Hyperparasitism of Streptomyces albus on a Destructive Mycoparasite Nectria inventa

Using scanning electron microscopy, Streptomyces albus was proved to be a hyperparasite of Nectria inventa, itself a well-known mycoparasite on many fungi. The hyperparasite had no apparent antagonistic activity against N. inventa, but did have intense tropic response toward it. Upon contact with the host, the hyperparasite grew along, and formed appressorium-like structures on the host hyphae. The parasitism that led to the eventual collapse of the host cells was not necessarily accompanied by actual hyphal penetration. The hyperparasite could, however, readily penetrate the host hyphae, and its hyphae were frequently found inside the host hyphae.     

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.     

Die Parasitierung des Bohnenrostes Uromyces appendiculatus var. appendiculatus durch den Hyperparasiten Verticillium lecanii: Untersuchungen zur Wirt-Erkennung, Penetration und Abbau der Rostpilzsporen

Hyperparasitism of Uromyces appendiculatus var. appendiculatus by Verticillium lecanii Host Recognition, Penetration and Degradation of Spores Culture filtrates of the hyperparasite Verticillium lecanii contain numerous lytic enzymes. When specific substrates were added to the filtrate, degradation of chitin is increased by a factor of 2,25 and degradation of starch is increased by a factor of 1,5. The degradation of uredo- and teliospores of Uromyces appendiculatus var. appendiculatus is documented cytologically. Appressoria-like structures initiated direct penetration of the spore walls. Additional routes of penetration through the germpores of both sporetypes and the pedicles of the teliospores were observed. Degradation of the spore cytoplasm is described. Sugars on the surface of uredo- and teliospores and on hyphae of the hyperparasite were characterized using the gold-marked lectins Con A and WGA. Their role in the host-parasite recognition process is discussed.     

Hyperparasitic Attack of Trichothecium roseum on Conidia of Pestalotiopsis funerea

Trichothecium roseum (Tr) has been shown to be a highly effective hyperparasite on conidia of Pestalotiopsis funerea (Pf) in vivo and in vitro. The stages of this spore parasitism are: positive tropism of Tr towards Pf conidia, contact between Tr and Pf, formation of simple or lobed appressoria of Tr on the host conidial surface, penetration of the attacked host cells from the base of the appressoria, development of host-internal, mostly branching parasitic hyphae by Tr, desintegration, lysis and death of the parasitized host cells, exit of Tr from the destroyed host cells and its intensive sporulation over Pf remnants. Pf did not show any defence reactions against the attack by Tr. In addition to the antagonistic activities of Tr against Pf reported previously, which are due to extracellular toxins released by Tr, direct hyperparasitism is a second mechanism of antagonism, which contributes to the successful competitive ability of Tr in this fungal interaction.     

Anatomical study on the interaction between the root endophytic fungus <Emphasis Type="Italic">Heteroconium chaetospira</Emphasis> and Chinese cabbage

Chinese cabbage roots colonized by the dematiaceous fungal taxon Heteroconium chaetospira were previously found to become highly resistant to clubroot and Verticillium yellows. The dematiaceous fungus possesses an endophytic nature, but no detailed anatomical studies on endophyte–host plant interactions have so far been provided. Light and electron microscopy revealed that hyphae of H. chaetospira were abundant on and inside the root epidermal cells by 3 weeks following inoculation. The penetration pegs easily breached into epidermal cells, and the infection hyphae penetrated into cortical cells. Some appressorium-like swollen structures formed from intracellular hyphae, but no visible degradation of the host cell walls was evident where the hyphae contacted. No visible signs of host reactions and no invagination of the host plasma membrane around the hyphae were seen in the host cells. By 8 weeks following inoculation, masses of closely packed fungal cells had been formed in some cells of the epidermis and cortical layers, but further hyphal ingress was halted, mostly in the inner cortical cell layer. Thus, root vascular cylinders remained intact.     

Mycoparasitism of Endophytic Fungi Isolated From Reed on Soilborne Phytopathogenic Fungi and Production of Cell Wall-Degrading Enzymes In Vitro

Antagonism of three endophytic fungi isolated from common reed (Phragmites australis) against eight soilborne pathogenic fungi was investigated on potato dextrose agar by light microscopy, scanning electron microscopy, and transmission electron microscopy. Inhibitory zones were not observed. The microscopical studies suggested that the endophytes inhibit growth of soilborne pathogens by means of coiling around hyphae and, after penetration, the degradation of hyphal cytoplasm. Since penetration of hyphae seems to play a major role in parasitism, we studied the production of cell wall degrading enzymes by the three endophytes. Choiromyces aboriginum produced higher activities of β-1,3-glucanases compared to Stachybotrys elegans and Cylindrocarpon sp. For C. aboriginum and S. elegans, colloidal chitin was the best substrate for the induction of β-1,3-glucanases and chitinases, respectively. This result suggests that mycoparasitism by endophytes on soilborne plant pathogens can be explained by their mycoparasitic activity.     

Morphological Alterations of Metarhizium anisopliae During Penetration of Boophilus microplus Ticks

Chronological histological alterations of Metarhizium anisopliae during interaction with the cattle tick Boophilus microplus were investigated by light and scanning electron microscopy. M. anisopliae invades B. microplus by a process which involves adhesion of conidia to the cuticle, conidia germination, formation of appressoria and penetration through the cuticle. Twenty-four hours post-infection conidia are adhered and germination starts on the surface of the tick. At this time, the conidia differentiate to form appressoria exerting mechanical pressure and trigger hydrolytic enzyme secretion leading to penetration. Massive penetration is observed 72 h post-inoculation, and after 96 h, the hyphae start to emerge from the cuticle surface to form conidia. The intense invasion of adjacent tissues by hyphae was observed by light microscopy, confirming the ability of M. anisopliae to produce significant morphological alterations in the cuticle, and its infective effectiveness in B. microplus.     

Fine structure of the host-fungus interface in orchid mycorrhiza

Summary Electron microscopy of protocorms of Dactylorhiza purpurella infected with a symbiotic Rhizoctonia sp. showed that the intracellular hyphae examined did not penetrate the plasmalemma of the host cell. Walls of hyphae within cells bore many hemispherical protuberances over which the host plasmalemma was closely pressed. we estimate that these protuberances would increase the area of contact between hyphae and host plasmalemma by about 15%. They were not found on hyphae growing on agar. Except for these protuberances, and some vesicles or tubules which invaginated the fungus plasmalemma, no other structures were seen which could be suggested to be adaptations to transport across the living fungus-host interface.     

Studies on turfgrass snow mold caused byTyphula ishikariensis. II. Microscopical observation of infected bentgrass leaves

Light and transmission electron microscopy revealed thatTyphula ishikariensis penetrated into bentgrass leaves either through cuticles or stomata either by single hyphae or infection cushions formed on host surfaces. Time course study on infected leaves showed that penetration through stomatal subsidiary cells and their adjacent cells seemed to occur earlier than that through epidermal cells located farther from stomata. More than 30% of epidermal cells were infected by 10 days after inoculation. When hyphae penetrated through an intact cuticle of epidermal cells, they seemed to dissolve host cell walls enzymatically at penetration sites. Physical pressure also seemed to be involved in penetration.     

A morphological study of the mycorrhiza ofEntoloma clypeatum f.hybridum onRosa multiflora

Mycorrhizas ofEntoloma clypeatum f.hybridum onRosa multiflora in the field in Japan were studied by stereo, light and electron microscopy. In most mycorrhizas, the root cap, meristem, and apical region of the cortex disappeared, but in a few mycorrhizas, these tissues remained. Fungal hyphae of the mycorrhizas invaded root tissues and branched palmately. Hyphae in contact with cortical cells were larger than those far from the root cells and contained many mitochondria, cisternae of endoplasmic reticulum and transitional vesicles. Invading hyphae were undulate in the apical part of the mycorrhiza, and some of them lacked distinct organelles. Electron-dense granules accumulated in the root cells adjacent to the fungal hyphae. Both the remnants of the plant cells and the fungal hyphae were included in the amorphous materials on the tip of the stele. These observations suggest the destructive infection by fungal hyphae of the root cells and their collapse near the tip of the stele.     

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。     

In vitro antagonism of Thielaviopsis paradoxa by Trichoderma longibrachiatum

Seventy-nine Trichoderma strains were isolated from soil taken from 28 commercial plantations of Agave tequilana cv. ‘Azul’ in the State of Jalisco, Mexico. Nine of these isolates produced nonvolatile metabolites that completely inhibited the growth of Thielaviopsis paradoxa on potato dextrose agar plates. These isolates were identified as Trichoderma longibrachiatum on the basis of their morphology and DNA sequence analysis of two genes (ITS rDNA and translation elongation factor EF-1α). Mycoparasitism of Th. paradoxa by T. longibrachiatum strains in dual cultures was examined by scanning electron microscopy. The Trichoderma hyphae grew alongside the Th. paradoxa hyphae, but penetration of Thielaviopsis hyphae by Trichoderma was no apparent. Aleurioconidia of Th. paradoxa were parasitized by Trichoderma. Both hyphae and aleurioconidia of Th. paradoxa lost turgor pressure, wrinkled, collapsed and finally disintegrated. In liquid cultures, all nine Trichoderma isolates produced proteases, β-1,3-glucanases and chitinases that would be responsible for the degradation of Thielaviopsis hyphae. These results demonstrate that the modes of action of T. longibrachiatum involved against Th. paradoxa in vitro experiments are mycoparasitism and the production of nonvolatile toxic metabolites.     

Structure and Development of the Endophyte in the Parasitic Angiosperm <Emphasis Type="Italic">Cuscuta japonica</Emphasis>

The endophyte, that is, the haustorial part within the tissues of the host plant Impatiens balsamina, of the parasitic angiosperm Cuscuta japonica was studied with light and electron microscopy. The endophyte consisted mainly of vacuolated parenchymatous axial cells and elongate, superficial (epidermal) cells. Then the elongate, epidermal cells separated from each other and transformed into filamentous cells, called searching hyphae. The hyphae grew independently either intercellularly or intracellularly in the host parenchyma. The apical end of the hyphal cells was characterized by conspicuous, large nuclei with enlarged nucleoli and very dense cytoplasm with abundant organelles, suggesting that the hyphal cells penetrating host tissue were metabolically very active. Numerous osmiophilic particles and chloroplasts were noted in the hyphae. The osmiophilic particles were assumed to be associated with elongation of the growing hyphe. Plasmodemata connections between the searching hyphal cells of the parasite and the host parenchyma cells were not detected. Hyphal cells that reached the host xylem differentiated into water-conducting xylic hyphae by thickening of the secondary walls. A xylem bridge connecting the parasite and the host was confirmed from serial sections. Some hyphal cells that reached the host phloem differentiated into nutrient-conducting phloic hyphae. Phloic hyphae had a thin layer of peripheral cytoplasm with typical features of sieve-tube members in autotrophic angiosperms, i.e., parallel arrays of smooth endoplasmic reticulum, mitochondria, and plastids with starch granules. Interspecific open connections via the sieve pores of the host sieve elements and plasmodesmata of the parasite phloic hyphae were very rarely observed, indicating that the symplastic translocation of assimilate to the parasite from the host occurred.     

Host recognition by the gypsy moth [Lymantria dispar] [Lep.: Lymantriidae] hyperparasite,Eurytoma appendigaster [Hym.: Eurytomidae] of cocoons ofCotesia melanoscela [Hym.: Braconidae]

Artificial and modified natural hosts were exposed to females of the gypsy moth [Lymantria dispar (L.)] hyperparasite,Eurytoma appendigaster (Swederus), to investigate its host recognition behavior on the primary host, which are cocooned larvae of the gypsy moth parasite,Cotesia melanoscela (Ratzeburg). Material(s) which caused drilling behavior by the hyperparasite on host cocoons were extracted with both polar and non-polar solvents. However, cocoons washed with large volumes of solvent still caused substantial drilling activities by females, suggesting that additional cues may be important. Results suggest that host recognition in this hyperparasite involves a variety of host characteristics.      

A comparative study of the searching efficiencies of a parasite and a hyperparasite

The searching efficiencies of a primary parasite (Diaeretiella rapae (McIntosh )) and a hyperparasite (Alloxysta brassicae (Ash. )) were investigated and compared. In both species, at all parasite densities, there was a curvilinear relationship (P<0.001) between the number of hosts parasitised and the host density. A linear regression (log a=log Q−m log P) was fitted for log area of discovery against log parasite density (P<0.001). The area of discovery for its immediate (i.e. primary) host (viz. Diaeretiella for the hyperparasite and aphid for Diaeretiella) is lower in the hyperparasite than in the primary parasite. In Diaeretialla both the searching efficiency and the mutual interference constant increased (but not significantly, P>0.05) in the presence of its males.     

Streptomyces alni as a biocontrol agent to root-rot of grapevine and increasing their efficiency by biofertilisers inocula

Root-rotted samples of grapevine cv. superior were collected from Nobaria province, Beheira Governorate, Egypt. Fusarium oxysporum Schlech. was the most fungal causing root-rot syndrome of grapevine and directly affected the yield productivity. Seven isolates of Streptomyces were isolated from grapevine rhizospheric soil and screened for antagonistic activities against F. oxysporum on dual culture plate. All isolates showed antifungal activity, but isolate No. 1 exhibited the highest activity. It was identified as Streptomyces alni according to morphological, cultural, physiological and biochemical studies. The properties of the antagonism were revealed by scanning electron microscopy (SEM) examination of F. oxysporum and S. alni on PDA medium. The forms of antagonism found in this study according to the interaction between the S. alni and the pathogen indicated a hyperparasite, including inhibition of fungal growth and colonisation of S. alni over F. oxysporum hyphae. Also, malformation and lysis of F. oxysporum hyphae and conidiophores were observed. Conidia and normal branches of fungal hyphae were absent. Greenhouse and field studies were performed to evaluate the ability of S. alni and some commercial biofertilisers incorporated into the soil for root-rot control. Pot trails indicated that antagonistic S. alni isolate and biofertilisers i.e. blue green algae, phosphoren and rhizobacterin reduced the root-rot incidence of grapevine plants Cv. superior. Soil treatment before sowing with 50 ml of S. alni suspension (1 × 10⁸ spore/ml) + 50 g of rhizobacterin for each pot was the best and significant treatment reduced root-rot of grapevine plants. Also, the total count of F. oxysporum in rhizosphere soil of grapevine treated plants was reduced compared with control. Under field conditions, drenching soil of diseased grape trees with a spore suspension of S. alni (1 × 10⁸ spore/ml) 200 ml/tree + 250 g/tree of rhizobacterien caused a significant reduction in root rot of treated grapevine trees as well as high fruit yield/tree when compared with other treatments. The obtained results suggest that S. alni could be used successfully in combination with biofertilisers, as environmentally safe, for controlling root-rot of grapevine and other soil-borne plant pathogens especially with organic farming systems.     

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.     

Einsatz von V. lecanii als biologisches Schädlingsbekämpfungsmittel gegen den Bohnenrostpilz U. appendiculatus var. appendiculatus im Feld und im Gewächshaus

The use of Verticillium lecanii as a biological control agent against the bean rust fungus Uromyces appendiculatus var. appendiculatus in the field and in the glasshouse The deuteromycete V. lecanii parasites uredo- and teliospores of the bean-rust-fungus U. appendiculatus var. appendiculatus. We investigated the conditions for the use of the hyperparasite as biological control agent in the field and in glasshouses. The growth rate of the hyperparasite was 0,3 cm per day at 25 °C. Under suitable conditions in the lab (25 °C, 100 % r. h.) it took about 20 days to invade 100 % of uredospores and 65 % of teliospores. We failed to prevent the spread of bean-rust-fungus spores in the field, but we succeeded in the glasshouse by 68 %, compared to the untreated controls, using the hyperparasite V. lecanii as biological control agent.     

Scanning electron and light microscopy of appressorium development in Piptocephalis unispora (Mucorales)

Appressorium development in the mycoparasite Piptocephalis unispora was studied by means of scanning electron microscopy using the techniques of critical point drying, sputter coating and light microscopy. The germ tube which contacts both the young host hypha or a germinating spore swells at the tip to form an appressorium closely adpressed to the surface of the host. Lateral proliferation of hyphae may occur from the mature appressorium. Factors affecting the sites of appressorium development are suggested and their significance discussed.     

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.