Nematode-destroying Fungi Research Articles

Characterization and Effect of a Nematophagous Fungus Talaromyces cystophila sp. nov. for the Biological Control of Corn Cyst Nematode.

The dynamic of plant-parasitic nematode populations in soil is closely related to soil microorganisms. Fungi from Heterodera zeae cysts were isolated to explore the phenomenon of decline in the H. zeae population in the soil. Phylogenetic study of partial ITS, BenA, CaM, and RPB2 gene sequences, in addition to morphological investigations, was utilized to identify a nematode-destroyingfungus. The nematicidal activity of a novel strain GX1 against H. zeae was assessed in vitro and in the greenhouse. Our findings revealed that strain GX1 is a new species of Talaromyces, named Talaromyces cystophila. It has a strong parasitic and lethal effect on H. zeae cysts, with 91.11% parasitism on cysts at 3 days after treatment. The contents of second-stage juveniles (J2s) and eggs inside the cysts were degraded and formed dense vacuoles, and the damaged eggs could not hatch normally. The spore suspension exhibited high nematophagous activity against nematodes, and fermentation filtrate exhibited marked inhibition of egg hatching and nematicidal activities on J2s. The hatching inhibition rates of eggs exposed to 1 × 108 CFU/ml spore suspensions or 20% 1-week fermentation filtrate (1-WF) for 15 days were 98.56 and 100%, respectively. The mortality of J2s exposed to 1 × 108 CFU/ml spore suspension reached 100% at 24 h; exposure to 50% 2-WF was 98.65 and 100% at 24 and 48 h, respectively. Greenhouse experiments revealed that the spore suspension and fermentation broth considerably decreased H. zeae reproduction by 56.17 to 78.76%. T. cystophila is a potential biocontrol strain with nematophagous and nematicidal activity that deserves attention and application.

Comparative Genomic Analysis of Drechmeria coniospora Reveals Core and Specific Genetic Requirements for Fungal Endoparasitism of Nematodes.

Drechmeria coniospora is an obligate fungal pathogen that infects nematodes via the adhesion of specialized spores to the host cuticle. D. coniospora is frequently found associated with Caenorhabditis elegans in environmental samples. It is used in the study of the nematode’s response to fungal infection. Full understanding of this bi-partite interaction requires knowledge of the pathogen’s genome, analysis of its gene expression program and a capacity for genetic engineering. The acquisition of all three is reported here. A phylogenetic analysis placed D. coniospora close to the truffle parasite Tolypocladium ophioglossoides, and Hirsutella minnesotensis, another nematophagous fungus. Ascomycete nematopathogenicity is polyphyletic; D. coniospora represents a branch that has not been molecularly characterized. A detailed in silico functional analysis, comparing D. coniospora to 11 fungal species, revealed genes and gene families potentially involved in virulence and showed it to be a highly specialized pathogen. A targeted comparison with nematophagous fungi highlighted D. coniospora-specific genes and a core set of genes associated with nematode parasitism. A comparative gene expression analysis of samples from fungal spores and mycelia, and infected C. elegans, gave a molecular view of the different stages of the D. coniospora lifecycle. Transformation of D. coniospora allowed targeted gene knock-out and the production of fungus that expresses fluorescent reporter genes. It also permitted the initial characterisation of a potential fungal counter-defensive strategy, involving interference with a host antimicrobial mechanism. This high-quality annotated genome for D. coniospora gives insights into the evolution and virulence of nematode-destroying fungi. Coupled with genetic transformation, it opens the way for molecular dissection of D. coniospora physiology, and will allow both sides of the interaction between D. coniospora and C. elegans, as well as the evolutionary arms race that exists between pathogen and host, to be studied.

Survey of Nematode-Destroying Fungi from Selected Vegetable-Growing Areas in Kenya

Plant-parasitic nematodes cause severe damage to a wide range of economic crops, causing upto 5% yield losses globally. In Kenya, vegetables are affected, among other pests, by parasitic nematodes, causing upto 80% loss in yield. Nematode control is very difficult and relies heavily on use of chemical nematicides. Use of these chemical nematicides leads to biological magnification, and elimination of natural enemies of other pathogens, thus creating a need for greater application of pesticides, increased production costs, and development of insecticide-resistance. These factors have led to a growing interest in search for alternate management strategies. The objective of this study was, therefore, to document nematode-destroying fungi in selected, major vegetable-growing areas in Kenya as a step towards developing a self-sustaining system for management of plant-parasitic nematodes. Soil samples were collected from five vegetable-production zones, viz., Kinare, Kabete, Athi-river, Machakos and Kibwezi, and transported to the laboratory for extraction of nematode-destroying fungi. The soil-sprinkle technique described by Jaffee et al (1996) was used for isolating the nematode-destroying fungi from soil, while, their identification was done using identification keys described by Soto Barrientos et al (2001). From this study, a total of 171 fungal isolates were identified as nematodedestroying. The highest population was recorded in Kabete, at 33.9% of the total, followed by Machakos, Kibwezi, Athi-river, with the least in Kinare, at 24.6, 22.2, 11.7 and 7.6% of the total population, in that order. Arthrobotrys was the most frequent genus, with mean occurrence of 7.3, followed by Monacrosporium with 6 and Stylophage with 5.2. A. dactyloides was significantly (P=0.002) affected by the agro-ecological zone, with the highest occurrence recorded in Kabete, and the least in Athi-river. Kibwezi recorded highest diversity index, with a mean of 1.017, while, Athi-river recorded the least, with a mean of 0.333. Kibwezi had the highest species richness, recording a mean of 3.4, while, the least mean of 1.6 was recorded in Athi-river. Mean species richness of 2.2 was recorded for both Kabete and Machakos, and 1.8 for Kinare. From the three genera recorded, Arthrobotrys was more effective at trapping nematodes compared to Monocrosporium and Stylopage. The genus Arthrobotrys had the highest number of trapped nematodes, with a total population of 57, followed by Monacrosporium, the least being Stylopage, with 45 and 36, respectively, in a period of 104 hours. From the study, it is evident that agricultural practices affect occurrence and diversity of nematodedestroying fungi, and, Arthrobotrys can be used as a bio-control agent for managing plant-parasitic nematodes.

Stimulation of Nematode-Destroying Fungi by Organic Amendments Applied in Management of Plant Parasitic Nematode

Stimulation of Nematode-Destroying Fungi by Organic Amendments Applied in Management of Plant Parasitic Nematode

Costa Rican species ofNematoctonus(anamorphic Pleurotaceae)

Nematoctonus (Hyphomycetes) and Hohenbuehelia (Agaricales, Pleurotaceae) are the names for the asexual and sexual stages of a genus of nematode-destroying fungi (Basidiomycota). Six morphospecies of Nematoctonus, all previously described, were isolated from fruiting bodies of Hohenbuehelia and from 439 samples of soil and organic debris collected in all 12 Holdridge life zones in Costa Rica. Nematoctonus was recorded in all but three life zones at the lowest and highest altitudes: tropical dry forest, tropical moist forest, and subalpine rain paramó. Isolates of Nematoctonus were identified by the micromorphology of their conidia and adhesive knobs, which are usually an hourglass-shaped secretory cell surrounded by a drop of mucilage. Adhesive knobs were found either exclusively on hyphae in predatory species ( Nematoctonus robustus F.R. Jones), exclusively on germinated conidia in parasitoid species ( Nematoctonus leptosporus Drechsler, Nematoctonus pachysporus Drechsler, and Nematoctonus tylosporus Drechsler) or on both hyphae and germinated conidia in a group we term “intermediate predators” ( Nematoctonus angustatus Thorn & G.L. Barron, Nematoctonus geogenius Thorn & G.L. Barron, and one monokaryotic isolate of N. robustus). Teleomorph–anamorph connections, made by culturing the anamorph from a teleomorph fruiting body, were made for N. angustatus (Hohenbuehelia angustata (Berk.) Singer), N. geogenius (Hohenbuehelia petalodes (Fr.) Schulz.), N. leptosporus (an unidentified Hohenbuehelia), and N. robustus ( Hohenbuehelia grisea (Peck) Singer).

Phylogenetic analyses of Nematoctonus and Hohenbuehelia (Pleurotaceae)

Hohenbuehelia (Agaricales, Pleurotaceae) and Nematoctonus (Hyphomycetes) are the names for the sexual and asexual stages of a genus of nematode-destroying fungi (Basidiomycota). We obtained partial sequences of nuclear ribosomal DNA, including the internal transcribed spacer region and the 5′ end of the large subunit, of 37 isolates of Hohenbuehelia and Nematoctonus representing 13 of the 16 described species in Nematoctonus. Phylogenetic analyses support Hohenbuehelia–Nematoctonus as a monophyletic clade of the Pleurotaceae, within which the species were placed in five main subclades. Exclusively predatory species ( Nematoctonus brevisporus Thorn & G.L. Barron, Nematoctonus campylosporus Drechsler, Nematoctonus robustus F.R. Jones, and Nematoctonus sp. UAMH 5317) appear to be basal. In these species, adhesive knobs to capture prey are produced on their hyphae but not on their conidia. A single mycelial individual may feed on many nematodes. From these have arisen both exclusively parasitoid species ( Nematoctonus cylindrosporus Thorn & G.L. Barron, Nematoctonus leiosporus Drechsler, Nematoctonus leptosporus Drechsler, Nematoctonus pachysporus Drechsler, Nematoctonus tylosporus Drechsler), and species that we call intermediate predators ( Nematoctonus angustatus Thorn & G.L. Barron, Nematoctonus concurrens Drechsler, Nematoctonus geogenius Thorn & GL. Barron, Nematoctonus hamatus Thorn & G.L. Barron, and Nematoctonus subreniformis Thorn & G.L. Barron). Exclusively parasitoid species have conidia that germinate to form sticky knobs that attach to passing nematodes but lack adhesive knobs on the hyphae. Each mycelial individual feeds on only one nematode. Intermediate predators have adhesive knobs both on hyphae and on germinated conidia and can act in both predatory and parasitoid modes. Most morphospecies are resolved as monophyletic, but sequences of additional gene regions are required to clarify species limits within the N. angustatus – N. geogenius group.

Coprinus comatus: A Basidiomycete Fungus Forms Novel Spiny Structures and Infects Nematode

Nematophagous basidiomycete fungi kill nematodes by trapping, endoparasitizing and producing toxin. In our studies Coprinus comatus (O.F.Müll. : Fr.) Pers. is found to be a nematode-destroying fungus; this fungus immobilizes, kills and uses free-living nematode Panagrellus redivivus Goodey and root-knot nematode Meloidogyne arenaria Neal. C. comatus produces an unusual structure designated spiny ball. Set on a sporophore-like branch, the spiny ball is a burr-like structure assembled with a large number of tiny tubes. Purified spiny balls exhibit moderate nematicidal activity. Experiments show that spiny balls are not chlamydospores because of the absence of nuclei in the structures and quick formation within 3 d in a young colony. Nematodes added to C. comatus cultures on potato-dextrose agar (PDA) and cornmeal agar (CMA) become inactive in hours. Infection of nematodes by the fungus occurs only after the nematodes are immobilized (feeble or dead), probably by a toxin. Electron micrographs illustrate that C. comatus infect P. redivivus by producing penetration pegs with which hyphae colonize nematode bodies. An infected nematode is digested and consumed within days and hyphae grow out of the nematode.

Coprinus comatus: A basidiomycete fungus forms novel spiny structures and infects nematode

Nematophagous basidiomycete fungi kill nematodes by trapping, endoparasitizing and producing toxin. In our studies Coprinus comatus (O.F.Müll. : Fr.) Pers. is found to be a nematode-destroying fungus; this fungus immobilizes, kills and uses free-living nematode Panagrellus redivivus Goodey and root-knot nematode Meloidogyne arenaria Neal. C. comatus produces an unusual structure designated spiny ball. Set on a sporophore-like branch, the spiny ball is a burr-like structure assembled with a large number of tiny tubes. Purified spiny balls exhibit moderate nematicidal activity. Experiments show that spiny balls are not chlamydospores because of the absence of nuclei in the structures and quick formation within 3 d in a young colony. Nematodes added to C. comatus cultures on potato-dextrose agar (PDA) and cornmeal agar (CMA) become inactive in hours. Infection of nematodes by the fungus occurs only after the nematodes are immobilized (feeble or dead), probably by a toxin. Electron micrographs illustrate that C. comatus infect P. redivivus by producing penetration pegs with which hyphae colonize nematode bodies. An infected nematode is digested and consumed within days and hyphae grow out of the nematode.

Biological control of helminths

As a potential component in future integrated parasite-control strategies, biological control by means of predacious fungi seems to be moving from a promising possibility toward becoming a reality, and the netforming nematode-destroying fungus Duddingtonia flagrans appears to be the candidate of choice. Not only has this fungus been found in, and isolated from, fresh sheep, cattle and horse faeces, but it also appears to be the only fungus that is able to consistently and significantly reduce the number of infective trichostrongyle larvae in faeces from animals fed fungal spores. Results from the last few years have shown that D. flagrans is able to trap and destroy free-living stages of the most important and common trichostrongylid larvae with very similar external life-cycles, as well as larvae of parasites with a slightly different transmission biology ( Nematodirus spp., Dictyocaulus viviparus). The introduction of microfungi for biological control could be as part of a feed supplement or incorporated in feed-blocks presented to animals which are raised under relatively intensive conditions and constant surveillance. Apart from the special niche for organic farmers, such a product would be suited for horses, small ruminants (as either milking herds or housed daily for other reasons), cattle in special situations and free-roaming pigs. The most important constraint, still, for a major breakthrough in biological control in the latter is the lack of good antagonists against the long-lived and rather resistant infective stages of parasites, being transmitted as larvae inside the egg. Since the first Conference on Novel Approaches to the Control of Helminth Parasites of Livestock in Armidale, Australia, 1995, there has been a steady evolution within the area of biological control of parasitic nematodes. Today this principle is being exploited and tested out in almost all parts of the world, under various climatic conditions and production systems. Where, in the past, a large part of the work focused on cattle and to a lesser degree horse and sheep parasites, the focus of the research in many of the newly involved countries is on small ruminants, because of their importance to primarily small-scale farmers in local communities. Today research and trials are either on-going or being planned in many developing countries, as well as in countries in transition. The involvement of multinational agencies in addition to national and industrial interests is very welcome and should increase the chances and keep up the momentum for development and implementation of biological control in future animal production around the world.

Biological control of gastro-intestinal nematodes — facts, future, or fiction?

The potential of using fungi to prevent nematodosis caused by parasites with free-living larval stages is well documented today. In this respect Duddingtonia flagrans, a net-trapping, nematode-destroying fungus, appears to be the most promising candidate. Laboratory experiments and in-vivo studies, where fungal spores have survived passage through the gastro-intestinal tract of cattle and horses, plus field studies with cattle, horses and pigs, demonstrate significant reduction in the number of infective larvae that develop in the faecal environment. In field trials this reduction subsequently leads to reduced infectivity of herbage and also reduced worm burdens in grazing animals. A status of the present situation, primarily based upon work performed in Denmark within the last 6–8 years, plus an outlook for practical implementation of an integrated control strategy including the use of nematode-destroying fungi in the future is discussed.

The prospects for biological control of the free-living stages of nematode parasites of livestock

Control of nematode parasites of livestock is focused almost entirely on the parasitic stages within the host. Current methods rely on anthelmintic drugs, but these are under increasing threat with the development of resistance covering the whole spectrum of anthelmintics amongst the important nematode species of a range of livestock. However, invariably the greatest proportion of the parasite biomass resides not within the animal hosts, but in the external environment - commonly on pasture. It is in this environment that the free-living stages are vulnerable to a range of abiotic factors (extremes in temperature and desiccation) and biotic factors (macro- and micro-organisms) that may decimate their numbers. Of the latter, there are organisms, which exert their effects either indirectly by rendering faecal deposits inimical for the development of nematode eggs through to infective larvae, or directly by acting as pathogens or by exploiting the free-living stages as a food source. Within this vast assemblage of organisms, which include microarthropods, protozoa, viruses, bacteria and fungi, could well emerge a variety of biological control agents of nematode parasites. At present, greatest interest lies with the nematode-destroying fungi. Work has progressed from Petri dishes, to plots, to paddocks with several species of the genus Arthrobotrys and Duddingtonia flagrans. These studies indicate that the voracious nematophagous capabilities of these fungi, clearly demonstrated in vitro, translate to reductions in the number of infective larvae on pasture and indicate that levels of control, comparable to conventional schemes using anthelmintics, can be achieved. The challenge now lies in developing methods of administration of fungi to animals which can be applied under practical farm conditions. However, the pursuit of candidates for biological control of nematode parasites of livestock should not involve just a few species of nematophagous fungi. More than 100 species of fungi have been identified as possessing nematode destroying capabilities. These need to be more specifically investigated for their effects on free-living stages of nematode parasites of livestock, together with other classes of organisms, particularly bacteria, which have proved successful as biological control agents of arthropods.

The capacity of the fungus Duddingtonia flagrans to prevent strongyle infections in foals on pasture

A field trial was conducted to evaluate the potential of the nematode-destroying fungus Duddingtonia flagrans to control free-living stages of horse strongyles. In late Spring 2 groups of horses (yearlings) with mixed infections of strongyles were allowed to contaminate 2 equal-sized pastures. One of the groups (F) received a daily dose of D. flagrans mixed in a feed supplement, while the other (C) received a similar amount of supplement without fungus. During a 3-month contamination period strongyle egg counts in faeces and number of infective strongyle larvae harvested from faecal cultures were determined. Grass samples were collected fortnightly. After the contamination period the yearlings were removed and 2 groups of young tracer foals (TF and TC) grazed the fungus and control pastures respectively for 4 weeks, housed for another 15 weeks and then killed to determine their worm burdens. The number of larvae in cultures from group TF was significantly lower than that in TC and herbage infectivity was reduced to a very low level on the pasture grazed by horses fed fungi. The number of Strongylus vulgaris and Strongylus edentatus larvae was also significantly lowered in group TF. Cyathostome larvae recovered from the mucosa of the ventral and dorsal colon and from the caecum were significantly lowered in group TF foals. Also, the number of strongyles found in the gut contents of group TF foals were significantly reduced in the dorsal colon, but numbers of worms in the ventral colon and in the caecum were similar to those of the controls.

Predacious activity of the nematode-destroying fungus Arthrobotrys oligospora in dependence of the medium composition

The nematophagous fungus Arthrobotrys oligospora Fres. can live saprophytically as well as predatorily. As a predator it forms sticky reticulate traps in the presence of living nematodes which enable it to kill and consume the animals. In laboratory experiments the nutrient acquisition of the fungus was tested on agar media with various N-and C-sources in different concentrations. The intensity of predacious response was determined by counting the traps formed on the agar surface after induction by nematodes. Predacious activity (PA) served as a unit of the strength of nematophagous response. PA was defined as the number of traps/100 mm 2. In contrast to carnivorous higher plants, which consume insects in order to compensate their N-deficit, the fungus seems to kill nematodes to get both, N as well as C. If the agar substrate contains a certain concentration of N and C, the fungus does not form traps and lives as a saprophyte. The level which determines the switch towards pure saprophytism under laboratory conditions ranges around 0.12 M C at a N-concentration of 0.05 M. If no nitrogen is added to the medium the fungus forms nematode-induced traps even in the most concentrated C-media. On N-media without any carbon source the fungus shows a much stronger PA compared with N-free media. Furthermore, more chlamydospores were formed on N-media.

Nematophagous fungi: Prospective biological control agents of animal parasitic nematodes?

The rapidly escalating problem of anthelmintic resistance and the increasing concerns of chemical residues in livestock products and the environment pose serious threats to the future of chemotherapeutic control o f animal parasitic nematodes. One possible non-chemotherapeutic approach is biological control using nemotophagous fungi. Although Grønvold and colleagues recently described their experiments with two nematode-destroying fungi, Arthrobotrys and Duddingtonia, this alternate control option has received relatively little research attention. Here, Peter Waller broadens the discussion to consider the fundamental problems and prospects of this non-chemotheropeutic control strategy.

Interaction between the nematode-destroying fungus Arthrobotrys robusta (Hyphomycetales) and Haemonchus contortus infective larvae in vitro

In an in vitro trial, the effect of the nematode-destroying fungus Arthrobotrys robusta on Haemonchus contortus infective larvae was evaluated in petri dishes containing corn meal agar. After seven days incubation at 25°C, 92.33% (±4.1) predation was recorded.

Direct observation of trapping activities of nematode-destroying fungi in the soil using fluorescence microscopy

The nematophagous fungi (Arthrobotrys oligospora and Monacrosporium cionopagum) were observed under semi-natural conditions by fluorescence microscopy after fluochroming, using fluorescein-diacetate. The formation of 3-dimensional sticky networks and the capture of nematodes inside the soil could be demonstrated.

Direct observation of trapping activities of nematode-destroying fungi in the soil using fluorescence microscopy

Direct observation of trapping activities of nematode-destroying fungi in the soil using fluorescence microscopy

Direct observation of trapping activities of nematode-destroying fungi in the soil using fluorescence microscopy

Direct observation of trapping activities of nematode-destroying fungi in the soil using fluorescence microscopy

Effect of pH and Temperature on the Nematode Destroying Fungus, Paecilomyces lilacinus

The growth or Paecilomyces lilacinus (in buffered and unbuffered medium) in a pH range of 5-9; and its survival upon exposure to high temperatures (40-50°C for 0-10 days) was studied in Potato Dextrose Broth in vitro . P. lilacinus had the tendency to alter the initial pH (acidic or basic) towards neutrality. The fungus could grow at all pH levels. However, growth was better in unbuffered medium. P. lilacinus can survive exposure to 40°C for 10 days, 45°C for 6 days and 50°C for 24h. However, the resumption of normal growth at 28±1°C was delayed.

Activity of commercial Bacillus thuringiensis preparations against Trichostrongylus colubriformis and Nippostrongylus brasiliensis

Control of nematode parasites of livestock is focused almost entirely on the parasitic stages within the host. Current methods rely on anthelmintic drugs, but these are under increasing threat with the development of resistance covering the whole spectrum of anthelmintics amongst the important nematode species of a range of livestock. However, invariably the greatest proportion of the parasite biomass resides not within the animal hosts, but in the external environment - commonly on pasture. It is in this environment that the free-living stages are vulnerable to a range of abiotic factors (extremes in temperature and desiccation) and biotic factors (macro- and micro-organisms) that may decimate their numbers. Of the latter, there are organisms, which exert their effects either indirectly by rendering faecal deposits inimical for the development of nematode eggs through to infective larvae, or directly by acting as pathogens or by exploiting the free-living stages as a food source. Within this vast assemblage of organisms, which include microarthropods, protozoa, viruses, bacteria and fungi, could well emerge a variety of biological control agents of nematode parasites. At present, greatest interest lies with the nematode-destroying fungi. Work has progressed from Petri dishes, to plots, to paddocks with several species of the genus Arthrobotrys and Duddingtonia flagrans. These studies indicate that the voracious nematophagous capabilities of these fungi, clearly demonstrated in vitro, translate to reductions in the number of infective larvae on pasture and indicate that levels of control, comparable to conventional schemes using anthelmintics, can be achieved. The challenge now lies in developing methods of administration of fungi to animals which can be applied under practical farm conditions.However, the pursuit of candidates for biological control of nematode parasites of livestock should not involve just a few species of nematophagous fungi. More than 100 species of fungi have been identified as possessing nematode destroying capabilities. These need to be more specifically investigated for their effects on free-living stages of nematode parasites of livestock, together with other classes of organisms, particularly bacteria, which have proved successful as biological control agents of arthropods.