Electron microscopy of infection of nematodes byDactylaria haptotyla

Infection of nematodes byDactylaria haptotyla, a nematode-trapping hyphomycete, was studied by electron microscopy. The cytoplasm of the adhesive knob in the fungus contained a number of electron-dense, membrane-bound vesicles, 0.2–0.5 µm in diam. The vesicles were rarely seen in the stalk cell or vegetative cell cytoplasm. When the adhesive knob came into contact with the nematode's cuticle, it secreted an adhesive which was seen in ultrathin sections between the knob and the cuticle as an amorphous mass. At the same time, electron-dense vesicles in the cytoplasm were reduced in number and many small vacuoles developed. Soon after capture of a nematode, the cell wall of the adhesive knob became obscure at the prospective site of penetration, where a vesicle, 0.7 µm in diam, was found in serial thin sections of the knob's cytoplasm. At the site facing the vesicle, the peripheral part of the nematode's cell exhibited a high electron density. The vesicle, which appeared to be derived from smaller electron-dense vesicles coalesced with each other, released its enzymic contents toward the captured nematodes before penetration by the fungus.     

Time-lapse photomicrography and electron microscopy on initiation of infection of nematodes by Dactylella ellipsospora

 Infection of nematodes by two strains of Datylella ellipsospora was observed using video photomicrography and electron microscopy. By light microscopy, each cell with adhesive knobs contained a number of particles that were distributed evenly before capture of a nematode. The cytoplasmic particles moved to and fro at random. At the moment when the knob cell came into contact with a nematode, the particles accumulated at the place where the cell wall of the knob stuck firmly to the nematode cuticle and exuded adhesive at the same time. The adhesive can be seen near the point of contact between the cell wall of the knob and the cuticle of the nematode. At that point, the knob cell produced an infection peg in most cases, and the cell showed a preference to invade the body of the nematode rather than the tail and head. During capture, accumulation of cytoplasmic particles was seen until infection-bulb formation began. In electron micrographs of ultrathin sections, most of the particles could be seen as electron-dense vesicles, 0.2–0.6 μm in diameter. After attachment of the knob cell to the nematode cuticle, the vesicles were found to fuse with plasmalemma one after another to exude adhesive seen as an amorphous electron-dense substance. Received: January 30, 2002 / Accepted: April 25, 2002     

Scanning electron microscope-analysis of the protrusions (knobs) present on the surface of Plasmodium falciparum-infected erythrocytes

The nature of the surface deformations of erythrocytes infected with the human malaria parasite Plasmodium falciparum was analyzed using scanning electron microscopy at two stages of the 48-h parasite maturation cycle. Infected cells bearing trophozoite-stage parasites (24-36 h) had small protrusions (knobs), with diameters varying from 160 to 110 nm, and a density ranging from 10 to 35 knobs X micron-2. When parasites were fully mature (schizont stage, 40-44 h), knob size decreased (100-70 nm), whereas density increased (45-70 knobs X micron-2). Size and density of the knobs varied inversely, suggesting that knob production (a) occurred throughout intraerythrocytic parasite development from trophozoite to schizont and (b) was related to dynamic changes of the erythrocyte membrane. Variation in the distribution of the knobs over the red cell surface was observed during parasite maturation. At the early trophozoite stage of parasite development, knobs appeared to be formed in particular domains of the cell surface. As the density of knobs increased and they covered the entire cell surface, their lateral distribution was dispersive (more-than-random); this was particularly evident at the schizont stage. Regional surface patterns of knobs (rows, circles) were seen throughout parasite development. The nature of the dynamic changes that occurred at the red cell surface during knob formation, as well as the nonrandom distribution of knobs, suggested that the red cell cytoskeleton may have played a key role in knob formation and patterning.     

Development of Aspergillus parasiticus and formation of aflatoxin B1 under the influence of conidiogenesis affecting compounds

The influence of various inhibitors of hyphal growth, sporulation and biosynthesis of aflatoxin B₁ in Aspergillus parasiticus NRRL 2999 was studied. 6-Thioguanine, dl-ethionine, fluoroacetic acid and phenylboric acid, inhibitors of maturation of fungal conidiophores and of conidiogenesis, were added at various concentrations to malt extract agar. Lower concentrations of 6-thioguanine and dl-ethionine did not inhibit the growth of hyphae and the sporulation. Phenylboric acid reduced conidiogenesis more than hyphal growth. The yields of aflatoxin B₁ were significantly reduced. Additions of fluoroacetic acid did not greatly affect the growth of hyphae but totally inhibited the production of conidia and concurrently significantly reduced the formation of aflatoxin B₁. An interrelation between conidiogenesis and onset of secondary metabolism in A. parasiticus is evident.     

Two new species of nematode-trapping fungi: relationships inferred from morphology,rDNA and protein gene sequence analyses

Two new nematode-trapping fungi, Dactylellina sichuanensis and D. varietas from China, which capture nematodes by both adhesive knobs and non-constricting rings, are described and illustrated. D. sichuanensis is characterized by both adhesive knobs and non-constricting rings, solitary conidiophores and 3-(4)-6 septate conidia, as compared with species such as D. appendiculata, D. candida, D. leptospora and D. lysipaga. Although D. sichuanensis shares similar types of trapping devices, the presence of simple conidiophores and spindle-shaped conidia with these species, it can be distinguished by its larger conidia and presence of more than four septa. In D. sichuanensis, a single conidium is born at the tip of conidiophore, while in D. candida, 3–10 conidia are born near the apex of conidiophore in a capitate arrangement. It produces spindle-shaped conidia with 3–6 septa, whereas D. leptospora produces cylindrical-shaped conidia with 5–15 septa. D. appendiculata captures nematodes by adhesive knobs whereas D. sichuanensis captures nematodes by both adhesive knobs and non-constricting rings. They also differ in conidial size (35–82.5 μm in D. sichuanensis as compared with 57–108 μm in D. appendiculata). D. varietas is characterized by conidiophores that are branched at right-angles, and elongate to fusoid conidia, with 7–8 septa (more than 25% of which are curved). D. varietas resembles D. asthenopaga, Dactylella oxyspora and Monacrosporium multiseptatum, but has elongate-fusoid conidia, whereas D. asthenopaga possesses obconical or clavate conidia. D. varietas forms both adhesive knobs and non-constricting rings whereas Dactylella oxyspora does not produce any trapping device. M. multiseptatum differs from D. varietas in having larger conidia with an inflated middle cell. Phylogenetic analyses based on nuclear and protein coding DNA sequences (18 S, and a combined 28 S + 5.8 S + β-tubulin dataset) indicate that these two taxa should be assigned to the family Orbiliaceae.     

Conidia of the nematophagous fungus Drechmeria coniospora adhere to but barely infect Acrobeloides buetschilii

Abstract Conidia of the endoparasitic fungus Drechmeria coniospora were able to adhere to the nematode Acrobeloides buetschilii ; approximately 70% of the nematodes were carrying spores 16 h after their addition to the fungal culture. Young nematodes were preferentially affected; adhesion mainly took place in the head region of the animal. Full infection, characterized by colonization of the nematode body and production of conidiophores and conidia, was observed, albeit at low frequencies. In case of failure, a second adhesive knob was often formed on the germinated spore. The low frequencies of infection are not related to a loss of virulence, due to prolonged in vitro culturing of the fungus. Fresh spores, obtained from cultures which were passed several times through the host, did not show higher infection frequencies.     

Consecutive monitoring of lifelong production of conidia by individual conidiophores of Blumeria graminis f. sp. hordei on barley leaves by digital microscopic techniques with electrostatic micromanipulation

Conidial formation and secession by living conidiophores of Blumeria graminis f. sp. hordei on barley leaves were consecutively monitored using a high-fidelity digital microscopic technique combined with electrostatic micromanipulation to trap the released conidia. Conidial chains formed on conidiophores through a series of septum-mediated division and growth of generative cells. Apical conidial cells on the conidiophores were abstricted after the conidial chains developed ten conidial cells. The conidia were electrically conductive, and a positive charge was induced in the cells by a negatively polarized insulator probe (ebonite). The electrostatic force between the conidia and the insulator was used to attract the abstricted conidia from the conidiophores on leaves. This conidium movement from the targeted conidiophore to the rod was directly viewed under the digital microscope, and the length of the interval between conidial septation and secession, the total number of the conidia produced by a single conidiophore, and the modes of conidiogenesis were clarified. During the stage of conidial secession, the generative cells pushed new conidial cells upwards by repeated division and growth. The successive release of two apical conidia was synchronized with the successive septation and growth of a generative cell. The release ceased after 4-5 conidia were released without division and growth of the generative cell. Thus, the life of an individual conidiophore (from the erection of the conidiophore to the release of the final conidium) was shown to be 107 h and to produce an average of 33 conidia. To our knowledge, this is the first report on the direct estimation of life-long conidial production by a powdery mildew on host leaves.     

A NEMATODE-CAPTURING PHYCOMYCETE WITH DISTALLY ADHESIVE BRANCHES AND PROXIMALLY IMBEDDED FUSIFORM CONIDIA

Drechsler , Charles . (U.S.D.A., Plant Industry Sta., Beltsville, Md.) A nematode-capturing phycomycete with distally adhesive branches and proximally imbedded fusiform conidia. Amer. Jour. Bot. 49(10): 1089–1095. Illus. 1962.—A fungus newly described as Acaubpage pectospora, though some uncertainty remains as to its taxonomic relation, captures nematodes (Bunonema sp.) by means of small sticky knobs disposed on the surface of the substratum, where they are supported on upcurved tips of continuous subsurface hyphae and of short subsurface hyphal branches. After effecting capture, the knob grows out at its apex and produces a clavate or dolioform appressorium together with an infection-bulb broadly intruded into the animal. From the infection-bulb assimilative hyphae are extended lengthwise through the fleshy interior. Asexual reproduction ensues through development of solitary, colorless, slenderly fusiform conidia, gradually but markedly tapered toward both ends, mostly about 200 μ long and 11 μ wide. Each conidium is connected with a subsurface hypha by an isthmus about 1 μ wide. While its cornuate proximal end is securely imbedded in the substratum, the conidium for about seven-eights of its length extends erectly into the air, thereby contrasting with the submerse elongated “gemmae” of Zoophagus tentaclum and of some other robust rotifer-capturing fungi that have been referred to Z. insidians.     

Protoplast Fusion of Trichoderma reesei, Using Immature Conidia

Protoplast fusion of strains derived from Trichoderma reesei QM9414 and QM9136 and the segregation of the resulting fusants were studied. Combinations of protoplasts prepared from young conidia with double amino acid requirements, one of which was a common requirement and the other uncommon, were fused in the presence of polyethylene glycol 6000. Fusants were selected as regenerant colonies requiring only the commonly deficient amino acid. The frequency of fusion was 0.9 × 10⁻⁴ to 4.0 × 10⁻⁴ for the starting conidia and 3.0 × 10⁻² to 4.9 × 10⁻² for the regenerated protoplasts, which was significantly higher than the expected reversion frequencies by mutation. Conidia generated on the fusant colonies showed diverse phenotypes, i.e., parental types (40 to 80%) and nonparental types (20 to 60%). Colonies developed from single conidia of the nonparental phenotype contained special spots called “knobs” that have a higher density of mycelia. The phenotype of the knobs was again varied among prototrophs, parental types, and recombinant types; and their traits were inherited stably. The phenotype of the mycelia in the nonknob part was essentially the same as that of the original conidia and again formed knobs in colonies upon transfer of a piece of mycelia to a fresh medium. The conidial DNA content of the knob clone was almost the same as that of the parents, but that of the fusants was 1.2 to 2.0 times higher than that of the parents. From these results, we conclude that knobs are the segregants from the fusants. One knob clone showed twice the carboxymethyl cellulose hydrolyzing activity of the parents, suggesting the possibility of breeding T. reesei cells by the protoplast fusion technique.     

Phylogenetics and evolution of nematode-trapping fungi (Orbiliales) estimated from nuclear and protein coding genes

The systematic classification of nematode-trapping fungi is redefined based on phylogenies inferred from sequence analyses of 28S rDNA, 5.8S rDNA and beta-tubulin genes. Molecular data were analyzed with maximum parsimony, maximum likelihood and Bayesian analysis. An emended generic concept of nematode-trapping fungi is provided. Arthrobotrys is characterized by adhesive networks, Dactylellina by adhesive knobs, and Drechslerella by constricting-rings. Phylogenetic placement of taxa characterized by stalked adhesive knobs and non-constricting rings also is confirmed in Dactylellina. Species that produce unstalked adhesive knobs that grow out to form loops are transferred from Gamsylella to Dactylellina, and those that produce unstalked adhesive knobs that grow out to form networks are transferred from Gamsylella to Arthrobotrys. Gamsylella as currently circumscribed cannot be treated as a valid genus. A hypothesis for the evolution of trapping-devices is presented based on multiple gene data and morphological studies. Predatory and nonpredatory fungi appear to have been derived from nonpredatory members of Orbilia. The adhesive knob is considered to be the ancestral type of trapping device from which constricting rings and networks were derived via two pathways. In the first pathway adhesive knobs retained their adhesive material forming simple two-dimension networks, eventually forming complex three-dimension networks. In the second pathway adhesive knobs lost their adhesive materials, with their ends meeting to form nonconstricting rings and they in turn formed constricting rings with three inflated-cells.     

High infectivity of an endoparasitic fungus strain, <Emphasis Type="Italic">Esteya vermicola</Emphasis>, against nematodes

Esteya vermicola, as the first recorded endoparasitic fungus of pinewood nematodes, exhibits great potential as a biological agent against nematodes. However, only two strains of this species have been described so far. In this study, we identified a novel endoparasitic fungal strain, CNU 120806, isolated from infected nematodes in forest soil samples during a survey of nematophagous fungi in Korea. This strain showed similar morphological characteristics and infection mode with the two previously described strains of E. vermicola. All strains are characterized by the ability to produce two types of conidiogenous cells and conidia, and to parasitize nematodes with lunate adhesive conidia. Moreover, the CNU 120806 strain showed 100% identity with E. vermicola CBS 115803 when their partial sequences of 28S rRNA gene were compared. Molecular phylogenetic analysis further identified CNU 120806 as a strain of E. vermicola, by clustering CNU 120806 and E. vermicola CBS 115803 into a single subclade. Culture medium influenced the proportion of dimorphic CNU 120806 conidia, and further changed the adhesive and mortality rates of nematodes. The CNU 120806 strain exhibits high infection activity against nematodes on nutrient-rich PDA medium. Almost all tested nematodes were killed within 8 approximately 10 days after inoculation. This study provides justification for further research of E. vermicola, and the application and formulation of this fungus as a bio-control agent against nematodes.     

<Emphasis Type="Italic">Lecophagus vermicola</Emphasis> sp. nov., a nematophagous hyphomycete with an unusual hunting strategy

Lecophagus vermicola sp. nov. is described and illustrated as a predacious (carnivorous) hyphomycete living in bark fissures of living trees of Platanus and other angiosperm and gymnosperm trees, recorded in Hungary, Luxembourg and France. The fungus captures nematodes unlike other Lecophagus species, which are predators of rotifers and tardigrades. The morphology of the sessile, adhesive knobs differ from all previously described species of the genus which form adhesive pegs. Molecular data confirms that the new species belongs to the Lecophagus clade but without matching existing sequences. The fungus captures victims with adhesive knobs and colonizes its prey with a mycelium of rather broad hyphae on which, again, adhesive knobs are formed which penetrate the cuticule of the victim. Clusters of colonized nematodes form a network utilized to capture more prey. The fungus lives in the xeric, ephemerally aquatic habitat of bark fissures of standing, living or dead, corticated trunks and branches. The genus Haptocara is compared, which has similar adhesive knobs capturing nematodes and similar broad hyphae, but for which no molecular data was available.     

Plasmodium falciparum-infected erythrocytes: qualitative and quantitative analyses of parasite-induced knobs by atomic force microscopy

We used the combination of an atomic force microscope and a light microscope equipped with epifluorescence to serially image Plasmodium falciparum-infected erythrocytes. This procedure allowed us to determine unambiguously the presence and developmental stage of the malaria parasite as well as the number and size of knobs in singly, doubly, and triply infected erythrocytes. Knobs are not present during the ring stage of a malaria infection but a lesion resulting from invasion by a merozoite is clearly visible on the erythrocyte surface. This lesion is visible into the late trophozoite stage of infection. Knobs begin to form during the early trophozoite stage of infection and have a single-unit structure. Our data suggest the possibility that a two-unit structure of knobs, which was reported by Aikawa et al. (1996, Exp. Parasitol. 84, 339-343) using atomic force microscopy, appears to be a double-tipped image. The number of knobs per unit of host cell surface area is directly proportional to parasite number in both early and late trophozoite stages. These results indicate that knob formation by one parasite does not influence knob formation by other parasites in a multiply infected erythrocyte. In addition, knob volume is not influenced by either parasite stage or number at the late trophozoite stage, indicating that the number of component molecules per knob is constant throughout the parasite maturation process.     

Ecology of nematophagous fungi: vertical distribution in a deciduous woodland

Summary The distribution of nematophagous fungi in soil collected from a deciduous woodland is compared to various biotic and abiotic soil factors. The microfungi are isolated at all depths down to a maximum of 35 cm. Predators forming constricting rings, adhesive branches and adhesive knobs are restricted to the upper litter and humus layers. The net forming predators and endoparasites are isolated at all depths, although they are significantly more abundant in the lower mineral rich soils. A much greater species diversity of nematophagous fungi is recorded in the upper organic zones.Preliminary soil analysis indicates thatCephalosporium balanoides is independent of all soil variables, while predators able to form traps spontaneously are restricted to the organic soils which are rich in nematodes. Non-spontaneous trap forming predators, which are excellent saprophytes, are isolated from the deeper soils which are low in nutrients. The ecological significance of these results is discussed.     

Effects of chelators (EGTA, EDTA) and calcium ions on nematode capture by the nematode-trapping fungusArthrobotrys ellipsospora

The nematode-trapping fungusArthrobotrys ellipsospora developed an adhesive knob and trapped nematodes when cultured on a low-nutrient medium. It also trapped polystyrene beads in the same way. The adhesive knob produced mucus that was stained with alcian blue, while mycelium of the fungus was stained with periodic acid/Schiff (PAS). The amount of mucus increased with in days after culturing in the low-nutrient media. The fungus completely lost its ability to trap nematodes when treated with EDTA and EGTA, but it recovered the ability after incubation in the presence of a low concentration of Ca (10⁻⁶–10⁻⁷ M) for 1 h. Calmodulin inhibitor W-7 also inhibited the trapping ability of the fungus, and there was a significant (p<0.05) difference between the effects of W-7 and W-5. Ca-binding protein was also detected in the fungus.     

Nematophagous fungi from the maritime antarctic: factors affecting distribution

Soil collected from throughout the maritime Antarctic was analyzed to determine the effects of a range of abiotic and biotic soil parameters on the distribution of nematophagous fungi. Endoparasites were far more abundant than predatory fungi due to the greater efficiency of endoparasites in attracting and infecting nematodes, which resulted in a rapid completion of the infection cycle. This allowed endoparasites to colonise even the most exposed soil habitats where conditions favourable for nematode activity were restricted to only a few hours daily. 76% of all endoparasites isolated formed adhesive conidia. The success of this group of endoparasites was due to the ability of the conidia to attract nematodes.Spontaneous trap forming predators were far more abundant than nonspontaneous trap formers. The former have a clear competitive advantage over the latter, as they are able to attract nematodes to the conidia which subsequently germinate and form traps only when induced by direct chemical stimulation of nematodes. These predators are able to use internal reserves only and so rapidly respond to short periods when the soil is unfrozen and the nematodes are active.Although nematodes were utilized by all the species isolated, many were also capable of using Protozoa and Rotifera as a food source. While endoparasites were capable of infecting Rotifera, they were never observed infecting, or were isolated from soil containing, Protozoa. The distribution of endoparasites and spontaneous trap forming predators was largely independent of abiotic soil parameters. They were however significantly associated with the presence and abundance of suitable prey. These species survive solely on nematodes and therefore do not require prolonged periods of suitable soil conditions in order to produce vegetative mycelium to adsorb nutrients saprophytically from the soil, which is a significant ecological advantage over the other groups of microfungi found in the Antarctic.     

A NEMATODE-DESTROYING HYPHOMYCETE FORMING PARALLEL MULTISEPTATE HYALINE CONIDIA IN CIRCULAR ARRANGEMENT

In several Petri plate cultures that had been inoculated with decaying plant detritus from central Maryland, a fungus developed which attacks nematodes by extending a rather wide infection-tube either from an adhesive hyphal protuberance, or, more often, from a subglobose adhesive cell produced terminally on its elongate-ellipsoid pluriseptate hyaline conidia. A number (sometimes three) of these conidia are borne simultaneously in peripheral positions on the broadly rounded tip of the colorless condiophore, and are directed upward, parallel to the conidiophore axis. From the segment immediately below its adhesive cell, each conidium puts forth laterally a flexible unicellular tubular appendage that grows downward approximately to the level of the spore attachment. The fungus is described as Haptocara latirostrum gen. n., sp. n. Although presumably belonging in the Moniliaceae, it shows no close kinship with the numerous interrelated clampless Hyphomycetes that have been made known as subsisting through capture of nematodes.     

Fine structure of the knobby spore type of Streptomyces torulosus

The type strain of Streptomyces torulosus Lyons and Pridham (1971) was studied by scanning- and transmission electron microscope. Spore chains were formed in spirals by aerial mycelium. The spores were connected by nozzles in which small channels could be observed. The knobby ornamentations of the spores arised on a thin fibrous sheath, enveloping the spore chains. These irregular blunt projections, called knobs, had varying diameters of 100 to 250 nm. The base of the knob, consisting of globose to flattened electron dense material, was sitting directly on the sheath. It was covered by several small vesicles of the same material. Each hollow vesicle beared a thin bowlshaped shell of electron transparent material. In general, the cupular bowls and their supporting vesicles became easily depressed on their base, but not detached from the surface of the spores. This type of knobby spore ornamentation was suggested to be designated as a verrucate spore type.     

Fusarium globosum from subtropical Japan and the effect of different light conditions on its conidiogenesis

The second report ofFusarium globosum is based on strains isolated from wheat in Ishigaki Island, Okinawa, in subtropical Japan. Morphological features of the Japanese isolates are described. These strains show different reactions in conidiogenesis to light conditions. Under continuous BLB light, falcate sporodochial conidia are typically induced, but production of aerial globose conidia is suppressed. In two of the strains, clavate conidia became longer under BLB light. Application of both, complete darkness and continuous BLB light, is recommended as standard light conditions to cultureFusarium isolates. Collaborator via fellowship under OECD Project on Biological Resource Management.