观察木耳菌种老化过程中菌丝细胞的形态变化。结果显示：接种后30d时,光镜下观察到菌丝结构均匀紧凑,细胞壁光滑;电镜下观察到细胞结构完整,内含物丰富,各种细胞器形态规整,没有老化现象。接种后60d时,光镜下菌丝部分肿胀,色泽加深;电镜下细胞壁疏松,线粒体和液泡肿大,细胞核不规则肿胀,核仁消失,脂肪滴和囊泡增多,并有少量电子致密度高的嗜锇性黑色颗粒状物质出现,表明菌丝开始老化。90d时,光镜下部分菌丝严重肿胀,且色泽更深;电镜下线粒体和液泡肿胀明显,部分细胞核破裂,脂肪滴、囊泡和电子致密度高的嗜锇性黑色颗粒状物质显著增多,细胞壁更加疏松。120d时,光镜下许多菌丝开始断裂,色泽进一步加深;电镜下细胞壁塌陷,膜系统也随之解体,线粒体等细胞器部分溶解消失。150d时,光镜下大部分菌丝完全断裂,并失去菌丝形态;电镜下细胞膜及其内含物已基本消失,只剩部分严重塌陷的细胞壁残骸。由此表明,木耳菌丝的老化是一个由个别向整体逐渐过渡的不可逆的过程。