The fine structure of Culicinomyces clavisporus invading mosquito larvae

Electron microscope observations were made of the Australian and U.S. strains of Culicinomyces clavisporus infecting mosquito larvae. The wall of the conidium is composed of an inner (primary) layer, an outer (secondary) layer, and an exterior coating of a mucopolysaccharide substance believed responsible for conidial adhesion to the host cuticle prior to germination and penetration. In some instances the wall of the conidium is ruptured during germination and new wall layers and mucoid coating form around the germ tube whereas in other specimens the conidial wall layers extend around the germ tube without fracturing. The most common invasion site is through the larval foregut following ingestion of conidia. The apex of the germ tube presses tightly against the surface of the foregut cuticle and the mucilaginous coating is stripped away. There is evidence to suggest that the host epicuticle, which disappears across the zone of contact with the germ tube, is utilized for nutrition of the invading fungus. A collar of cuticle forms around the germ tube apex and a narrow penetrant hyphae extends into the procuticle. It is believed that cuticular penetration is primarily enzymatic assisted by mechanical pressure. The penetrant hypha swells into an oval cell in the hypodermal region and vegetative hypha then invade the hemocoel. The cells of the hypodermis develop signs of degeneration presumably due to the secretion of toxic substances from the invading hyphae. Host reactions, involving melanization of the host tissues, are sometimes evident among the invading penetrant hyphae in the cuticle or in the hypodermal cells in contact with the fungus. Melanized capsules form around some of the hyphae within the hemocoel. These latter reactions do not directly involve host blood cells and are examples of “humoral encapsulation” similar to that described by other authors during invasion of pathogenic organisms into mosquito larvae and chironomid larvae.     

Phenol oxidation in soft cuticle and blood of crayfish compared with that in other arthropods and activation of the phenol oxidases by fungal and other cell walls

Phenol oxidase from blood as well as from soft cuticle of different crayfish was highly activated by cell walls of a number of fungi and other plants, but the enzymes from other crustaceans tested and from some insects were not. Attachment of the phenol oxidase on blood and soft cuticle to the cell wall surface of certain plants and subsequent heavy melanization of the walls with natural or added substrate may be a unique property of crayfish since it did not occur in any other arthropods tested. Interestingly, blood and cuticle of crayfish showed the same specificity in these processes and the mechanisms might therefore be similar, if not the same, in both. The activity of the nonadsorbed enzyme dispersed in the blood was higher in a crayfish species resistant to fungal attack than in susceptible ones, but the relative degree of activation by adding cell walls was greater in the latter.The natural substrate for the phenol-oxidase activity in crayfish blood was released or mobilized by the blood cells. The substrate for the activity in cuticle was not available in the intact cuticle itself. It appeared only after a “signal” had been transmitted through the cuticle to the cells inside. The possibility that these mechanisms are involved in the cuticular as well as the blood defense system against fungal and other microbial parasites is discussed.     

Ultrastructure of the penetration of the crayfish integument by the fungal parasite, Aphanomyces astaci, Oomycetes

In the crayfish, Astacus astacus, susceptible to the crayfish plague fungus, penetration of the cuticle by the parasite occurred in the soft cuticle. The zoospore lysed the surface lipid layer, tore it away, and formed an infection peg (germ tube) that penetrated through the epicuticle. A septum was formed in the infection peg, and a hypha was formed below the inner epicuticular surface. In the endocuticle, hyphae grew preferrentially parallel to the surface, occassionally perpendicular to it. Growth direction in relation to cuticle architecture is discussed. Subsequently, some hyphae started to penetrate out through the epicuticle. This process was preceded by the swelling of the hyphal tip touching the inner side of the epicuticle. The hypha penetrating out through the epicuticle was much thicker than the infection peg. Histolytic activity, combined with mechanical penetration, seems to be evident in all stages and levels except in the outward penetration of the epicuticular lipid surface layer, where only mechanical rupture could be seen. Differences in the protoplasmic ultrastructure were found between the spore and the penetrant hyphae. Penetration of the cuticle of a resistant crayfish was essentially identical to that in susceptible ones. However, inward penetration of intact epicuticle was too scarce to allow for ultrastructural studies.     

Ultrastructural Study on the Interactions Between Three Respectively Different Disease- resistant Cucumber Cultivars and Phytophthora melonis

Ultrastructural changes during the process of interactions between three different diseaseresistant, susceptible, moderately resistant and resistant cucumber (Cucumis sativus L. ) cuhivars and Phytophthora melonis Katsura were respectively compared and the associated factors in the observed differences in colonization were determined. During the process of interaction in the susceptible cultivar, both inter- and intracellular growth of hyphae associated with extensive colonization of healthy hyphae and marked alteration of mesophyll cells, such as aggregation of cytoplasm and disintegration of cellular organelle were demonstrated. The general disorganization of host cell was observed in areas directly adjacent to the fungal hyphae and in cells several layers from the nearest visible hyphae. In the moderately resistant cultivar during interaction the host showed limited hypha growth with responses of resistance including 1 ) breakage of plasmodesmata to prevent the host cells to contact the hyphae, 2) increasing the quantity of endoplasmic reticulum and Golgi apparatus so as to increase the quantity of secretory protein, 3) a plasmalemma invagination containing granular material at the site in contact with the intercellular hyphae. The interaction in the resistant cuhivar was characterized as a hypersensitive reaction (HR) which means a rapid necrosis of the infected cells and the cells surrounding the invaded hyphae, resulting in death of the invaded hyphae. Formation of cell wall appositions right at the penetration sites of hyphae and haustorium-like structure was observed. The results indicate that the mechanism of host response to P. melonis invasion was differ- ent between the moderately resistant and the resistant cucumber cultivars.     

黄瓜不同抗病品种与疫霉菌相互作用的超微结构研究

电镜观察发现,黄瓜（ＣｕｃｕｍｉｓｓａｔｉｖｕｓＬ．）的不同抗病品种在与疫霉菌（ＰｈｙｔｏｐｈｔｈｏｒａｍｅｌｏｎｉｓＫａｔｓｕｒａ）的相互作用过程中有不同的表现。感病性品种易被疫霉菌侵染,被侵染的叶肉组织及周围细胞中胶层解离、胞质凝聚、细胞器解体,叶片组织内有大量胞间及胞内生长菌丝。中抗病性品种也被疫霉菌侵染,但表现出胞间连丝断裂、内质网和高尔基体增多等抗病性反应,与菌丝相接触的细胞出现质膜内陷。抗病性品种出现过敏性坏死反应,叶肉细胞与入侵菌丝一同解体死亡,胞间菌丝向细胞内穿透处形成壁附加物。中抗病性品种和抗病性品种在与疫霉菌的相互作用过程中表现出不同的抗性机制。     

Wound reactions and Aphanomyces astaci growth in crayfish cuticle

Superficial wounding of crayfish soft cuticle by removal of the epicuticle caused hemocyte aggregation beneath the wound. The aggregation seemed to be induced by osmotic factors. Subsequently, melanin was only formed in parts of the wound region. Swimming zoospores of the fungal parasite Aphanomyces astaci preferentially encysted in the near vicinity of fresh superficial wounds. Cysts attached onto the naked endocuticle became encapsulated by melanin and were mostly killed during the process. The induced melanization reactions may have antifungal effects. The intermolt cuticle of a highly resistant crayfish species, Pacifastacus leniusculus, was easily penetrated by the fungus only via wounds. The cell walls of the penetrating hyphae became heavily melanized, growth was visibly disturbed, and they grew sparsely in the wound region. As soon as they reached outside the wound region they grew profusely. In susceptible crayfish Astacus astacus melanization was slower and the wound reactions prevented fungal growth much less as evidenced by microscopy. The preferential direction of hyphal growth is parallel to the chitin fibrils in both the resistant crayfish P. leniusculus and the susceptible A. astacus.     

Response ofZea mays roots to infection withPhytophthora cinnamomi

Summary Zea mays is a non-host ofPhytophthora cinnamomi; plants survive contact with this fungus both in the field and in pot trials. TheZ. mays-P. cinnamomi interaction has been studied by light and electron microscopy. In the epidermal layer, fungal hyphae grow intercellularly through the middle lamella. This is always the case for the first hyphal contact with any cell. Hyphae making second or subsequent contacts with a cell grow preferentially between the cell wall and plasma membrane of the infected cell rather than through the middle lamella.Papillae (callose deposits) are formed in response to some, but not all, regions of contact between the plant cell and the hypha. They do not completely encase the hypha and do not stop hyphal growth. The plasma membrane-cell wall interface of the host cell must be intact for effective papilla formation, as papillae are rarely formed when the hyphae grow between the plasma membrane and the cell wall.     

Morphological and cytological aspects of oidium formation in a basidiomycete,Pholiota nameko

Pholiota nameko produced abundant oidia on aerial hyphae from monokaryotic and dikaryotic test stocks, but oidia were rare on submerged hyphae. The oidia from the former stocks had a layer of hydrophobic protein between the cell wall and the inner cell membrane which was absent in the oidia from the latter. The only remarkable differences in the morphological features of the oidia from monokaryotic and dikaryotic mycelia was the slightly larger size of the latter. Observation of various test stocks on slide cultures revealed that about 80% of oidia were produced from the secondary branched hypha, and about 20% from the terminal hyphal, cell of the main hypha. In the former, the secondary hyphae were segmented to form several oidium cells; in the latter, a single or several oidia were formed at the terminal end of the main hypha. Most oidia from monokaryons and dikaryons had only one haploid nucleus, while the remainders were multinucleate. Among the stocks tested, most oidia had a DNA content with a haploid amount at the G1 phase of the cell cycle, but a few contained twice that amount corresponding to the G2 phase     

Ultrastructure of Dimorphic Transformation in Paracoccidioides brasiliensis

The fine structure of Paracoccidioides brasiliensis undergoing temperature-dependent transformation from mycelium to yeast and vice versa (M right harpoon over left harpoon Y) was studied. The transitional form to mycelium from the yeast appears as an elongated bud that extends from the yeast and which has a mixture of characteristics from both the yeast and the mycelium. The transitional form to yeast from the mycelium starts with enlargement of the interseptal spaces and cracking of the outer electron-dense layer of the cell wall of the hypha. Later the interseptal spaces tend to become round and separate. In M --> Y only few interseptal spaces seem to transform. The yeast is produced by self-transformation of the hypha. In Y --> M a new structure is formed and the yeast dies. Intrahyphal hyphae are observed during the transformation from M --> Y, and intrayeast hyphae during the Y --> M. Due to the high mortality and breakage observed in both types of transformations, we believe that wound of the yeast or the mycelium could elicit this phenomenon.     

蜜环菌侵染天麻皮层过程中酸性磷酸酶的细胞化学研究

被蜜环菌(Armillaria mellea)侵染的天麻(Gastrodia elata B1.)皮层中,由外至内形成三种类型的染菌细胞:菌丝结细胞、空腔细胞和消化细胞。外部两类细胞中的酸性磷酸酶定位显示,一些位于空腔细胞或衰老的菌丝结细胞中的菌丝内部逐渐产生大量酸性磷酸酶,随后菌丝发生自溶。这两类细胞中未发现明显的释放水解酶消化菌丝的现象。当菌丝进入消化细胞以后,情况与此不同,大量包含酸性磷酸酶的微小颗粒出现在菌丝周围,随后这些酶颗粒相互融合,形成包围菌丝的消化泡,菌丝被溶酶体水解酶所消化。最后消化泡变为包含代谢废物的残体。     

葡萄座腔菌Botryosphaeria dothidea侵染及其对苹果果实影响的组织细胞学研究

利用光镜和电镜技术系统研究了苹果轮纹病菌葡萄座腔菌在成熟果实上的侵染扩展过程及其细胞学特征。扫描电镜观察发现,接种后3h位于皮孔处的分生孢子开始萌发,萌发后的孢子从一端或两端产生芽管直接侵入皮孔细胞,接种后9h完成侵入。30d后果面接种部位表现症状,45d后产生子实体。对接种部位取样进行光镜和透射电镜观察发现,病菌菌丝主要存在于寄主细胞壁、细胞内、细胞间隙及细胞壁与细胞膜之间。菌丝呈丝状,分枝,具隔膜。菌丝细胞内含有细胞核、线粒体、液泡等细胞器;菌丝外散发出一些高电子密度的颗粒物质,这些物质以菌丝为中心,呈放射状分布。病菌在果肉细胞生长扩展过程中,果肉细胞发生一系列变化。果肉细胞壁膨胀、变形,胞间层分离、破裂。与菌丝接触或相邻的果肉细胞细胞壁电子致密度降低,被降解成为如散发状的胞壁纤维束丝。果肉细胞的液泡破裂,质壁分离,细胞质凝结坏死并沉积于细胞壁周围,或通过受损的细胞壁胞间连丝从一个细胞转移到另一个细胞。后期菌丝在表皮下聚集生长、发育成分生孢子器。分生孢子器内壁细胞排列紧密,细胞中含有由数条丝状物平行排列而成的细胞器。该细胞器形状多样,周围总是分布着丰富的脂肪粒,推测可能与营养的运输与积累有关。     

On-line study of growth kinetics of single hyphae of Aspergillus oryzae in a flow-through cell

Using image analysis the growth kinetics of the single hyphae of the filamentous fungus Aspergillus oryzae has been determined on-line in a flow-through cell at different glucose concentrations in the range from 26 mg L-1 to 20 g L-1. The tip extension rate of the individual hyphae can be described with saturation type kinetics with respect to the length of the hyphae. The maximum tip extension rate is constant for all hyphae measured at the same glucose concentration, whereas the saturation constant for the hyphae varies significantly between the hyphae even within the same hyphal element. When apical branching occurs, it is observed that the tip extension rate decreases temporarily. The number of branches formed on a hypha is proportional to the length of the hypha that exceeds a certain minimum length required to support the growth of a new branch. The observed kinetics has been used to simulate the outgrowth of a hyphal element from a single spore using a Monte Carlo simulation technique. The simulations shows that the observed kinetics for the individual hyphae result in an experimentally verified growth pattern with exponential growth in both total hyphal length and number of tips.     

A Cytochenfical Study of Acid Phosphatas in the Mycorrhizal Cells of Gastrodia elata Seedling

A cytochemical study has been made to examine the activity of acid β-glycerophosphatase in the mycorrhizal cells of the seedling of Gastrodia elata BI. using thin sectioning technique in which sections were embedded in glycol mathacrylate (GMA). After the seedling was invaded by the hyphae of Mycena osmundicola Lange, two different kinds of infected cells were formed in its root cortex.the outer 1–2 cell layers namely the hyphae-containing cells (or host cells) contained many coiled hyphae pelotons; the inner comparativly large cell layer or fungus-digesting cells contained a few straight hyphae. Localization of acid phosphatase in hyphae-containing cells showed that only a few senescent hyphae retained the enzyme activity and the plant cells did not release hydrolytic enzyme. So it is considered that the hyphal lysis in hyphae-containing cell may be due to autolysis. In contrast, higher acid phosphatase activity was visualized in many vesicles and small vacuoles of the fungus-digesting cells. When a hypha entered a fungus-digesting cell through a hyphae-containing cell, a number of enzyme granules (i. e, enzymecontaining vesicles) gathered around it. Later on the enzyme granules expanded gradually and became small enzyme vacuoles of 1.6–2.0 μm in diameter. Still later the small enzyme vacuoles fused with each other to form a large vacuole in which a part of an invading hypha was enclosed and gradually digested by hydrolytic enzymes. Finally,the digesting vacuole changed into a residual body containing some metabolic waste. The above results suggest that fungus-digesting cells can actively release hydrolytic enzymes by lysosomal vesicles to digest the invading hyphae, but such function is not present in the hyphae-containing cells,the role of which may be attributed to attracting and controling the invading hyphae.     

Role of Lysosomal Vesicles in the Digestive Process of Armillaria mellea by the Large Cells of Gastrodia elata

The hyphae of Armillaria mellea Fr. invade the large ceils of Gastrodia elata BI. Through the wall pits of cortical cells. During early stage the plasmalemma of large cell invaginates and the cell wall forms papillary thickenings to restrain the hyphae from invading. When a hypha enters a large cell, it is encircled tightly by the invaginated plasmalemma which is surrounded by a large number of vesicles coated by a unit membrane. As these vesicles fusing with their membranes to the plasmalemma and discharging their contents into the space around the hypha, the space lined by the invaginated plasmalemma enlarges gradually and becomes a digestive vacuole in which a hypha is completely digested. Reaction product form acid phosphatase activities in the vesicles and digestive vacuoles testifies that the vesicles and digestive vacuoles are identical with primary and secondary lysosomes of plant lysosomal system respectively.     

天麻大型细胞消化蜜环菌过程中溶酶体小泡的作用

蜜环菌(Armillaria mellea Fr.)菌丝由天麻(Gastrodia elata Bl.)皮层细胞经纹孔侵入大型细胞。初期大型细胞的原生质膜凹陷,同时细胞壁产生乳突状加厚阻止菌丝侵入。当菌丝侵入大型细胞以后,凹陷的质膜将菌丝紧密包围,大量由单位膜围成的小泡聚集在其周围。随后这些小泡的膜与质膜融合并将其内含物释放到菌丝周围的空间中,凹陷质膜逐渐膨大成为一个包围菌丝的消化泡。小泡和消化泡中均具酸性磷酸酶活性反应产物,证实其分别相当于植物溶酶体系统中的初级和次级溶酶体。菌丝在消化泡中被彻底消化。     

Ultrastructural Studies of the Interaction Between Trichoderma spp. and Plant Pathogenic Fungi

The uhrastructural changes during parasitism of the biocontrol agents Trichoderma harzianum and T. hamatum, were observed under a transmission electron microscope. Electron micrographs show that during the interaction of Trichoderma spp. with either Sclerotium rolfsii or Rhizoctonia solani the hyphae of the parasites contact their host, and then enzymatically digest their cell walls. Extracellular fibrillar material is deposited between the interacting cells. Parasite organelles, e.g. mitochondria, vesicles and dark osmiophilic inclusions, accumulate in the parasitizing cells. In response to the invasion, the host produces a sheath matrix which encapasulates the penetrating hypha and the host cells become empty of cytoplasm.     

Mycelium development and architecture, and spore production of Scutellospora reticulata in monoxenic culture with Ri T-DNA transformed carrot roots

Mycelium development and architecture and spore production were studied in Scutellospora reticulata from single-spore isolates grown with Ri T-DNA transformed carrot root-organ culture in monoxenic system. Culture establishment, anastomosis occurrence and auxiliary cell development also were examined. Seventy percent of the pregerminated disinfected spores colonized the transformed carrot roots. After 8 mo, the average spore production was 56 (24-130) per 30 cm(3) of medium. Of the spores produced, 75% germinated and produced new generations in monoxenic culture. The mycelium network was formed by thick light-brown hyphae, which exhibit two major architecture patterns related to either root colonization or resource exploitation, and lower-order hyphae, bearing auxiliary cells, branched absorbing structures (BAS), hyphal swellings (HS) and forming anastomoses. BAS were formed abundantly in extramatrical mycelium and frequently had HS resembling vesicles, a feature not previously reported in the Gigasporaceae, to the best of our knowledge. Few anastomosis were observed within the mycelium and most often corresponded to a healing mechanism that form hypha bridges to reconnect broken hyphae or overcoming obstructed areas within a hypha. Numerous auxiliary cells were produced during culture development and their role was inferred.     

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     

Scanning electron microscopy of the Alnus crispa var. mollis Fern. root nodule endophyte

Nitrogen-fixing root nodules of the Alnus crispa var. mollis Fern. were studied by scanning electron microscopy (SEM). The critical point drying of glutaraldehyde-osmium fixed nodular tissue permitted an excellent morphological preservation of the three-dimensional structures of the host and endophyte cells. The nodule endophyte was observed as two forms: the hypha which can be branched, and the vesicle which developed at the parental hypha tip. The actinomycetal endophyte penetrated through the host cortical cell wall and became enveloped by a membrane. This enclosing membrane is suggested to be the invaginated host plasmalemma. Perforations of the cell wall of the host infected cell were observed. These perforations are suggested to be the result of an enzymatic degradation process, probably regulated by the penetrating endophyte hyphae. In addition to the polymorphic endophyte, endogenous bacterial contaminants were observed in the nodular tissue. The present SEM study confirms previous light microscopy and transmission electron microscopy studies of the same species of root nodule symbiosis.     

Der Anschluß derCuscuta-Hyphen an die Siebröhren ihrer Wirtspflanzen

Zusammenfassung Morphologische und feinstrukturelle Differenzierung der Kontakthyphe des HoloparasitenCuscuta odorata werden beschrieben. Der apikale Bereich einer Suchhyphe bildet nach Anschluß an ein Siebelement des Wirtes einen sogenannten Kontaktfuß aus — einer Hand vergleichbar, die mit zahlreichen Fingern die Wirtszelle umfaßt. Lokal an den dem Siebelement anliegenden Wandpartien wird ein ausgedehntcs Protuberanzensystem von der Parasitenzelle gebildet, wodurch deren absorbierende Oberfläche um ein Vielfaches (6–20fach) vergrößert wird. Die Umstrukturierung des Protoplasten von der Suchhyphe zur Kontakthyphe hat grundsätzliche Gemeinsamkeiten mit der Siebelement-Differenzierung: Kernverlust, Auflösung des Tonoplasten und Ausbildung charakteristischer Plasmafilamente sind die wesentlichen Merkmale. Darüber hinaus ist im Bereich des Kontaktfußes ein komplexes glattes Endomembransystem entwickelt, wie es bisher von anderen Pflanzenzellen nicht bekannt war. Die beschriebenen Feinstrukturen werden im Zusammenhang mit der Funktion der Parasitenzelle als Absorptionsorgan und Leitelement diskutiert.

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The contact ofCuscuta hyphae with the sieve tubes of its host plants

Summary Morphological and fine structural differentiation of the contact hypha are described. Attaching to the sieve element of the host the apical region of a searching hypha forms a special contact cell, resembling a hand which grasps around the host cells with many fingers. At those parts of the wall adjacent to the sieve element, the parasitic cell developes a conspicuous wall labyrinth, thus enlarging the absorbing surface many (6–20) times. Changing from searching hypha to contact hypha the differentiation of the protoplast shows fundamental similarities to the development of sieve elements: enucleation, disintegration of the tonoplast, and occurrence of typical plasmatic filaments. Besides that an extended smooth surfaced ER is found within the contact hypha, unknown in plant cells until now. The fine structures described are discussed in view of the function of the parasitic cell as an absorbing and a conducting element.

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Mein besonderer Dank gilt Herrn Professor Dr. R.Kollmann für alle wertvollen Diskussionen und seine jederzeitige Hilfe. Die Arbeit wurde am Botanischen Institut Bonn begonnen.