Occurrence and metabolic significance of microbodies in trophic hyphae of the nematophagous fungus Arthrobotrys oligospora

This paper describes the results of an ultrastructural study on the subcellular events occurring in nematode-infecting (trophic) hyphae of the nematophagous fungus Arthrobotrys oligospora. In early stages of the infection process (30 min-4 h), the infection bulb and developing trophic hyphae are characterized by a highly proliferated endoplasmic reticulum (ER). Its membranes often appeared vesiculated and occur in close association with the cell membrane of the cells. Upon further invasion of the nematode, lipid droplets developed in the trophic hyphae; these droplets were first observed 4–5 h after the infection but were abundantly present after 24–36 h. Along with the formation of lipid droplets proliferation of microbodies was observed. These organeles were characterized by the presence of catalase and thiolase and were frequently observed in close association with the lipid droplets. Later on the lipid droplets disappeared. During this period new vegetative mycelium developed from the trap that had originally captured the nematode. Our results suggest that part of the nutrients released from the nematode are first converted into lipids by the fungus which in turn are degraded via the -oxidation pathway and further metabolized to support growth of new vegetative hyphae.     

Occurrence, characterization and development of two different types of microbodies in the nematophagous fungus Arthrobotrys oligospora

Abstract The occurrence of microbodies in different cells of the nematophagous fungus Arthrobotrys oligospora has been investigated. In the predacious phase this organism forms complex 3-dimensional network traps. Mature trap cells generally were crowded with "special" microbodies which possessed an electron dense matrix and were surrounded by a membrane of approx. 9 nm. These organelles developed during the early stages of trap formation and were derived from specialized regions of the endoplasmic reticulum. Cytochemical staining experiments revealed that the electron-dense microbodies contained catalase and d -amino acid oxidase and thus must be considered peroxisomal in nature. Electron-dense bodies were absent in normal vegetative cells of the fungus. These cells contained "normal" microbodies which developed from each other by the separation of small organelles from mature ones. As in yeasts, the metabolic function of these latter organelles was dependent upon environmental conditions.     

Hyphal fusion during initial stages of trap formation in Arthrobotrys oligospora

Hyphal fusion during initial stages of trap formation by Arthrobotrys oligospora was studied by video-enhanced contrast and electron microscopy. Trap initials grew perpendicularly to the parent hypha, then curved around and anastomosed with a peg that developed on the hypha. Trap initials usually developed 40–140 m apart while the anastomosis occurred 20–25 m from the initial. Vigorous cytoplasmic movements in trap initials and developed traps corresponded to intense staining with fluorescein diacetate (FDA) of these cells. In addition, bundles of microfilaments were seen in developing loops of traps. On fusion organelle migration took place from the tip cell of the trap into the peg. Later on a septum was formed at the site of fusion.     

Improvement on genetic transformation in the nematode-trapping fungus Arthrobotrys oligospora and its quantification on dung samples

An improved DNA-mediated transformation system for nematode-trapping fungus Arthrobotrys oligospora based on hygromycin B resistance was developed. The transformation frequency varied between 34 and 175 transformants per μg linearized DNA and 93% of the transformants were stable for drug resistance when tested 100 randomly selected transformants. More than 2000 transformants were obtained by transformation of the fungus with pBChygro in the presence of HindIII and among them, one, YMF1.00110, which lost its ability of forming predacious structure, was isolated. Southern analysis showed that the plasmid DNA had integrated into the genome of all tested transformants (including YMF 1.00110) except one. The transformant tagged with hph gene could be re-isolated and quantified from dung samples based on the resistance of hygromycin B. All the results suggested that the method of restriction enzyme mediated integration (REMI) should facilitate not only the insertional mutagenesis for tagging and analysis genes of interest but also the ecological investigation of tagged fungi in a given environment.     

An endogenous rhythm of trap formation in the nematophagous fungus Arthrobotrys oligospora

In the predacious fungus Arthrobotrys oligospora Fres., the number and distribution of traps formed after the addition of living nematodes to the colonies were determined. At 21°C the traps were formed periodically; the mean period was 42.3±0.8 h. The periodicity was independent of light-dark (LD) cycles of 24 h (10:14). Temperature influenced the hyphal elongation but did not affect the periodic trap formation; at lower temperatures the peaks of trap formation were close together, showing partial overlapping. Induction of rhythmic mycelial growth and conidiation by chemical means was effective only in LD-cycles. The latter diurnal rhythm was weakly correlated with the trap formation and did not affect the endogenous period of approximately 42 h.Abbreviations LD light-dark - DD continuous darkness - LNM low-nutrient medium - CMA corn meal agar     

Proliferation and function of microbodies in the nematophagous fungus Arthrobotrys oligospora during growth on oleic acid or d-alanine as the sole carbon source

Abstract The nematophagous fungus Arthrobotrys oligospora is able to grow on oleic acid or d-alanine as the sole carbon source. During growth on oleic acid, activities of enzymes of the β-oxidation pathway, but not catalase, were induced. In the presence of d-alanine, both d-amino acid oxidase and catalase activities were enhanced. Biochemically and cytochemically, the activities of the above enzymes were assigned to microbodies. The significance of these results in relation to the function of microbodies in trophic hyphae, which are formed during nematode infection, is discussed.     

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.     

Development and fate of electron-dense microbodies in trap cells of the nematophagous fungusArthrobotrys oligospora

The development of electron-dense microbodies in cells of capture organs of the nematophagous fungus Arthrobotrys oligospora was studied with different ultrastructural techniques. Kinetic experiments revealed that the synthesis of these microbodies started in a very early stage of trap formation; the organelles originated from special regions of endoplasmic reticulum by budding. Mature organelles were surrounded by a single membrane of approximately 9 nm (KMnO4-fixation) and lacked crystalline inclusions. The presence of the electron-dense microbodies was independent of the conditions during which the traps had developed. The organelles remained intact during aging of the trap cells. They were also observed in the trophic hyphae after capture and penetration of nematodes. However, the distribution patterns of these organelles in the trophic hyphae, which were identical to those observed after germination of isolated traps on different cultivation media, suggested that their presence must be explained by dilution of organelles in newly formed cells.     

Do Organic Amendments Enhance the Nematode-Trapping Fungi Dactylellina haptotyla and Arthrobotrys oligospora?

Soil cages (polyvinyl chloride pipe with mesh-covered ends) were used to determine how the quantity of two organic amendments affected the nematode-trapping fungi Dactylellina haptotyla and Arthrobotrys oligospora, which were studied independently in two different vineyards. Each cage contained 80 cm³ of field soil (120 g dry weight equivalent), fungal inoculum (two alginate pellets, each weighing 1.9 mg and containing assimilative hyphae of one fungus), and dried grape or alfalfa leaves (0, 360, or 720 mg equivalent to 0, 4,500, or 9,000 kg/ha) with a C:N of 28:1 and 8:1, respectively. Cages were buried in the vineyards, recovered after 25 to 39 days, and returned to the laboratory where fungus population density and trapping were quantified. Dactylellina haptotyla population density and trapping were most enhanced by the smaller quantity of alfalfa amendment and were not enhanced by the larger quantity of alfalfa amendment. Arthrobotrys oligospora population density was most enhanced by the larger quantity of alfalfa amendment, but A. oligospora trapped few or no nematodes, regardless of amendment. Trapping and population density were correlated for D. haptotyla but not for A. oligospora.     

Isolation,identification, and in vitro predatory activity of nematophagous fungus Arthrobotrys musiformis and its interaction with nematodes using scanning electron microscopy

We isolated and identified nematophagous fungus Arthrobotrys musiformis from materials derived from cattle and sheep. We also molecularly characterised the native fungal isolates and evaluated the nematophagous activity of the isolates. A total of 19 isolates of A. musiformis were isolated from 1532 samples, and the detection rate of A. musiformis in all samples was 1.24%. These isolates were identified using a light microscope and their 5.8S rDNA, 18S rDNA, 28S rDNA, and the internal transcribed spacers 1 and 2 region. Interaction of the isolate (NPS045) with the nematode targets of the infective larvae (L₃) of Haemonchus contortus and the free-living nematode Caenorhabditis elegans was observed by scanning electron microscopy (SEM). SEM result showed that the two species of nematodes were initially captured at 5?h after being added to the isolates. L₃ was penetrated at 22?h after capture and completely destroyed by the fungus at 68?h. C. elegans was penetrated at 14?h post-capture and was completely digested by the fungus at 24?h. In vitro experimental assay of samples in 24-well plates showed that for the three fungal isolates, the L₃s of Trichostrongylus colubriforms were reduced by 94.80%, 90.17%, and 89.02%.     

Taxonomic revision of the nematode-trapping fungus Arthrobotrys multisecundaria

The gene encoding an extracellular serine protease was cloned from Arthrobotrys multisecundaria using degenerate primers. The gene was highly similar (99.26%) to protease Mix from Monacrosporium microscaphoides. To clarify the taxonomic relationship between these species, genes encoding the internal transcribed spacer (ITS) and β-tubulin were also cloned and sequenced from A. multisecundaria and M. microscaphoides, respectively. Homologous analysis of the nuclear (ITS) and protein (β-tubulin) encoding genes showed that the two species of nematode-trapping fungi also shared extensive identity (99.82 and 99.63%, respectively), although they exhibited obvious differences in secondary conidia morphology. Accordingly, a taxonomic revision is recommended, with A. multisecundaria being revised as A. microscaphoides var. multisecundaria. In addition, the identified mutation may better facilitate the study of the sporulation of nematode-trapping fungi. These authors contributed equally to this work.     

The pH sensing receptor AopalH plays important roles in the nematophagous fungus Arthrobotrys oligospora

There is well-conserved PacC/Rim101 signaling among ascomycete fungi to mediate environmental pH sensing. For pathogenic fungi, this pathway not only enables fungi to grow over a wide pH range, but it also determines whether these fungi can successfully colonize and invade the targeted host. Within the pal/PacC pathway, palH is a putative ambient pH sensor with a seven-transmembrane domain. To characterize the function of a palH homolog, AopalH, in the nematophagous fungus Arthrobotrys oligospora, we knocked out the encoding gene of AopalH through homologous recombination, and the transformants exhibited slower growth rates, greater sensitivities to cationic and hyperoxidation stresses, as well as reduced conidiation and reduced trap formation, suggesting that the pH regulatory system has critical functions in nematophagous fungi. Our results provide novel insights into the mechanisms of pH response and regulation in fungi.     

Quantification of mycoparasitism by the nematode-trapping fungus Arthrobotrys oligospora on Rhizoctonia solani and the influence of nutrient levels

Abstract The influence of nutrient level, type of carbon source and nitrogen concentration on the parasitism of Arthrobotrys oligospora on Rhizoctonia solani were investigated by quantification of coiling frequency. Changes in coiling frequency were also compared with changes in hyphal density and colony radial growth rate. Increasing concentrations of corn meal agar gave increasing coiling frequency up to a concentration of half the recomended strength. At higher concentrations the coiling frequency was constant, although the hyphal density of both fungi increased over the whole concentration range. Coiling frequency was positively correlated with the probability of hyphal encounter, calculated as the product of the hyphal densities of the two fungi, except at high CMA concentrations. Amongst several carbohydrates tested, glucose resulted in the highest, and sucrose the lowest, coiling frequency. The effect of the different carbohydrates on coiling frequency was not correlated with the hyphal densities of the fungi. Addition of a nitrogen source, NaNO₃, removed the differences in coiling frequency between glucose and sucrose and increased coiling frequencies on both sugars.     

Isolation,identification and characterisation of the nematophagous fungus Arthrobotrys thaumasia (Monacrosporium thaumasium) from China

The objective of this study is to select a native isolate of Arthrobotrys thaumasia (Monacrosporium thaumasium), a nematophagous fungus that shows potential for use in biocontrol programmes for ruminants. First, we looked for native isolates of A. thaumasia and characterised them using light microscopy and molecular markers. Then, we determined the effect of temperature, pH and nutrition on the growth rate and trap formation of a representative isolate. Of the 1532 samples of different types related to sheep and cattle, 11 isolates of A. thaumasia were isolated, and their occurrence frequency in the samples was 0.71% (11/1532). We sequenced the rDNA internal transcribed spacer of isolate NBS005, submitted it to GenBank (ID: KX640093) and then sequenced it using BLAST. The NBS005 could not grow at 37.5°C but could grow from 11°C to 35°C, and it exhibited its optimum growth at 30°C on 1% corn meal agar (CMA). Over 4 days, the fungus did not grow in the pH interval from 1 to 3 or from 13 to 14 but did grow in the pH interval from 4 to 12 and exhibited its optimum growth between pH 9 and 10 on 2% CMA. The factors responsible for the trap formation of NBS005 in liquid culture were identified. Trap formation was induced only by contact with L3 lysate. The concentrations of sucrose, ammonium chloride and tryptone of 0.4, 0.2 and 0.2%, respectively, promoted trap formation, and there were higher numbers of trapping nets in 2% wheat bran liquid medium containing L3 lysate.     

An improved method for light- and electron microscopial studies of nematode/fungal interactions

A method is presented that enables studies to be made of single nematode-fungal interactions under conditions where fungal growth at the expense of external nutrients is prevented. The nematophagous fungus Arthrobotrys ologospora was used as a model organism in these studies. The method is based on removal of the traps from the vegetative mycelium, immediately after a nematode was captured and transfer of the trap with the captured nematode into a droplet of sterile distilled water placed in a moisture chamber. In the absence of external nutrients, such isolated traps of A. oligospora were fully effective in penetrating and subsequently digesting the captured nematode. Solely vegetative mycelium was formed at the expense of the digested nematode; this developed from the trap that originally had captured the nematode. One advantage of the present method is that studies on various stages of the nematode-fungal interaction can now be performed under conditions that exclude major influences of external nutrients which otherwise could be communicated to the trap cells by way of the vegetative mycelium.     

Biological control of sheep parasite nematodes by nematode-trapping fungi: <Emphasis Type="Italic">in vitro</Emphasis> activity and after passage through the gastrointestinal tract

Summary The main method used for the control of gastrointestinal nematodes in sheep production is the application of chemotherapeutic agents, which often lead to the selection of parasites resistant to given active principles. Biological control can be considered a promising alternative, contributing to an increase in the efficacy of verminous control. We determined the in vitro activity and in situ survival of the predatory fungi Arthrobotrys musiformis and Arthrobotrys conoides during passage through the gastrointestinal tract of sheep after oral administration of conidia in microencapsulated form and as a liquid in natura. Initial in vitro tests showed that both fungi were efficient in the predation of trichostrongylid L3 larvae present in the faeces of sheep naturally infected with gastrointestinal nematodes. The fungi presented high nematophagous activity, which was 99.3% for A. conoides and 73.7% for A. musiformis. A. conoides did not survive passage through the gastrointestinal tract under the conditions of the present experiment. On the other hand, A. musiformis was reisolated after administration in either microencapsulated or liquid form, suggesting that this species is a promising alternative for the control of nematodes in sheep since it survives without any protection (in natura).     

Effects of carbon and nitrogen sources,carbon-to-nitrogen ratio,and initial pH on the growth of nematophagous fungus <Emphasis Type="Italic">Pochonia chlamydosporia</Emphasis> in liquid culture

The effects of carbon and nitrogen sources, carbon-to-nitrogen ratio (C:N) and initial pH value on the growth and sporulation of the nematophagous fungus Pochonia chlamydosporia in liquid culture were examined. Among the 21 carbon sources and 15 nitrogen compounds tested, the optimal carbon and nitrogen sources for mycelial growth were sweet potato and L-tyrosine, and for sporulation were sweet potato and casein peptone. A C:N ratio of 10:1 at pH 3.7 gave the maximum yield of conidia and a C:N ratio of 40:1 at pH 6.8 gave the maximum biomass. The initial pH value had a significant effect on mycelial growth and conidial production, with the optimal ranges being 3.5–4.5 for sporulation and 5–6 for growth. Maximum conidial production was obtained at an initial pH of 4.0 and the maximum biomass at pH 6.0. The results also showed that the final pH after 7 days cultivation was always higher than the initial value. The variability in growth and sporulation of seven strains of P. chlamydosporia in liquid culture was also compared and discussed.     

Trophic interactions between bacterial-feeding nematodes in plant rhizospheres and the nematophagous fungus Hirsutella rhossiliensis to suppress Heterodera schachtii

Trophic exchanges in soil food webs may suppress populations of pest organisms. We hypothesize that the suppressive condition of soils might be enhanced by manipulating components of the food web. Specifically, by enhancing populations of bacterial-feeding nematodes, propagule density of the nematophagous fungus Hirsutella rhossiliensis should increase and constrain populations of Heterodera schachtii, a plant-parasitic nematode. The rhizospheres of Crotalaria juncea and Vicia villosa stimulated population growth of the bacterial-feeding nematode, Acrobeloides bodenheimeri, but not of the nematodes Caenorhabditis elegans or Rhabditis cucumeris. The rhizospheres of Tagetes patula, Eragrostis curvula, and Sesamum indicum had no effect on any of the bacterial-feeding nematodes investigated. Acrobeloides bodenheimeri was most susceptible to parasitism by the nematophagous fungus H. rhossiliensis with 35% of individuals being parasitized in a laboratory assay. In three separate trials, parasitism of H. schachtii by H. rhossiliensis was not enhanced when populations of A. bodenheimeri were amplified in a suitable rhizosphere.