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.     

The fine structure of developing microsclerotia of Verticillium dahliae Kleb.

Summary Electron micrographs of the early stages in microsclerotial development in v. dahliae Kleb. showed that hyphae became swollen and vacuolate and extruded melanizing particles into the interhyphal spaces of the microsclerotium. Peripheral microsclerotial cells were killed either by a process of autoparasitisation from adjacent hyphae or by autolysis. Variations in the thickness of the melanized material surrounding cells gave the superficial appearance of variations in cell wall thickness between individual cells though actual changes in cell wall thickness were not observed.     

内吸杀菌剂甲霜灵对向日葵霜霉菌发育的影响

利用电镜技术研究了内吸杀菌剂甲霜灵对向日葵霜霉菌在寄主向日葵上发育的影响。观察发现甲霜灵引起病菌发生一系列的变化。病菌胞间菌丝细胞内液泡、电子致密体增加,线粒体肿胀畸形;菌丝细胞壁不规则地加厚,部分极度加厚的细胞壁构成了假隔膜,并且在菌丝上还有不规则的突起形成。病菌吸器外间质明显不规则地变宽,其中充满大量电子致密度高的物质：部分吸器体不能正常扩张膨大,发育受阻而成畸形。向日葵霜霉菌的以上变化导致菌丝细胞和吸器的坏死,从而抑制了病菌的进一步发育。     

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。     

三唑酮对玉米弯孢病菌超微结构和细胞化学的影响

三唑酮(triadimenfon)属于麦角甾醇类生物合成抑制剂(ergosterol biosynthesis inhibitors．EBI),具有较广的抗真菌谱,明确其对玉米弯孢菌发育的影响可为该杀菌剂的田间应用提供理论依据。利用电镜技术和细胞化学技术观察的结果表明,玉米率孢菌经三唑酮处理后,菌丝生长明显受到抑制,表现为菌落生长速度减慢、菌丝分枝增多,且不观则地肿大和缢缩,出现许多瘤状突起,处理菌丝明显畸形。透射电镜观察结果表明,三唑酮可引起菌丝细胞壁不规则增厚,特别是菌丝顶端细胞壁增厚尤为明显:菌丝细胞隔膜发育受阴而表现畸形;菌丝细胞外有大量电子染色深的外渗物质。细胞化学标记定位结果表明,真菌细胞壁主要成分β-1,3-葡聚糖和几丁质的含量在药剂处理后发生很大变化,其标记密度明显低于未处理的对照菌丝,表明病菌细胞壁的结构和功能受到明显的不利影响。论文对弯孢菌受三唑酮影响后胞壁成份变化与其它真菌不同的原因进行了讨论。     

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.     

柿树炭疽菌侵染寄主的细胞学研究*

超微结构研究表明,柿树炭疽菌(Colletotrichum gloeosporioides)侵染后在寄主细胞中形成初生菌丝和次生菌丝,寄主细胞膜外沉积了一层厚的电子不透明物质,初生菌丝与具有沉积物的寄主原生质膜之间有一层界面基质(interfacial matrix)。当初生菌丝扩张并侵染相邻细胞时, 围绕着初生菌丝层的界面基质消失,具有沉积物的原生质膜被逐步降解。初生菌丝在穿透寄主细胞壁过程中形成一个漏斗状的菌丝锥,然后穿透寄主细胞壁并迅速膨大, 然后形成厚壁的初生菌丝。初生菌丝在寄主细胞壁中收缩狭窄处产生一个隔膜,隔膜两边菌丝中细胞质的电子密度明显不同,菌丝锥中有浓密的电子密度。死体营养的次生菌丝在死的细胞中繁殖和扩展,并产生分枝。次生菌丝可直接穿透较薄的寄主细胞壁,无缢缩或任何变形现象,菌丝顶端部分未见隔膜产生;在穿透较厚的细胞壁时,靠近顶端处产生隔膜,顶端细胞膨大,使寄主细胞壁撕裂。接种90h后分生孢子盘在枝条表面形成。柿树炭疽菌其侵染过程有两个阶段,即初生菌丝的活体营养阶段和次生菌丝的死体营养阶段。     

戊唑醇对赤霉病菌生长发育影响的细胞学和免疫细胞化学研究

本文采用电子显微镜和免疫细胞化学技术研究了三唑类杀菌剂戊唑醇 (Tebuconazole) 对小麦赤霉病菌Fusarium graminearum菌丝的形态结构、细胞壁成份和毒素产生的影响。结果表明,戊唑醇不但强烈抑制了培养基上菌丝的生长,而且可引起菌丝形态和结构的明显畸形。电镜观察发现,药剂处理后菌丝呈现不规则的肿胀、过度分枝;菌丝细胞壁不规则加厚,尤其是菌丝顶端部位加厚明显;菌丝形成的不完整隔膜增多,且隔膜壁不规则增厚;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。有时在坏死菌丝内可发现新的子菌丝,但子菌丝细胞壁不规则加厚、细胞质坏死、也呈不正常状态,并可再度形成新的菌丝。免疫细胞化学标记表明,药剂处理后菌丝细胞中毒素的标记密度明显低于对照菌丝,表明毒素的产生受到了抑制;而菌丝细胞壁的主要成份几丁质和-1,3-葡聚糖的标记密度明显高于对照处理,表明药剂处理可引起菌丝细胞壁中几丁质和-1,3-葡聚糖的过度累积。     

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.     

The Effects of Griseofulvin on Certain Phytopathogenic Fungi

In vitro and in vivo investigations have been carried out intothe reaction of actively growing hyphae of Botrytis spp. onencountering an environment containing griseofulvin. In vitro, the reactions noted were slowing of the growth rate,stimulation to branching, abnormal appearance of hyphal protoplasm,and loss of rigidity of the hyphal membrane in the region ofthe tip. The antibiotic is not translocated within the mycelium. In vivo, Botrytis tulipae was incapable of penetrating cellmembranes in the stem of tulips watered with griseofulvin. Theantibiotic was shown to delay or to prohibit the penetrationof onion epithelium by B. allii, depending on concentration.The characteristic curling and stunting of hyphae caused bygriseofulvin have both been observed to occur in plant cells.     

Wall Structure and spore Germination in Fusarium culmorum

The conidial and germ-tube walls of Fusarium culmorum (W. G.Smith) Sacc. have been examined by various chemical and electron-microscopetechniques. On the basis of these results and hypothesis isproposed for the organization of these walls. Microchemicaltests indicate the presence of chitin in the walls and suggestthat the mucilaginous layer around the conidium is mainly composedof xylan. Chemical analyses of isolated wall material confirmthe presence of chitin constituents in the wall, and a rylanlayer around the conidium. Furthermore, the wall contains apolypeptide moiety which has a different amino acid compositionfrom the rest of the protein of the cell. Electron microscopestudies of replicas and sections of conidia, germ tubes, andhyphae reveal a layered structure for the wall. The centrallayer is non-microfibrillar and is overlaid on both sides witha layer of randomly orientated microfibrils. The mucilaginouslayer of the conidium obscures the microfibrillar structurebeneath it unless the mucilage is removed by hydrolysis. Theproblem of hyphal growth is discussed on the basis of the structureof germ-tube tips and mature hyphae observed.     

Cytochemistry and Ultrastructure of Hyphae and Haustoria of Peronospora parasitica (Pers. ex Fr.) Fr.

The uniform distribution of nuclei, mitochondria, lipid material,protein, and RNA in the hyphae and haustoria of Peronosporaparasitica was demonstrated by staining techniques. Glycogenwas not detected, the only insoluble carbohydrate material detectedby the periodic acid-Schiff reaction being in the fungal wall.The host cell walls reacted more intensely to this stain thanthe hyphal walls. The reaction of haustorial walls varied betweenthe slight staining reaction characteristic of the fungal walls,and the strong reaction of the host cell wall. Callose sheathswere occasionally seen. Fine structure was found to be similar to that of other Oomycetes.Welldeveloped sheaths of a vesicular nature, possibly synonymouswith callose sheaths, were occasionally seen partly surroundinghaustoria.     

抗病品种中小麦条锈菌细胞的超微结构变化过程

本文就寄主抗病性表达过程中,小麦条锈菌细胞的超微结构变化进行了系统地观察研究。结果表明:胞间菌丝的细胞壁染色逐渐加深,厚度加宽,结构疏松,形成小空洞,并逐渐解体;细胞质逐渐凝聚、脂肪粒的数量增多、有黑色颗粒状沉积物积累;细胞质中小囊泡数目增多并逐渐融合成大液泡,线粒体数目增多,并逐渐肿胀和解体。次生吸器畸形,初生吸器体呈圆球形。吸器壁加厚,染色加深;在吸器的中央,细胞质逐渐分解而形成空泡;线粒体数目增多,并逐渐肿胀和解体;吸器外质膜呈皱褶状,吸器外间质加宽,其中有大量的丝状或颗粒状内含物形成;吸器形态结构的变化均早于其胞间菌丝。     

种衣剂17号包衣对小麦条锈菌发育影响的超微结构研究

本研究采用电镜技术研究了种衣剂17号对小麦条锈菌发育的影响。观察结果表明,该种衣剂引起病菌和寄主细胞内发生了一系列变化。病菌菌丝和吸器内脂肪粒和液泡明显增加;菌丝壁和吸器壁呈不规则加厚;菌丝分枝处无隔膜产生或隔膜畸形;有的吸器母细胞产生的畸形入侵栓,大都不能穿透寄主细胞壁,初生吸器外间质内沉积有染色较深的物质,次生吸器可产生多个不规则分枝,但不能扩张膨大;菌丝外渗的物质可能引起寄主细胞的坏死;大多数受侵寄主细胞可分泌形成较大的胼胝质,有时寄主细胞分泌的物质可将吸器体完全包围起来。上述结果表明,种衣剂17号不仅可直接作用于条锈菌,而且也可通过影响寄主而间接地影响病菌。     

Observations on the Responses of Lentil Root Cells to Hyphae of Fusarium oxysporum

The infection of lentil roots by Fusarium oxysporum Schlecht and the responses of the host cells to invading hyphae were examined by light microscopy. Hyphae from inoculum placed on the zone of cell elongation entered the roots at the juncture of epidermal cells within 8 h after inoculation. Although swollen hyphal apices were observed on the epidermal cells, root penetration occurred without formation of these structures or appressoria. The sheath of material found on the surface of uninoculated roots was absent from inoculated roots penetrated by hyphae. Prior to penetration, the epidermal cells became irregular in shape and their cytoplasm appeared to be plasmolysed or granular. Hyphae were observed in the cortex 10—12 h after inoculation and non–penetrated cortical cells were distinctly lobate. Often these lobed cells had a broad, peripheral band of diffuse cytoplasm. When hyphae were first observed in the cortical cells, the walls were ruptured and only slightly stained or unstained by toluidine blue. The inability of such walls to bind the stain may have been the result of the removal of wall components by fungal enzymes. Although extensive proliferation of hyphae was evident throughout the cortex after 24 h of incubation, the endodermis and vascular cylinder were free of hyphae for at least 72 h. Hyphae from inoculum placed on the root hairs or the root apex failed to penetrate the roots during the first 24 h of incubation. The cytological results herein are discussed in relation to the infection of field plantings by this pathogen.     

The Anatomy, Histochemistry and Ultrastructure of Stigmas and Styles in Commelinaceae

The anatomy and ultrastructure of stigmas in 37 species of 13genera of Commelinaceae are described. The stigmas are papillate,papillae forming a dense fringe of cells around the mouth ofthe stylar canal in most species. The papillar cell wall iscovered by an unstructured cuticle of variable thickness andis of variable thickness because of small wall ingrowths. Thecuticle and the external surface of the papillar cell wall arevariably disrupted, particularly in the mid and basal regionsof the cell. This was not found in species of the genus Aploleiaor Callisia. The cell cytoplasm possesses all major organellesexcept chloroplasts and each cell is vacuolate. In all species except Aploleia mulitiflora the style comprisesan epidermis, a cortex and a hollow, tripartite canal whichis continuous into the ovary cavity. The three vascular strandsare positioned at the apex of each canal lobe. The canal cellsare elongate and tabular and the wall abutting the canal hasingrowths. The style in Aploleia is solid and the transmittingtissue comprises cells whose walls are electron opaque. Thecytoplasms of both types of cell are similar in content althoughthere is a single, large vacuole in canal cells and many smallvacuoles in transmitting tissue. The morphology, position and histochemistry of stigmatic andstylar exudate was similar in all ‘wet’ stigmas.Most of the exudate originates from the stylar canal althoughsignificant contributions are made by the papillae in stigmasof Coleotrype, Dichorisandra and Thyrsanthemum. There is no apparent relationship between stigma structure andthe presence of self-incompatibility. Stigma papillae, stylar canal, transmitting tissue, Commelinaceae     

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.     

Effects of nocodazole and brefeldin A on microtubule cytoskeleton and membrane organization in the homobasidiomyceteSchizophyllum commune

Summary The effects of nocodazole and brefeldin A (BFA) on the growth of dikaryotic hyphae inSchizophyllum commune corresponded with the development of abnormal structures in the apical region of treated hyphae. Microtubules (MTs) were totally depolymerized after 1 h nocodazole treatment, which correlated with strong branch formation in the apical cells. One reason for branching could be the shift in the position of apical vesicles from the center to the side of the tip, observed in some nocodazole-treated hyphae. After 2 h growth in the presence of nocodazole the apical cells had malformed or swollen tips, or tips of normal shape but containing only a few apical vesicles. After 0.5 h treatment with BFA, almost all the leading hyphae had swollen apical parts in which the endoplasmic reticulum (ER) formed an interconnected network and perturbed Golgi particles were found. The orientation of MTs in the BFA-treated hyphae often followed that of the interconnected ER network, which suggested an association between MTs and ER. The results of the experiments with nocodazole suggest that, in filamentous homobasidiomycetes the subtle organization of cytoplasm necessary for the polar growth at the apex is maintained only in the presence of an intact MT cytoskeleton. The BFA experiments indicated that the secretion pathway inS. commune is sensitive to BFA. In addition rapid change in apical morphology in the BFA-treated hyphae emphasizes the importance of correct orientation of components of the secretory pathway for normal apical growth to continue.Abbreviations BFA brefeldin A - EM electron microscopy - ER endoplasmic reticulum - IIF indirect immunofluorescence - MBC methylbenzimidazole-2-ylcarbamate - MT microtubule - MVB multivesicular body - RER rough endoplasmic reticulum     

Dodder hyphae invade the host: a structural and immunocytochemical characterization

Summary.  Dodder (Cuscuta pentagona) hyphae are unique amongst the parasitic weeds for their ability to apparently grow through the walls of the host plant. Closer examination reveals, however, that the hyphae do not grow through the host but rather induce the host to form a new cell wall (or extend the existing wall) to coat the growing hypha. This chimeric wall composed of walls from two species is even traversed by plasmodesmata that connect the two cytoplasms. Compositionally, the chimeric wall is quite different from the walls of either the host or in other cells of the dodder plant, on the basis of immunocytochemical labeling. The most striking differences were in the pectins, with much stronger labeling present in the chimeric wall than in either the host or other dodder walls. Interestingly, labeling with monoclonal antibodies specific to arabinan side chains of rhamnogalacturonan I pectin fraction was highly enriched in the chimeric wall, but antibodies to galactan side chains revealed no labeling. Arabinogalactan protein antibodies labeled the plasma membrane and vesicles at the tips of the hyphae and the complementary host wall, although the JIM8-reactive epitope, associated with very lipophilic arabinogalactan proteins, was found only in dodder cells and not the host. Callose was found in the plasmodesmata and along the forming hyphal wall but was found at low levels in the host wall. The low level of host wall labeling with anticallose indicates that a typical woundlike response was not induced by the dodder. When dodder infects leaf lamina, which have more abundant intercellular spaces than petioles or shoots, the hyphae grew both intra- and extracellularly. In the latter condition, a host wall did not ensheath the parasite and there was clear degradation of the host middle lamellae by the growing hyphae, allowing the dodder to pass between cells. These data indicate that the chimeric walls formed from the growth of the host cell wall in concert with the developing hyphae are unique in composition and structure and represent an induction of a wall type in the host that is not noted in surrounding walls. Received February 1, 2002; accepted July 8, 2002; published online November 29, 2002     

Aspects of the dimorphism of Histoplasma farciminosum: a light and electron microscopic study.

Within 48h following the induction of mycelial to yeast-like phase conversion of Histoplasma farcininosum, randomly occurring hyphal cells were observed to contain multiple nuclei and markedly increased numbers of mitochondria. Yeast-like cells arose as buds from swollen tips of terminal hyphae, as sessile buds along the hyphae, and as buds from chlamydospores. Yeast-like cells were characterized by the presence of numerous buds over the surface of the mother cell. Bud scars were evident in the cell wall of the mother cell following abscission of the bud cell. Little similarity was noted between the fine structure of yeast-like H. farciminosum and that reported for H. capsulatum. The yeast-like cells of H. frciminosum underwent rapid transformation to the mycelial phase at 25 degrees C. The hyphal cell wall originated from the inner layer of cell wall of the yeast-like form. The cytoplasm of the hyphal cell usually contained a single nucleus, scattered mitochondria and occasional lipid storage bodies. Occasionally, Woronin bodies were observed at the septal pore.