An ultrastructural study of cell-cell interactions in capture organs of the nematophagous fungus Arthrobotrys oligospora

Abstract A detailed ultrastructural analysis was made of interactions between individual cells within the same adhesive network (trap) of the nematophagous fungus Arthrobotrys oligospora . These interactions were confined to traps which had captured nematodes, and occurred concurrently with the fungus-nematode interactions. The process was initiated by the anchoring of 2 cells of different loops constituting the trap network, by means of the adhesive coating of these cells. Subsequently, penetration tubes were formed. As in nematode-fungus interactions, the walls of these tubes arose from underneath the original trap cell walls. Two response were observed: either only one of the anchored cells formed a penetration tube, which penetrated the opposite cell and subsequently digested its contents; or both cells formed penetration tubes simultaneously, which were directed against each other. In the latter case, no penetration of other cells was observed, and elongated tubes were occasionally formed. The above mechanism differed from 2 other modes of interaction also observed, namely fusion of intact cells (anastomosis) and development of new hyphae inside dead hyphal cells. In the latter case the newly formed cells developed from the cross-wall of the neighbouring intact cell.     

<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.     

Effect of different level of inoculums of root knot nematode on predacity of Arthrobotrys oligospora

Arthrobotrys oligospora is one of the most important predaceous fungi occurring widely in different types of soil. This fungus produces the hyphal bails and network compound. This hyphal nets as trapping structure which may be one dimensional to two or three dimensional and are formed through repeated hyphal anastomosis, which capture nematodes either through the adhesive material present on the surface of hyphal nets or due to physical entanglement. When moving nematodes pass through such hyphal bails, they are captured by sticky substances present on the bails. Such nematodes struggle violently to escape the capture but they are usually entangled at several places of their body. They absorb the nutrients and finally kill the nematodes. Irrespective of the different levels of inoculums of root knot nematode, capturing of nematodes increased with increasing period of incubation. After 5 days of inoculation, 93–97.33% nematodes were captured irrespective of the nematode number.     

Predation of entomopathogenic nematodes by <Emphasis Type="Italic">Sancassania</Emphasis> sp. (Acari: Acaridae)

Predation of the entomopathogenic nematode, Steinernema feltiae (Rhabditida: Steinernematidae), by Sancassania sp. (Acari: Acaridae) isolated from field-collected scarab larvae was examined under laboratory conditions. Adult female mites consumed more than 80% of the infective juvenile (IJ) stage of S. feltiae within 24 h. When S. feltiae IJs were exposed to the mites for 24 h and then exposed to Galleria mellonella (Lepidoptera: Pyralidae) larvae, the number of nematodes penetrating into the larvae was significantly lower compared to S. feltiae IJs that were not exposed to mites (control). Soil type significantly affected the predation rate of IJs by the mites. Mites preyed more on nematodes in sandy soil than in loamy soil. We also observed that the mites consumed more S. feltiae IJs than Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae). No phoretic relationship was observed between mites and nematodes and the nematodes did not infect the mites.     

Consumption of nematodes by fungivorous mites,Tyrophagus spp. (Acarina: Astigmata: Acaridae)

Summary Free-living nematodes may be attacked and eaten by soil mites which are normally considered fungivores or saprophages. Three species in the genus Tyrophagus, common inhabitants of grassland soils and also common pests in stored products, museums and laboratories, are predators of nematodes. All active stages of the mites will voraciously consume nematodes. When offered nematodes and a choice of other food (baker's yeast and algae), 11% of the Tyrophagus putrescentiae, 23% of the T. zachvatkini, and 56% of the T. similis tested fed on nematodes. Tyrophagus zechvatkini and similis were reared on a diet consisting entirely of nematodes, and developed at rates similar to a fungal diet and produced viable offspring. Agar cultures of Aphelenchus avenae which were inoculated with five mating pairs of Tyrophagus zachvatkini had populations that were one-third less than mite-free controls. Observations indicate that nematodes may be attacked by tyrophagid mites when in a dry, anhydrobiotic state.     

Experimental study on the impact of dinoflagellate Alexandrium species on populations of the rotifer Brachionus plicatilis

To investigate harmful effects of the dinoflagellate Alexandrium species on microzooplankton, the rotifer Brachionus plicatilis was chosen as an assay species, and tested with 10 strains of Alexandrium including one known non-PSP-producer (Alexandrium tamarense, AT-6). HPLC analysis confirmed the PSP-content of the various strains: Alexandrium lusitanicum, Alexandrium minutum and Alexandrium tamarense (ATHK, AT5-1, AT5-3, ATCI02, ATCI03) used in the experiment were PSP-producers. No PSP toxins were detected in the strains Alexandrium sp1, Alexandrium sp2.Exposing rotifer populations to the densities of 2000 cells ml⁻¹ of each of these 10 Alexandrium strains revealed that the (non-PSP) A. tamarense (AT-6) and two other PSP-producing algae: A. lusitanicum, A. minutum, did not appear to adversely impact rotifer populations. Rotifers exposed to these three strains were able to maintain their population numbers, and in some cases, increase them. Although some increases in rotifer population growth following exposures to these three algal species were noted, the rate was less than for the non-exposed control rotifer groups.In contrast, the remaining seven algal strains (A. tamarense ATHK, AT5-1, AT5-3, ATCI02, ATCI03; also Alexandrium sp1 and Alexandrium sp2) all have adverse effects on the rotifers. Dosing rotifers with respective algal cell densities of 2000 cells ml⁻¹ each, for Alexandrium sp1, Alexandrium sp2, and A. tamarense strains ATHK and ATCI03 showed mean lethal time (LT₅₀) on rotifer populations of 21, 28, 29, and 36h, respectively. The remaining three species (A. tamarense strains AT5-1, AT5-3, ATCI02) caused respective mean rotifer LT₅₀s of 56, 56, and 71 h, compared to 160 h for the unexposed “starved control” rotifers. Experiments to determine ingestion rates for the rotifers, based on changes in their Chlorophyll a content, showed that the rotifers could feed on A. lusitanicum, A. minutum and A. tamarense strain AT-6, but could graze to little or no extent upon algal cells of the other seven strains. The effects on rotifers exposed to different cell densities, fractions, and growth phases of A. tamarense algal culture were respectively compared. It was found that only the whole algal cells had lethal effects, with strongest impact being shown by the early exponential growth phase of A. tamarense. The results indicate that some toxic mechanism(s), other than PSP and present in whole algal cells, might be responsible for the adverse effects on the exposed rotifers.     

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.     

An electron-microscopical analysis of capture and initial stages of penetration of nematodes byArthrobotrys oligospora

A detailed analysis was made of the capture and subsequent penetration of nematodes by the nematophagous fungusArthrobotrys oligospora using different electron-microscopical techniques. Capture of nematodes by this fungus occurred on complex hyphal structures (traps) and was effectuated by an adhesive coating, present on these trap cells. The adhesive layer was largely fibrillar in nature and was absent on cells of normal hyphae. Following capture, penetration hyphae were formed at those sites where the trap cell wall was anchored to the nematode cuticle by the adhesive. New walls of these hyphae were formed underneath the original trap cell walls, which were partly hydrolysed to allow growth and development of the penetration tubes through the adhesive coating towards the cuticle. Our observations indicated that the cuticle of the nematode was subsequently penetrated by the penetration tubes by mechanical means. After penetration a large infection bulb was formed from which trophic hyphae arose. Cytochemical experiments indicated that the sites of penetration of the cuticle were intensely stained for acid phosphatase activity. At later stages of infection activity of this enzyme was present throughout the nematode contents; the enzyme was most probably secreted by complex membranous structures associated with the cytoplasmic membrane of the infection bulb and the trophic hyphae.     

Genetic variability within isolates of Colletotrichum lindemuthianum belonging to race 65 from the state of Minas Gerais, Brazil

Colletotrichum lindemuthianum, the causal agent of anthracnose in the common bean (Phaseolus vulgaris), presents a wide genetic and pathogenic variability that gives rise to complications in the development of resistant bean cultivars. The aim of this study was to identify the variability within race 65 of C. lindemuthianum, the race most commonly encountered in Brazil, through randomly amplified polymorphic DNA (RAPD) and anastomosis analyses. Thirteen isolates of race 65, collected in different years and from various host cultivars located in diverse areas of the state of Minas Gerais, Brazil, were investigated. Twenty-four RAPD primers were employed and 83 polymorphic bands amplified. Genetic similarities were estimated from the Sorensen-Dice coefficient and ranged from 0.54 to 0.82. The dendrogram obtained by cluster analysis classified the isolates into 11 separate groups. For the purposes of the analysis of anastomosis, isolates were considered to be compatible when the fusion of hyphae from different isolates could be observed. The proportion of compatible reactions for each isolate was estimated and similarity estimates, based on the Russel & Rao coefficient, ranged from 0.28 to 0.85. Isolates were classified into 11 anastomosis groups, 10 of which were formed by only one isolate. Although isolates LV61, LV73 and LV58 were classified in the same anastomosis group, they were genetically distinct according to RAPD analysis. Results from both RAPD and anastomosis analyses revealed great variability within C. lindemuthianum race 65.     

Characterization of the nematocidal toxocyst in <Emphasis Type="Italic">Pleurotus</Emphasis> subgen. <Emphasis Type="Italic">Coremiopleurotus</Emphasis>

Toxocysts of the genus Pleurotus are blastoconidia-like ovoid structures surrounded by a liquid droplet containing a toxin that paralyzes nematodes. This study investigated toxocyst development using a strain S396 of Pleurotus cystidiosus subsp. abalonus (subgen. Coremiopleurotus). The surface of the liquid droplet was found to be an elastic envelope. When a nematode touches the toxocyst, the envelope adheres to the worm and bursts. Toxocysts are induced simultaneously with coremia formation in the absence of nematodes and developed only from aerial hyphae in which nuclear division had ceased. In the early stage of toxocyst development, liquid springs repeatedly from the tip of the sterigma-like stipe before ovoid (blastoconidium-like structure) formation. A certain substance in the liquid might polymerize to form the envelope while the ovoid simultaneously budded in the droplet. The nucleus tends to locate near the toxocyst, especially in early stage of toxocyst development. Each dikaryotic cell predominantly formed one or two toxocyst(s) while each monokaryotic cell predominantly formed one. In rare cases, a nucleus existed in the toxocyst, suggesting the possibility that the toxocyst is a vestigial blastoconidium.     

Vacuolar Reticulum in Oomycete Hyphal Tips: An Additional Component of the CaRegulatory System?

Cultures ofAchlyasp.,Phytophthora cinnamomi, Saprolegnia diclina, S. ferax,andS. parasitica,treated with 6-carboxyfluorescein diacetate solution, accumulate 6-carboxyfluorescein in a reticulate system of fine tubules. The network shows longitudinal polarity within the hyphae, tubules being finest toward the hyphal tips. In more mature subapical regions the network is connected with large vacuoles that also accumulate 6-carboxyfluorescein. A morphologically similar system has also been identified in freeze-substituted hyphae ofS. ferax.The network is considered to be vacuolar, but differs from the tubular vacuole system of true fungi in that tubules are less motile, more frequently branched, and do not alternate with clusters of spherical vacuoles. The appearance of the network resembles patterns of calcium-sensitive dye staining and it is suggested that the vacuolar reticulum in the tip region of oomycete hyphae may act as a Ca²⁺sink. The tubular reticulum in oomycetes is very fragile and can be shown with 6-carboxyfluorescein in only those hyphal tips with a motility and organelle distribution characteristic of growing hyphae with normal morphology. Diverse abnormal hyphae show a range of other fluorochrome localizations. These include large irregular compartments filled with fluorochrome, and fluorescent cytoplasm with organelles and vacuoles standing out in negative contrast. These localizations in abnormal hyphae are correlated with other structural changes indicative of damage. Special care is required in experiments with oomycetes to avoid such artefacts of localization.     

Aerial dispersal of European red mites (Acari: Tetranychidae) in commercial apple orchards

Aerial dispersal of European red mite, Panonychus ulmi (Koch), in commercial apple orchards was estimated by trapping windborne mites. Studies were conducted at four orchards in eastern New York during 1989 and 1990 and at three orchards in western New York during 1989. In each orchard mites were trapped in three locations; the interior of the orchard, at the border of the orchard and in a field or woodlot beyond the orchard. Large numbers of mites were captured, even when the numbers of mites on apple foliage were well below levels where mite injury to leaves was visible (less than five per leaf). The log numbers of mites trapped were linearly related to the log density of mites on leaves and this relationship was consistent for each year and region the study was conducted. The trap captures among the three locations in and outside an orchard were highly correlated. The implications these findings may have on metapopulation dynamics and resistance to acaricide dynamics are discussed.     

Parasexual analysis of common-A crosses in the basidiomycete Agrocybe aegerita

Summary Crosses were performed between homokaryons of Agrocybe aegerita having the same allele at the A incompatibility gene but different B alleles. Heterokaryotic mycelia originating from crosses between two complementary auxotrophs were characterized by their instability on complete medium and extensive anastomosis between hyphae. Diploid mycelia were selected by plating oidia recovered from these heterokaryons onto minimal medium. These mycelia were characterized by the production of larger oidia than those of homokaryons, the release of a brown pigment when growing on complete medium and extensive hyphal anastomoses. Diploids retained the two B incompatibility functions of their homokaryotic parents and gave rise to a diploid/haploid dikaryon when crossed with a compatible homokaryon. Nearly 1% of the oidia recovered from heterokaryons were diploid. These nuclear fusion frequencies as well as the production of brown pigments enabled the identification of diploid strains on complete medium. In this way, crosses between wild prototrophic strains were successfully performed. Somatic recombination was induced following the treatment of diploid mycelia with haploidizing compounds. Selection based on the inability of mycelia to produce the brown pigments on complete medium led to selection of strains homoallelic at the B locus.     

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.     

A novel approach to biological control with entomopathogenic nematodes: Prophylactic control of the peachtree borer, Synanthedon exitiosa

Generally, microbial control agents such as entomopathogenic nematodes are applied in a curative manner for achieving pest suppression; prophylactic applications are rare. In this study, we determined the ability of two Steinernema carpocapsae strains (All and Hybrid) to prophylactically protect peach trees from damage caused by the peachtree borer, Synanthedon exitiosa, which is a major pest of stone fruit trees in North America. In prior studies, the entomopathogenic nematodes S. carpocapsae and Heterorhabditis bacteriophora caused field suppression when applied in a curative manner to established S. exitiosa populations. In our current study, nematodes were applied three times (at 150,000–300,000 infective juveniles/tree) during September and October of 2005, 2006, and 2007. A control (water only) and a single application of chlorpyrifos (at the labeled rate) were also made each year. The presence of S. exitiosa damage was assessed each year in the spring following the treatment applications. Following applications in 2006, we did not detect any differences among treatments or the control (possibly due to a low and variable S. exitiosa infestation of that orchard). Following applications in 2005 and 2007, however, the nematode and chemical treatments caused significant damage suppression. The percentage of trees with S. exitiosa damage in treated plots ranged from 0% damage in 2005 to 16% in plots treated with S. carpocapsae (Hybrid) in 2007. In control plots damage ranged from 25% (2005) to 41% (2007). Our results indicate that nematodes applied in a preventative manner during S. exitios’s oviposition period can reduce insect damage to levels similar to what is achieved with recommended chemical insecticide treatments.     

Trichoderma species form endophytic associations within Theobroma cacao trichomes

Trichoderma species are usually considered soil organisms that colonize plant roots, sometimes forming a symbiotic relationship. Recent studies demonstrate that Trichoderma species are also capable of colonizing the above ground tissues of Theobroma cacao (cacao) in what has been characterized as an endophytic relationship. Trichoderma species can be re-isolated from surface sterilized cacao stem tissue, including the bark and xylem, the apical meristem, and to a lesser degree from leaves. SEM analysis of cacao stems colonized by strains of four Trichoderma species (Trichoderma ovalisporum-DIS 70a, Trichoderma hamatum-DIS 219b, Trichoderma koningiopsis-DIS 172ai, or Trichoderma harzianum-DIS 219f) showed a preference for surface colonization of glandular trichomes versus non-glandular trichomes. The Trichoderma strains colonized the glandular trichome tips and formed swellings resembling appresoria. Hyphae were observed emerging from the glandular trichomes on surface sterilized stems from cacao seedlings that had been inoculated with each of the four Trichoderma strains. Fungal hyphae were observed under the microscope emerging from the trichomes as soon as 6 h after their isolation from surface sterilized cacao seedling stems. Hyphae were also observed, in some cases, emerging from stalk cells opposite the trichome head. Repeated single trichome/hyphae isolations verified that the emerging hyphae were the Trichoderma strains with which the cacao seedlings had been inoculated. Strains of four Trichoderma species were able to enter glandular trichomes during the colonization of cacao stems where they survived surface sterilization and could be re-isolated. The penetration of cacao trichomes may provide the entry point for Trichoderma species into the cacao stem allowing systemic colonization of this tissue.     

Influence of temperature and body weight on mosquito predation by the dragonfly nymph Mesogomphus lineatus

Population densities, migrations and food composition of Echinogammarus veneris in Lake Kinneret were studied during march–September 1976. Migrations are affected by changes of water level and littoral currents. Peridinium was abundant in the gut content during its blooming period in the lake while cladocerans, benthic copepods, rotifers and other algae were abundantly found in other months. Metabolic parameters that were calculated for the gammarid population indicated that its contribution to the carbon-flow system in Lake Kinneret is negligible in comparison with nematodes, molluscans or planktonic copepods and cladocerans.     

Development of Dactylaria eudermata Drechsler a predaceous fungi in Meloidogyne incognita

Dactylaria eudermata Drechsler is one of the important predaceous fungi occurring widely in different soils. The fungus produces hyphal bails and network compound in which nematodes are entangled. This hyphal nets as trapping structure which may be single dimensional to two or three dimensional and are formed through repeated hyphal anastomosis, which capture nematodes either through the adhesive material present on the surface of hyphal nets or due to physical entanglement. In the experiment it was observed that inflation of hyphal bails takes 30 to 50 minutes and capturing and killing the nematode by a single conidia in water takes 35–55 hours, were as time required for inflation of hyphal bails and real trapping and killing of a nematode in maize meal (MMA: water (1:10) medium) takes one minute and 30–50 hours respectively.     

Lipid classes and fatty acid composition of rotifers (Brachionus plicatilis) fed two algal diets

Rotifers (Brachionus plicatilis), maintained on baker's yeast, were fed for 24h upon two algal diets, Isochrysis galbana (diet A) and Isochrysis galbana + Nannochloropsis gaditana (diet B). (These algal diets were selected for their potential use as essential fatty acid (EFA) boosters, taking into account the requirements of fish larvae). The effect of these algal diets on total lipid content, lipid classes and fatty acid composition was studied. The total lipid content increased after feeding upon both diets but no significant differences were found between the two types. Neutral lipid and polar lipid contents increased and a positive correlation was observed between the neutral lipids content of rotifers and that of the food supplied. However, the content of polar lipids in rotifers did not depend upon that of the diet. The increase in neutral lipid content was found to be higher in rotifers fed upon diet B, compared to diet A which increased the phospholipid content. Non-enriched rotifers contained only small amounts of polyenoic fatty acids, i.e. 18:3n-6, 18:3n-3, 20:4n-6, 20:5n-3 and 22:6n-3, the contents of which increased significantly by feeding both diets. The EFA composition (20:4n-6, 20:5n-3 and 22:6n-3) of neutral lipids and phopholipids in rotifers reflected the EFA composition of each diet. Diet B-fed rotifers had the highest content in 20:4n-6 and 20:5n-3, whereas rotifers fed diet A and the highest 22:6n-3 content. The mixed diet I. galbana + N. gaditana enhanced substantially the composition of lipid classes i.e. neutral lipids and of n-3 PUFA of rotifers in comparison with Isochrysis or yeast diets.     

Potentiation of Mycovirus Transmission by Zinc Compounds via Attenuation of Heterogenic Incompatibility in Rosellinia necatrix

Heterogenic incompatibility is considered a defense mechanism against deleterious intruders such as mycovirus. Rosellinia necatrix shows strong heterogenic incompatibility. In the heterogenic incompatibility reaction, the approaching hyphae hardly anastomosed, a distinctive barrage line formed, and green fluorescent protein (GFP)-labeled hyphae quickly lost their fluorescence when encountering incompatible hyphae. In this study, transmission of a hypovirulence-conferring mycovirus to strains with different genetic backgrounds was attempted. Various chemical reagents considered to affect the programmed cell death pathway or cell wall modification were examined. Treatment with zinc compounds was shown to aid in transmission of mycoviruses to strains with different genetic backgrounds. In incompatible pairings, treatment with zinc compounds accelerated hyphal anastomosis; moreover, cytosolic GFP was transmitted to the newly joined hyphae. These results suggest that zinc compounds not only increase hyphal anastomosis but also attenuate heterogenic incompatibility.