Heavy trap formation by Arthrobotrys oligospora in liquid culture

Abstract The nematode-trapping fungus Arthrobotrys oligospora was grown in liquid culture in modified separatory funnels with vigorous air bubbling. The growth medium was a dilute soya peptone broth supplemented with a trap-inducing substance. The dipeptide l -phenylalanyl- l -valine or its constituents phenylalanine and valine were used as inducers of trap formation. Trap formation started within 2 days after inoculation and the traps increased in number and size during a 7-day period. The traps formed in liquid culture were fully functional in trapping nematodes. At the ultrastructural level they were characterized by the presence of many electron-dense microbodies similar to those in trap cells grown on solid media. Biomass increase, amounting to 6–7 mg dry weight · ml⁻¹, and trap formation were highly synchronized. The mycelial mass was homogeneously dispersed in the growth vessel during the entire growth period. Heavy trap formation in liquid culture by this fungus has not been reported previously.     

Transformation of the nematode-trapping fungus Arthrobotrys oligospora

The nematode-trapping fungus Arthrobotrys oligospora was transformed to hygromycin resistance using the hygromycin-B phosphotransferase gene from Escherichia coli under the control of various heterologous fungal promoters. Plasmid DNA was introduced into fungal protoplasts by polyethylene glycol/CaCl2 treatment. Transformation frequencies varied between 1-6 transformants per microgram DNA. Seven out of 13 integration events analyzed from transformants were single copy integrations, whereas the remaining were multiple and more complex integrations. The addition of restriction enzymes during transformations increased the frequency of single copy integrations. Co-transformation, using the E. coli uidA gene encoding the beta-glucuronidase reporter gene under the control of an Aspergillus nidulans promoter, occurred at frequencies of up to 63%.     

Surface polymers of the nematode-trapping fungus Arthrobotrys oligospora

The nematophagous fungus Arthrobotrys oligospora captures nematodes using adhesive polymers present on special hyphae (traps) which form a three-dimensional network. To understand further the adhesion mechanisms, A. oligospora surface polymers were visualized by transmission electron microscopy and characterized by chemical methods. Both traps and hyphae were surrounded by a fibrillar layer of extracellular polymers which stained with ruthenium red. The polymer layer was resistant to most of the chemicals and enzymes tested. However, part of the layer was removed by sonication in a Tris-buffer or by extraction in a chaotropic salt solution (LiCl), and the structure of the polymers was modified by treatment with Pronase E. Chemical analysis showed that the crude extracts of surface polymers removed by sonication or LiCl solution contained neutral sugars, uronic acids and proteins. Gel chromatography of the extracts revealed that the major carbohydrate-containing polymer(s) had a molecular mass of at least 100 kDa, containing neutral sugars (75% by weight, including glucose, mannose and galactose), uronic acids (6%) and proteins (19%). There was more polymer in mycelium containing trap-bearing cells than in vegetative hyphae. SDS-PAGE of the extracted polymers showed that the trap-forming cells contained at least one protein, with a molecular mass of approx. 32 kDa, not present on vegetative hyphae. Examining the capture of nematodes by traps of A. oligospora in which the layer of surface polymers was modified, or removed by chemical or enzymic treatments, showed that both proteins and carbohydrate surface polymers were involved in the adhesion process.     

Role of N-Acetylgalactosamine-Specific Protein in Trapping of Nematodes by Arthrobotrys oligospora

An N-acetylgalactosamine-specific protein was purified from mycelial homogenates of the nematode-trapping fungus Arthrobotrys oligospora by using affinity chromatography. The molecular weight of the protein was estimated at 22,000 by its comparative mobility on sodium dodecyl sulfate-polyacrylamide slab gels. Pretreatment of nematodes with the purified protein reduced entrapment, indicating a role for the sugar-binding protein in recognition and capture of prey by the fungus.     

Purification and characterisation of two endoglucanases from Arthrobotrys oligospora

Two endoglucanases, designated Endo I and Endo II, were purified from the culture filtrates of a nematode trapping fungus, Arthrobotrys oligospora. The purification procedure entailed ammonium sulphate precipitation, gel filtration and preparative PAGE. Both the preparations (Endo I and Endo II) were homogeneous by PAGE, had molecular weights of 24,300 and 44,500 respectively as determined by non-denaturing PAGE, and yielded only cellobiose as the main product of CM-cellulose hydrolysis. The optimum pH and temperature for Endo I were 6.0 and 50 degrees C, and for Endo II, pH 5.6-6.4 and 50 degrees C. The two enzymes differed with respect to their Km (Endo I, 5.04 mg/ml; Endo II, 3.2 mg/ml) and energy of activation values (Endo I, 10.7 kCal; Endo II, 9.5 k Cal). Both enzymes were completely inhibited by 1.25 mH Hg2+ and partially by Pb2+, DTNB and p-HMB while DTT and GSH enhanced their activities.     

Biological Characteristics of Recombinant Arthrobotrys oligospora Chitinase AO-801

Chitinase AO-801 is a hydrolase secreted by Arthrobotrys oligospora during nematode feeding, while its role remained elusive. This study analyzed the molecular characteristics of recombinant chitinase of Arthrobotrys oligospora (reAO-801). AO-801 belongs to the typical glycoside hydrolase 18 family with conserved chitinase sequence and tertiary structure of (α/β)₈ triose-phosphate isomerase (TIM) barrel. The molecular weight of reAO-801 was 42 kDa. reAO-801 effectively degraded colloidal and powdered chitin, egg lysate, and stage I larval lysate of Caenorhabditis elegans. The activity of reAO-801 reached its peak at 40°C and pH values between 4–7. Enzyme activity was inhibited by Zn2⁺, Ca2⁺, and Fe3⁺, whereas Mg2⁺ and K⁺ potentiated its activity. In addition, urea, sodium dodecyl sulfate, and 2-mercaptoethanol significantly inhibited enzyme activity. reAO-801 showed complete nematicidal activity against C. elegans stage I larvae. reAO-801 broke down the C. elegans egg shells, causing them to die or die prematurely by hatching the eggs. It also invoked degradation of Haemonchus contortus eggs, resulting in apparent changes in the morphological structure. This study demonstrated the cytotoxic effect of reAO-801, which laid the foundation for further dissecting the mechanism of nematode infestation by A. oligospora.     

The influence of fungicides on Arthrobotrys oligospora in simulated putting green soil

Plant‐parasitic nematodes are destructive pests in bentgrass putting greens. Few chemical or nonchemical approaches for nematode management exist. Studies were conducted to determine: the in vitro tolerance of the nematophagous fungus Arthrobotrys oligospora, to the fungicides chlorothalonil and myclobutanil used to manage diseases on putting greens; the concentration of fungicides obtained from simulated putting green soil; and the ability of the fungus to reduce populations of the ring nematode, Criconemella ornata. Both fungicides reduced in vitro hyphal growth and germination of conidia above 10 mg kg^‐1. Soil concentrations of chlorothalonil were less than 5 mg kg^‐1 and concentrations of myclobutanil were below detection limits. Nematode populations were not affected by A. oligospora in simulated greens but nematode populations were lowest in pots inoculated with A. oligospora and receiving fungicide treatments. Results of these studies indicate that applications of chlorothalonil and myclobutanil used to manage fungal diseases of bentgrass may not adversely affect A. oligospora; however, the fungus may not reduce nematode populations below desired thresholds.     

Scanning Electron Microscopy of the Nematode-trapping Organs in Arthrobotrys oligospora

The three-dimensional capture organs of the nematodetrapping fungus Arthrobotrys oligospora are presented in scanning electron micrographs. The fungus was grown directly on agar substrates or on millipore filters or glass cover slips placed on agar substrates. To avoid shrinkage of the soft-walled mycelial material, a fixation in O_sO₄-vapour was performed, before freeze-drying and coating with Au-Pd alloy. When capture organs were prepared in this way, it was possible to visualize them in their erect position in the scanning electron microscope. Young capture organs, both spontaneously formed and nematode-induced, remained standing better than old ones. An adhesive substance on the capture organs, with the aid of which nematodes are trapped, seems to be visible in these pictures.     

Biological control of ruminant trichostrongylids by Arthrobotrys oligospora, a predacious fungus

Sheep and cattle are prey to many parasitic worms, including the trichostrongylid nematodes. Conventional control involves the use of anthelmintic drugs, but in this article Hadji Ahmad Hashmi and Roger Connan discuss the possible biological control of these nematodes by means of a 'living lasso', the predacious fungus Arthrobotrys.     

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.     

寡孢节丛孢菌对羊捻转血矛线虫幼虫捕食作用*

报道５株寡孢菌在体外对羊捻转血矛线虫第３期幼虫的捕食效果。结果表明：５株寡孢节丛孢菌对捻转血矛线虫幼虫的捕获率分别为８８．６５％、７９．００％、７４．７２％、８７．０９％、８０．９１％,具有较高的捕杀线虫效果。寡孢节丛孢菌对捻转血矛线虫的作用方式主要是通过在线虫周围形成捕食菌网。     

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     

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.     

Qualitative Characterization of Some Peptides Inducing Morphogenesis in the Nematode- Trapping Fungus Arthrobotrys oligospora

Some peptides inducing capture organ formation in Arthrobotrys oligospora were characterized with regard to their amino acid contents. The N-terminal, C-terminal and total amino acids were determined as their dinitrophenyl-derivatives by thin layer chromatography on silica gel in two different solvents. The amino acid composition was further confirmed by gas-liquid chromatography. The peptides investigated had a high proportion of non-polar and aromatic residues. Thus, leucine, isoleucine, valine, proline, and tyrosine were present in all the peptides. In addition, phenyl-alanine, glycine, and alanine occurred in some preparations. Tyrosine, valine, and phenylalanine were found in N-terminal position, and leucine or isoleucine were C-terminals.     

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.     

Genomic and proteomic analyses of the fungus Arthrobotrys oligospora provide insights into nematode-trap formation

Nematode-trapping fungi are "carnivorous" and attack their hosts using specialized trapping devices. The morphological development of these traps is the key indicator of their switch from saprophytic to predacious lifestyles. Here, the genome of the nematode-trapping fungus Arthrobotrys oligospora Fres. (ATCC24927) was reported. The genome contains 40.07 Mb assembled sequence with 11,479 predicted genes. Comparative analysis showed that A. oligospora shared many more genes with pathogenic fungi than with non-pathogenic fungi. Specifically, compared to several sequenced ascomycete fungi, the A. oligospora genome has a larger number of pathogenicity-related genes in the subtilisin, cellulase, cellobiohydrolase, and pectinesterase gene families. Searching against the pathogen-host interaction gene database identified 398 homologous genes involved in pathogenicity in other fungi. The analysis of repetitive sequences provided evidence for repeat-induced point mutations in A. oligospora. Proteomic and quantitative PCR (qPCR) analyses revealed that 90 genes were significantly up-regulated at the early stage of trap-formation by nematode extracts and most of these genes were involved in translation, amino acid metabolism, carbohydrate metabolism, cell wall and membrane biogenesis. Based on the combined genomic, proteomic and qPCR data, a model for the formation of nematode trapping device in this fungus was proposed. In this model, multiple fungal signal transduction pathways are activated by its nematode prey to further regulate downstream genes associated with diverse cellular processes such as energy metabolism, biosynthesis of the cell wall and adhesive proteins, cell division, glycerol accumulation and peroxisome biogenesis. This study will facilitate the identification of pathogenicity-related genes and provide a broad foundation for understanding the molecular and evolutionary mechanisms underlying fungi-nematodes interactions.