Morphogenesis and significance of hyphal coiling by nematode-trapping fungi in mycoparasitic relationships

Abstract The mycoparasitic behaviour of some nematode-trapping fungi was investigated. These organisms interacted with other soil fungi by hyphal coiling around the host hyphae. A detailed study with Arthrobotrys oligospora revealed that different fungi, representing all taxonomic groups, were attacked.
In dual cultures, the interaction between A. oligospora and Rhizoctonia solani occurred soon after hyphal contact, irrespective of the nutrient level of the medium. Coiling was also observed when the two organisms were grown in sterilized soil. The coils possessed a high metabolic activity compared to the surrounding hyphae, as was indicated by fluorescence microscopy of fluorescein diacetate (FDA)-stained preparations. On the ultrastructural level, developing coils showed an abundance of membranous vesicles which developed from tubular-shaped endoplasmic reticulum. At the site of coiling, a strong cell wall proliferation was observed in the Rhizoctonia cells. The cytoplasm of these cells subsequently disintegrated. The death of the cells was confirmed in vital staining experiments. Penetration of intact Rhizoctonia cells was not observed. The interaction between A. oligospora and R. solani is interpreted in terms of competition for nutrients.     

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.     

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.     

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%.     

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.     

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.     

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.     

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。     

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.     

Heterotrimeric G-protein and signal transduction in the nematode-trapping fungus Arthrobotrys dactyloides

The fungus Arthrobotrys dactyloides produces specialized constricting rings to trap and then consume nematodes. The signal transduction pathway involved in the nematode-trapping process was examined. Mastoparan, an activator of G-protein, had a stimulatory effect on the inflation of ring cells, whereas a G-protein inhibitor, pertussis toxin, prevented ring-cell expansion. The 40-kDa G_α of heterotrimeric G-proteins was specifically ADP-ribosylated by pertussis toxin. Using an antibody specific to the 35-kDa subunit G_β, we showed that immunogold-labeled G_β was more concentrated in ring cells than in the hyphae. In the absence of nematodes, the rings could be inflated by either pressurizing the culture in a syringe, raising intracellular Ca²⁺ concentrations, or adding warm water. We used these methods to reveal differences in responses to antagonists. The results support a model in which the pressure exerted by a nematode on the ring activates G-proteins in the ring cells. The activation leads to an increase in cytoplasmic Ca²⁺, activation of calmodulin, and finally the opening of water channels. The ring cells expand to constrict the ring and thus immobilize the nematode. Received: 13 April 2000 / Accepted: 22 June 2000     

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.     

立枯丝核菌营养菌丝多型性观察

采用了2种不同的方法对立枯丝核菌营养菌丝的形态进行了观察和比较,观察到2种不同的营养菌丝的形态,即菌核类和假分生孢子类。为以后进一步研究立枯丝核菌营养菌丝的多型性奠定了一定的基础。     

First Report of Rhizoctonia solani AG 8 on Wheat in Turkey

A survey was conducted in Ankara and Eskisehir provinces of Turkey for determining anastomosis groups and pathogenicity of Rhizoctonia species associated with root and crown rot of wheat. Pathogenicity tests revealed that Rhizoctonia solani AG 8 caused the common symptoms of damping‐off and stunting.     

Purification and characterization of a surface lectin from the nematode-trapping fungus Arthrobotrys oligospora.

Several studies have indicated that the capture of nematodes by the nematophagous fungus Arthrobotrys oligospora is mediated by a lectin on the fungal surface. One of the major surface proteins of this fungus showed haemagglutinating activity and was isolated by affinity chromatography using a mucin Sepharose column. Biochemical analysis showed that the protein was a dimeric glycoprotein with a molecular mass of 36 kDa and an isoelectric point of pH 6.5, and contained no sulphur amino acids. The protein was N-terminally blocked; four internal peptides were sequenced, and showed no significant similarity to sequences in the Swiss-Prot or PIR databases. The haemagglutinating activity of the isolated protein was not inhibited by any of the mono- or disaccharides tested, but it was inhibited by the glycoproteins fetuin and mucin. The haemagglutinating activity changed after incubating the protein in buffers of different pH, with maximal activity at pH 11.0 and no activity at pH 2.8. The lectin was tested for different enzymic activities but none were detected. Analysis of the haemagglutinating activity in various cell fractions indicated that the protein was associated with extracellular polymer layers and with the cell wall of the fungus. About the same amount of the haemagglutinating protein was recovered from samples of vegetative mycelium and of mycelium containing nematode-trapping cells.     

捕食线虫真菌少孢节丛孢Arthrobotrys oligospora线粒体基因组的异质性

捕食线虫真菌少孢节丛孢Arthrobotrys oligospora是子囊菌中的一个食肉性丝状真菌,其菌丝体可变态为各种结构精巧的捕食器官用于捕食线虫,在生物防治和进化上极具研究潜力.线粒体基因细胞色素c氧化酶亚基Ⅰ基因(COX1)在真菌物种鉴定中的效果饱受争议.本研究对A.oligospora的不同单孢培养的菌株进行...