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The in vitro effect of four isolates of the nematophagous fungi Duddingtonia flagrans (AC 001), Monacrosporium sinense (SF 53), and Pochonia chlamydosporia (VC 1 and VC 4) on eggs of Ascaris suum was evaluated. One hundred thousand A. suum eggs were plated on 2% water–agar with the grown isolates and control without fungus. After 7, 14, and 21 days, 100 eggs were removed and classified according to the following parameters: type 1, lytic effect without morphological damage to eggshell; type 2, lytic effect with morphological alteration of embryo and eggshell; and type 3, lytic effect with morphological alteration of embryo and eggshell, besides hyphal penetration and internal egg colonization. P. chlamydosporia showed ovicidal activity (p < 0.01), mainly of the type 3 effect, on A. suum eggs in the studied intervals of 13.3% (isolate VC 1) and 17.3% (isolate VC 4), 13.9% (VC 1) and 17.7% (VC 4), and 19% (VC 1) and 20% (VC4), respectively, at 7, 14, and 21 days. The other fungi showed no type 3 effect. P. chlamydosporia is a potential biological control agent of A. suum eggs.

Some aspects of nematodes regulation by Pasteuria penetrans and other endoparasitic Gram-negative bacteria are revised, together with application modeling tools, in reference to their biocontrol potentials. A review is given about general and more detailed epidemiological models and their applications. The models constants accounting for basic biological factors of the parasites and hosts biology and interactions, are also discussed. Some properties of applied models, including the phase plane representation, the identification of equilibrium points and their cyclic relationships are revised, in reference to the study of field and time series data. A modeling scheme for Pasteuria and nematode dynamics, accounting for the host life cycle and including its developmental stages, is also proposed. Finally, experimental and practical issues concerning nematodes biological control are also discussed.

Nematophagous fungi are potential candidates for biological control of plant-parasitic nematodes, and an important constituent in integrated pest management programs. In this chapter we describe various aspects on the biology of these fungi. Nematophagous species can be found in most fungal taxa, indicating that the nematophagous habit evolved independently in the different groups of nematophagous fungi. Regarding their mode of action we discuss recognition phenomena (e.g. chemotaxis and adhesion), signaling and differentiation, and penetration of the nematode cuticle/eggshell using mechanical, as well as enzymatic (protease and chitinase) means. The activities of nematophagous fungi in soil and rhizosphere is also discussed.

International audience; Pathozone dynamics were derived for approx. 50 Meloidogyne incognita juveniles infecting a single root of tomato under controlled conditions from a point source of inoculum and described using simple, non-linear models. The pathozone decayed sigmoidally with distance, but increased over time as progressively more nematodes were able to infect the root. Despite the reported ability of M. incognita juveniles to travel up to 50 cm in some conditions, the maximum width of the pathozone for a single tomato root was estimated as 18·1 mm. It is conjectured that this was because of (i) diffusion from a point source of inoculum, (ii) a small infection court (a single root tip) and (iii) the limited life span of the nematode. A second experiment was used to assess the effect of the nematophagous fungus Pochonia chlamydosporia on the pathozone dynamics of the nematode. This fungus is known to produce nematicidal products in vitro, which may affect invasion of roots by the free-living nematode. To examine the possibility of a change in the position of the site of infection, changes in the probability of gall formation along the root length were also examined. In the absence of P. chlamydosporia, the pathozone dynamics of M. incognita were very similar to those of the first experiment. It was shown that P. chlamydosporia did not significantly affect the pathozone dynamics of M. incognita nor the site of gall formation, which appear to have little importance for the role of the fungus as a biological control agent

The nematodes specificities and their interactions with plants are reviewed, considering host plants quality and compatibility. The potentials of nematode resistance and diversity of antagonists and parasitism are discussed, in relation to host specificity and obligate multitrophic relationships. The ecology and management of nematode communities are also reviewed, focusing on soil health approaches and new paradigms for plant protection.

SUMMARYRemoval of the sheath of the ensheathed infective juvenile ofTrichostrongylus colubriformisprevents capture by the nematophagous fungusArthrobotrys oligospora. Exposure of the trap hyphae to a variety of saccharides, which may block a recognition system based on lectin/carbohydrate binding, failed to prevent capture but some saccharides did inhibit penetration and invasion by the fungus. Capture and penetration thus appear to be two distinct processes with capture being less specific than penetration. Carbohydrate residues were absent from the outer surface of the cuticle and the sheath but were present on the inner surface of the sheath. The limited accessibility of these lectin-binding sites may explain the slow process of infection of the infective juvenile by the fungus. The sheath does not protect the infective juvenile against attack by this nematophagous fungus.

SUMMARY A freeze-substitution technique for preparing fungal specimens for scanning electron microscopy is described. This involves cryofixation in liquid nitrogen, freeze substitution in methanol at — 20°C and critical-point drying. The trapping complexes and conidiophores of the nematophagous fungus Arthrobotrys oligospora are well preserved and retain their normal three-dimensional arrangement.

Appressoria of the nematophagous fungus Verticillium suchlasporium infecting eggs of the beetroot cyst nematode Heterodera schachtii were studied by field emission scanning electron microscopy (FESEM). Protoappressoria and mature appressoria were found in both germ tubes and mycelial hyphae, and mucilaginous material was seen where the appressoria contacted the surface of the egg. The role of this material in the penetration of the eggs by the hyphae is discussed.

Aims: To investigate the toxic effect of chitosan on important root pathogenic and biocontrol fungi (nematophagous, entomopathogenic and mycoparasitic). Methods and Results: We have used standard bioassays to investigate the effect of chitosan on colony growth and developed bioassays to test spore germination. The results showed that the root pathogenic and mycoparasitic fungi tested were more sensitive to chitosan than nematophagous and entomopathogenic fungi. Chitosanases (and perhaps related enzymes) are involved in the resistance to chitosan. Two fungi, one sensitive to chitosan, Fusarium oxysporum f. sp. radicis-lycopersici, and one less sensitive, Pochonia chlamydosporia, were selected for ultrastructural investigations. Transmission electron microscopy revealed differences in the ultrastructural alterations caused by chitosan in the spores of the plant pathogenic fungus and in those of the nematophagous fungus. Confocal laser microscopy showed that Rhodamine-labelled chitosan enters rapidly into conidia of both fungi, in an energy-dependent process. Conclusions: Nematophagous and entomopathogenic fungi are rather resistant to the toxic effect of chitosan. Resistance of nematophagous and entomopathogenic fungi to chitosan could be associated with their high extracellular chitosanolytic activity. Furthermore, ultrastructural damage is much more severe in the chitosan sensitive fungus. Significance and impact of the study: The results of this paper suggest that biocontrol fungi tested could be combined with chitosan for biological control of plant pathogens and pests.

The aim of this study was to investigate the effect of the nematophagous fungus Duddingtonia flagrans applied orally to small ruminants in a field study in Germany. 20 female, pure breed goat kids and 20 female, pure breed lambs, all naturally infected with GIN, were kept on pasture and fed additionally with concentrates amended by 5 x 10(5) spores of D. flagrans per kilogram bodyweight daily for 3 months during pasture season. The equally sized control groups got the concentrate without spores. Every fortnight data of body weight, eggs per gram faeces and larval development in faecal cultures and on pasture were collected. Following 3 months of spore feeding the control goats showed arithmetic mean faecal egg counts of 1235 (+/-533) eggs per gram (epg) faeces, in comparison to 517 (+/-671) epg in the fungus-fed group (p0.001). No statistically significant difference was found between the two sheep groups. The maximum in larval reduction in faeces was found at the end of the fungus-feeding period (81.3% in the sheep groups and to 67.9% in the goat groups), but without statistical significance. At the end of the study the mean body weight gain in the fungus-treated groups tended to be higher than in the control groups, but not showing statistically significant differences. Only regarding the first-year-grazing-goats, the bodyweights of the post-feeding period revealed significant (p0.05) differences between fungus-fed and control group. However, no statistically significant differences were observed in qualitative faecal cultures, pasture larvae counts, serum pepsinogen-level and PCV. In the study presented here, no clear effect of fungus could be observed. Furthermore, climatic conditions during the study period may have demonstrated how vulnerable the fungus application is to such parameters.

The nematophagous fungus, Duddingtonia flagrans is a potential biocontrol agent against nematode parasites of ruminants. Improved methods for the rapid and accurate detection of D. flagrans would aid the evaluation of this fungus as a biocontrol agent and its suitability for environmental release. To date, detection and identification of D. flagrans is reliant on morphological methods, which can be laborious, time-consuming, and error prone. In this study, a PCR assay using species-specific primers located in the ITS regions was developed for the rapid and accurate identification of D. flagrans. The PCR assay was specific to five different isolates of D. flagrans and was capable of detecting a minimum concentration of 100 chlamydospores per gram of soil. In contrast to cultured-based detection and identification methods, this assay is amenable to high throughput screening of environmental samples. The assay detected D. flagrans in faecal, leaf litter, and soil samples collected from 80 % of the Irish farms tested indicating that the fungus is abundant in Ireland.

Nematophagous fungi constitute a group of fungal antagonists to nematodes. The latter arc small roundworms living in soil and water. Most nematodes are saprotrophic, but many species are parasites of plants and animals (Poinar 1983). The nematophagous fungi have been suggested as promising candidatesfor bioiogical control of parasitic nematodes (Stirling 1991), but so far no successful commercial products have been .presented. Many of the previous studies on these organisms have been concerned with the ecology and physiology of interactions between nematophagous fungi and nematodes. More recenrly, molecular techniques have been employed ( jansson and Lopez-Llorca 2001). Nernatophagous fungi also have the ability to infect and colonise other organisms, including other fungi and plant roots ( Jansson and Lopez-Llorca 2004). In the current review we will briefly describe the nematophagous fungi, with special emphasis on their interactions with plant roots.

This study was conducted to determine the effects of Hirsutella minnesotensis (Hm), an endoparasitic fungus, and N-Viro Soil® (NVS), a recycled municipal biosolid, on Meloidogyne hapla greenhouse populations from Rhode Island (RI), Connecticut (CT), Geneva, New York (NYG), Lyndonville, New York (NYL), and Michigan (MI). In a greenhouse experiment, tomato (cv. Rutgers) seedlings were inoculated with 0 or 600 eggs of each nematode population and exposed to Hm mycelium (0 or 0.1 g fresh) and NVS (0 or 1 g 0.1 L-1 of soil) in a factorial design. Hirsutella minnesotensis reduced nematode densities by 31-83% across nematode populations in one test, but only slightly reduced densities of NYG and CT populations in another test. NVS reduced nematode densities by 33-92% across populations in two repeated tests. The combination of the two agents resulted in greater nematode reduction compared with Hm alone, but not compared with NVS alone. Across all Hm and NVS treatments, reduction of nematode densities were generally greater in NYG, CT, and RI than in MI and NYL populations. This study demonstrated that Hm and NVS may be used to suppress different M. hapla populations. © 2007 Taylor & Francis. 02-51102-01948 We thank T. Kerchkof and R. Pereault for technical assistance, Drs G. Abawi (NY), J. A. LaMondia (CT), and N. A. Mitkowski (RI) for providing the nematode populations, Dr T. Powers, University of Nebraska, for confirming the populations of M. hapla by DNA analyses, and the two anonymous reviewers for their thorough review and thoughtful comments and suggestions for revising the manuscript. The project was funded in part by a grant from USDA/CGP/MBT Program (02-51102-01948) and N-Viro International, Toledo, OH, to the last author.

International audience; Gastrointestinal (GI) nematode infection is a major constraint for grazing livestock production. The increasing prevalence and severity of anthelmintic-resistant nematodes in many parts of the world has led to a search for non-chemical control options. Under experimental conditions, the nematophagous fungus Duddingtonia flagrans is emerging as an alternative to chemotherapy for the control of GI nematode infection in biological production systems. Also, recent information points to the role of energy nutrition to increase the immune response against GI nematode infection. In this study the effect of D. flagrans and energy supplementation on the epidemiology of GI nematode infections is explored on grazing kids. Four groups of 10, 4-month old goats were turned out on infected pasture in the early spring and allocated to four separate paddocks where they were rotationally grazed for 16 weeks. One of these groups (F) received $0.5\times 10^{6}$ D. flagrans spores/kg BW/d. Another group (S) was supplemented with 100 g barley grain per day. A third group (F+S) received both nematophagous fungi and barley supplement treatments simultaneously while the fourth group (C) was used as a non-treated control. Both nematophagous fungi and barley supplement had a significant effect ($P

The fungus Pochonia chlamydosporia is one of the most studied biological agents used to control plant-parasitic nematodes. This study found that the isolates Pc-3, Pc-10 and Pc-19 of this fungus promote the growth of tomato and lettuce seedlings. The isolate Pc-19 colonized the rhizoplane of tomato seedlings in only 15 days and produced a large quantity of chlamydospores. This isolate was able to use cellulose as a carbon source, in addition to glucose and sucrose. Scanning electron microscopy (SEM) revealed that hyphae of the P. chlamydosporia isolate Pc-10 penetrated the epidermal cells of the tomato roots. These three P. chlamydosporia isolates promote the growth of tomato and lettuce.

The biological control is an alternative method to reduce the population of parasites by the use of natural antagonist. In the present study, the efficacy of nematophagous fungus Duddingtonia flagrans was tested to control gastrointestinal nematodes parasites of cattle livestock in the field. Twenty calves were used, distributed equally in two distinct plots formed for native pasture. . Group A was treated with D. flagrans fungus, cultivated in sorghum, in a concentration with 1x106 clamidospores/kg body weight, mixed with maintenance ration, each day, during eight months. Group B served as a control and did not receive the fungus. Samples for faecal egg count (FEC), were collected each week. There were monthly counts in faecal cultures to identify the species of nematodes larvae, weight of the animals, collection of blood to determine red cell counts and collection of pasture to the counting of larvae. Temperature and rainfall data were registered daily. The FEC reduced around 56,84% in the last 3 months of the experiment, with a variation between 40,44 and 67,14% in the treated group (P

International audience; The nematophagous fungus Arthrobotrys oligospora was tested in vitro and in vivo for its ability to solubilize rock phosphate. Three types of rock phosphate (RP) from Burkina Faso (KRP), Senegal (TRP), and Mali (TIRP) were used at four concentrations for the in-vitro experiment. All three types of RP were solubilized by the fungus. The maximum quantity of P recovered in solution was obtained with TRP, 12.5% for an application of 1 g L-1. The effect of TRP and A. oligospora applied separately or in combination was tested in vivo on the growth of A. holosericea. In a P-deficient soil without addition of RP, P solubilization was increased by addition of A. oligospora. The P uptake by plants growing in soil amended with TRP and inoculated with A. oligospora was significantly higher compared to noninoculated controls, thus demonstrating the ability of the fungus to solubilize additional phosphate from RP in vivo.

WOS: 000233851700006 The effects of the endoparasitic fungus Hirsutella minnesotensis on populations of Meloidogyne hapla from Michigan (MI), Rhode Island (RI), Connecticut (CT), Lyndonville, New York (NYL), Geneva, New York (NYG), and Wisconsin (WI) were studied in the greenhouse. Twenty-day-old tomato (cv. Rutgers) seedlings were inoculated with either 0 or 600 eggs of each nematode population mixed with either 0, 0.02, or 0.1 g of fresh H. minnesotensis mycelium 0.1 L-1 of soil in pots containing 0.5 L of soil, and maintained at 25 +/- 2 degrees C for 2 months. No effect of the fungal treatments and nematode treatments on tomato plant heights and shoot dry weights was observed. While all M. hapla populations were suppressed by H. minnesotensis, the degree to which each population was affected varied slightly. Across fungal treatments and nematode populations, the fungus reduced total number of nematodes in roots by 61-98%, with the highest for NYG and RI, intermediate for NYL and CT, and lowest for MI and WI populations. The study demonstrated that H. minnesotensis may be used as a potential suppressor of M. hapla in vegetable production systems in the Great Lakes Region.

A field trial, conducted over two consecutive years, was aimed at assessing the efficacy of the administration of spores of the nematophagous fungus Duddingtonia flagrans to young goats for the control of nematode parasite infections on a French commercial dairy goat flock. For both years, the first-year grazing kids were divided into two similarly managed groups (fungus and control groups): in 2003 a daily dose rate of 5 x 10(5) spores/kg body weight was given to the fungus-group animals, while in 2004 a daily dose rate of 10(6) spores/kg body weight was used; the other half of the kids, acting as control, did not receive the spores. Parameters measured every 3 weeks included nematode egg excretion, larval development in faecal cultures and pasture larval counts. Additionally, at the beginning, the middle and the end of each grazing season, the goats were weighed and blood samples for pepsinogen determination were collected. In 2003, similar results were recorded for all the measured parameters in the control and fungus groups. In contrast, in 2004, the kids receiving the spores showed lower faecal egg counts and pepsinogen levels at the end of the season and higher growth rate compared to kids of the control group.

The gene encoding a cuticle-degrading serine protease was cloned from three isolates of Lecanicillium psalliotae (syn. Verticillium psalliotae) by 3′ and 5′ RACE (rapid amplification of cDNA ends) method. The gene encodes for 382 amino acids and the protein shares conserved motifs with subtilisin N and peptidase S8. Comparison of translated cDNA sequences of three isolates revealed one amino acid polymorphism at position 230. The deduced protease sequence shared high degree of similarities to other cuticle-degrading proteases from other nematophagous fungi.

The ability of the nematophagous fungus Duddingtonia flagrans to reduce the number of infective nematode larvae in coproculture was investigated in goats using different doses of chlamydospores (0, 1.25 × 10 5 , 2.5 × 10 5 , 5 × 10 5 chlamydospores/kg BW/day) given by oral administration or by voluntary consumption in feed during natural or experimental infections with nematodes. The kinetics of excretion of D. flagrans chlamydospores in the faeces was also determined using a dose of 5 × 10 5 chlamydospores/kg BW/day for five days. For all the trials, the faecal nematode egg outputs were determined by a modified McMaster method and standard coprocultures were set up (14 days, 25 °C) to determine the number of larvae emerging from culture in fungus treated and control faeces. When chlamydospores were orally administered, the number of larvae were reduced by 50 to 97% when compared to control cultures. No difference in the level of larval emergence from the culture was seen for experimental or natural infections at the different chlamydospore dose rates. In contrast, when chlamydospores were distributed in the feed, a dose- dependent relationship was observed 10 days after the start of administration, the larval development being 2.0%, 14.0% and 86.9% for 5 × 10 5 , 2.5 × 10 5 and 0 spores/kg BW/day, respectively. In addition, the kinetic study showed that the larval emergence from coproculture in the fungus group was statistically lower than in the control group from the second day of administration of the chlamydospores and remained lower until the second day after the last administration (p < 0.05). The results indicate that, for goats in farm conditions, a minimum daily dose of 5 × 10 5 chlamydospores/kg BW must be used to ensure a high treatment efficacy and that daily administration is preferable for maintenance of efficacy over time. goat / biological control / Duddingtonia flagrans / nematode parasite / dose trials

The nematophagous fungus Pochonia chlamydosporia is a potential biocontrol agent against root knot and cyst nematodes. Genetic transformation of the fungus to introduce visual marker genes, novel traits, or changes in expression levels of endogenous genes, would greatly enhance understanding of its behaviour on nematode-infested roots and of its interactions with other soil and rhizosphere microorganisms. A transformation system for the introduction of novel genes into P. chlamydosporia has been developed. Methods to generate protoplasts, introduce DNA and regenerate transformed viable fungal mycelium have been optimised, using plasmids carrying the green fluorescent protein marker gene gfp and the hygromycin resistance gene hph. Cultures of P. chlamydosporia were resistant to high levels of a range of fungal inhibitors, including hygromycin, that are commonly used with dominant selectable marker genes in the transformation of other fungi. However, regenerating protoplasts transformed with hph could be selected by their ability to grow through an agar overlay containing 1 mg ml(-1) hygromycin. Green fluorescence was observed in protoplasts and regenerating mycelium after transformation with gfp, but the GFP phenotype was lost on subculture. Maintenance of introduced genes was not stable, and during subculture, PCR assays indicated that the transformants lost both hph and gfp. When these genes were introduced on the same plasmid, segregation of hph and gfp was observed prior to their loss. It was unclear whether the introduced plasmids were able to replicate autonomously in P. chlamydosporia, or if they integrated transiently into the fungal genome. Possible reasons for the instability of the transformants are discussed.

Control of nematode parasites of small ruminants in a wet, tropical environment using the nematophagous fungus, Duddingtonia flagrans, was assessed in this study. Two methods of fungal delivery were tested, namely as a daily feed supplement, or incorporated into feed blocks. Initially, pen trials were conducted with individually penned groups of sheep and goats at dose rates of 125,000 spores and 250,000 spores/kg live weight per day. At the lower dose rate this reduction was between 80 and 90% compared with the pre-treatment levels. At the higher dose rate, there was virtually complete suppression (>99% reduction) of larval recovery. Trials using the fungal feed blocks, showed that when animals were individually penned, they consumed only small amounts of the block (particularly goats), hence little effect on larval recovery in faecal cultures was observed. Grouping animals according to species and dose rate induced satisfactory block consumption and subsequent high levels of larval reduction in faecal cultures. These larval reductions were mirrored by the presence of fungus in faecal cultures. This work was followed by a small paddock trial, whereby three groups of sheep were fed either a feed supplement without fungal spores, supplement with spores, or offered fungal blocks. The dose rate of spores in the latter two groups was 500,000 spores/kg live weight per day. Egg counts were significantly reduced in the two fungal groups, compared with the control group and the latter required two salvage anthelmintic treatments to prevent mortality due to haemonchosis. Pasture larval numbers on the two fungal group plots were also much lower than on the control plot.

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.

The mitosporic fungus Pochonia chlamydosporia is a potential biocontrol agent for cyst (Heterodera spp. and Globodera spp.) and root knot (Meloidogyne spp.) nematodes, which are important agricultural plant pests. 54 isolates from diverse geographical regions and several nematode hosts were used in this study. Genetic variation was examined using enterobacterial repetitive intergenic consensus (ERIC) primed PCR and sequences from the internal transcribed spacer (ITS) rRNA region. ERIC PCR yielded 35 scorable binary characters from all the fungi tested and cluster analysis of the data showed that isolates from cyst nematodes were more genetically variable than those from root knot nematodes. The ITS regions were highly conserved, the only significant difference being an extra thymidine in isolates from Meloidogyne spp. Assays with nematode eggs indicated that isolates differ in their ability to infect different nematode genera. The results indicate host related variation in P. chlamydosporia. This finding has significant implications for the application of P. chlamydosporia as a biocontrol agent.

The nematophagous fungus, Pochonia chlamydosporia (Goddard) Zare & Gams, has been investigated as a potential biological control agent for use in integrated pest management strategies for Meloidogyne incognita (Kof & White) Chitwood in vegetable crops. The release of the fungus as a biological control agent requires a diagnostic method to monitor its spread in the environment and to gain knowledge of its ecology. Only molecular methods are sufficiently discriminating to enable the detection of specific isolates of fungi in soil. A method to extract DNA from soil was developed to increase the efficacy of PCR-based diagnostic tests that use specific primers. A selected isolate of P chlamydosporia var catenulata was applied at densities similar to those that occur naturally in nematode-suppressive soils. The fungus significantly reduced nematode infestations in soil following a tomato crop, in a strategy that combined the use of the fungus with crop rotation. The survival of the fungus in soil was also examined in controlled conditions in which it remained in soil in densities significantly greater than its original application rate for at least 5 months. Hence, it seems that populations of this fungus may be built up in soil and have significant effects on the regulation of root-knot nematode populations.

Conídios de Monacrosporium thaumasium foram administrados por via oral a caprinos para verificar a viabilidade desse fungo nematófago após passagem pelo trato gastrintestinal. Foram realizadas amostragens de fezes de 18 a 30 horas após inoculação. Constatou-se crescimento do fungo e atividade predatória contra Panagrellus spp em placas confeccionadas com fezes coletadas 21 e 24 horas após a inoculação. Houve uma redução média de 79,24% no número de larvas infectantes de Haemonchus contortus provenientes de coproculturas preparadas com fezes coletadas 24 horas após a administração do fungo. O fungo M. thaumasium manteve atividade predatória sobre H. contortus, após passagem através do trato gastrintestinal de caprinos. In order to verify the viability after passage through the gastrointestinal tract, conidia of nematophagous fungus, Monacrosporium thaumasium, were orally administered to goats. Fecal samples were collected 18 to 30 hours after inoculation. It was verified fungus growth and predatory activity against Panagrellus spp in plates made with feces collected at 21 and 24 hours after inoculation. There was a 79.24% reduction of Haemonchus contortus infective larvae number from coprocultures prepared with feces collected at 24 hours after the fungus administration. The fungus M. thaumasium maintained predatory activity over H. contortus, after passage through the gastrointestinal tract of goat.

A three-year grazing experiment (1998-2000) was conducted with first-season grazing cattle (FSGC) on improved pastures in central-eastern Sweden. Comparison was made between five groups with 10 calves in each group where four of these were set stocked and either (1) untreated, (2) ivermectin bolus treated, (3) subjected to biological control with the nematophagous fungus Duddingtonia flagrans, or (4) treated with a copper wire particle bolus. The fifth treatment was an evasive grazing strategy, whereby untreated calves were turned out onto pasture used by older cattle the previous year and then these calves were moved to silage aftermath in mid-July. To introduce low-levels of parasite infection to the experiment, each animal received a 'priming dose' of approximately 5,000 Ostertagia ostertagi and 5,000 Cooperia oncophora infective third stage larvae immediately prior to the start of the first grazing year of the trial. Results showed that efficient and sustainable parasite control of FSGC was possible to achieve without the use of anthelmintics by using turnout pastures that the previous year had been grazed by older cattle, in combination with a mid-July move to aftermath leys. Biological control also proved beneficial but the efficacy was impaired if high faecal egg counts coincided with rapid dung pat degradation due to heavy rainfall. No indication of parasite control was observed with the copper wire particle bolus. It was also demonstrated that the impact of gastrointestinal (GI) parasitism varied between years and that the level of overwintering contamination is important but likewise, is unpredictable. Although faecal egg counts in 1999 were low, due both to a delayed turnout and drought for the major part of the grazing season, deposited eggs successfully developed to infective larvae and overwintered in large numbers. The population of overwintered infective larvae at the time of turnout in early May played an important role in the course of infection in 2000 and resulted in an average 65 kg advantage of the ivermectin treated calves compared with the untreated calves.Thus, this three-year grazing experiment has emphasised the importance of subclinical gastrointestinal nematode infections in FSGC in Sweden. In addition, the study has shown that adequate parasite control may be achievable without the use of anthelmintics.

International audience; Effective alternatives to anthelmintic treatment against nematode parasites of goats are required because of the high prevalence of benzimidazole resistance. Towards this objective, the nematophagous fungus, Duddingtonia flagrans (Df), was used in a plot study against two main parasitic nematode species of goats, Teladorsagia circumcincta (Tcir) and Trichostrongylus colubriformis (Tcol). Worm-free, culled goats were experimentally infected with strains of Tcir and Tcol to constitute donors. Half of the animals were periodically given Df chlamydospores at a daily dose of 2.5 $\times$ 10$^5$ spores/kg BW while the remaining animals were kept as controls. At 5 time periods i.e. March, May, July, September and November 2001, corresponding to the main grazing season in France for goats, faeces were collected from the 6th day of fungus administration for the following 2 days to obtain approximately 1 kg of faeces from each group of animals: Tcir/Control, Tcol/Control, Tcir/Fungus, Tcol/Fungus. For each period and each group, the faeces were deposited on a 1 m$^2$ grass plot and the grass was cut (3 replicates) on weeks 2, 4, 6, 8 ,12 after deposition, for infective larval recovery. Larvae were counted and the results were expressed as a ratio of larvae/eggs deposited. On the plots with the control faeces deposited in March, July and September, the grass infectivity due to Tcir and Tcol was similar and the maximum number occurred between 2 and 4 weeks post deposition. In May, the maximum numbers of larvae were not recorded until 8 weeks after deposition, due to high daily temperatures and dryness. In November, larval development took place only for Tcir. On the plots with the fungus treated faeces, a significant reduction in grass infectivity occurred for both nematodes and ranged from $50-60$% in May, July and November deposits to $80-90$% in the September deposit. On the contrary to these findings, no difference was recorded between the fungus and control plots for the March deposit. In conclusion, D. flagrans is suitable for reducing the number of infective larvae in the herbage during the main part of the grazing period for the most important digestive nematodes of goats.

The fungus Pochonia chlamydosporia is a biocontrol agent with commercial potential for root knot and cyst nematodes. It produces an alkaline serine protease, VCP1, during infection of nematode eggs. The gene encoding VCP1 was sequenced and the sequences of cDNAs from six isolates from different nematode hosts were compared. The gene encoding VCP1 was similar to PR1 from Metarhizium anisopliae with similar regulatory elements. Comparison of translated cDNA sequences revealed two amino acid polymorphisms at positions 65 and 99, indicating a difference between isolates from cyst and root nematodes. The positions and nature of the polymorphisms indicated that the two forms of VCP1 might have different properties and this was tested with five chromogenic polypeptide substrates. Enzyme assays revealed the two forms differed in their abilities to utilise Succ-Ala-Ala-Pro-Phe-pNa and Succ-Ala-Val-Pro-Phe-pNa, suggesting different amino acid affinities at the S3 binding region. This indicates host related genetic variation in VCP1 between isolates of P. chlamydosporia isolated from different nematode hosts, which might contribute to host preference. Such differences may be important in future exploitation of P. chlamydosporia as a nematode biocontrol agent.

Pooled faecal samples of Patanwadi sheep maintained were screened monthly from January to December 2011 for isolating nematophagous fungi. Two isolates of common nematophagous fungus, Arthrobotrys oligospora and one of Duddingtonia flagrans were isolated. One isolate of each fungus was evaluated for its nematode trapping ability by in vitro and in vivo studies. Of the two isolates evaluated, D. flagrans was found to be highly effective against Haemonchus contortus in sheep.

Biological control of parasitic nematodes of livestock is currently under development and represents another tool that may be integrated into helminth parasite control strategies. This paper presents a brief introduction to commercial sheep farming in South Africa and currently available nematode parasite control methods. These include the FAMACHA(r) clinical assay, strategies of pasture management, dilution of resistant worm species by introduction of susceptible worms, breed resistant sheep and nutritional supplementation. The purpose of this paper is to outline the principles of biological control using nematophagous fungi and how it may be applied on sheep farms in South Africa.

Studies showed that chlamydospores of the nematophagous fungus, Duddingtonia flagrans, are capable of surviving pressures of several tonnes when incorporated into matrices and pressed into tablets for the manufacture of prototype intraruminal controlled release devices (CRDs). They remain viable in this tabletted form for at least 9 months when stored at 4 degrees C. In vitro studies demonstrated that there was no effect on spore viability of prolonged exposure to either room or elevated temperature (40 degrees C) in air, or under an atmosphere of either of the major ruminal gases, carbon dioxide and methane. In vivo, studies showed that viable chlamydospores could be detected at the erosion surface of prototype CRDs recovered from the rumen and also in faeces of fistulated sheep, for up to 3 weeks after administration. Further studies have shown that chlamydospores released from such devices can substantially reduce the number of infective larvae that develop in cultures of faeces collected from sheep infected with the nematode parasite, Haemonchus contortus. This work demonstrates, in principle, that the deployment of chlamydospores of D. flagrans in intraruminal CRDs, is another possibility in the development of a range of methods for the biological control of parasites in livestock.

Barley roots were readily colonised by the nematophagous fungus Verticillium chlamydosporium. Light microscopy (LM) but also low temperature scanning electron microscopy (LTSEM) revealed details of the colonisation process. Hyphae were found on the rhizoplane often with dictyochlamydospores. Hyphae of V. chlamydosporium penetrated epidermal cells, often by means of appressoria. A hyphal network was formed in epidermal and cortical cells. Likewise, hyphal coils were found within root cells next to transverse cell walls. Cortical cells were the limits of fungal colonisation, since no hyphae were seen in the vascular cylinder. Modifications of root cell contents (phenolic droplets and callose appositions) were common three weeks after inoculation with V. chlamydosporium. These features may indicate induction of plant defence reactions in late stages of root colonisation by the fungus. Both LTSEM and LM have proved extremely useful to describe root colonisation by the fungus. The results found may have implications in the mode action of nematophagous fungi against plant parasitic nematodes.

Investigations were made into the timing of administration of Duddingtonia flagrans as a biological control agent against ovine parasitic nematodes including stongylid and Nematodirus spp. Faeces from 3-4 months old male lambs were deposited onto pasture plots that had never been grazed by sheep. The trial was conducted over two consecutive years (1998 and 1999). For both years, the following three plot types were involved: Sim plots had faeces containing nematode eggs and Duddingtonia flagrans spores deposited simultaneously; Post plots had faeces containing nematode eggs followed 2 weeks later by faeces containing D. flagrans spores alone; Control plots had faeces containing only nematode eggs; Prior plots (included in 1999) had faeces containing D. flagrans spores alone followed 2 weeks later by faeces containing nematode eggs. In each year, two deposition periods were involved: July and August in 1998 and June and July in 1999. During the first year pasture samples were collected at 2, 4, 6, 8 and 12 weeks after initial deposition. In 1999, additional samples were collected at 10, 16 and 20 weeks. Larvae were extracted from the pasture samples and counts performed to estimate the number and species of infective third-stage (L(3), larvae) present. The number of third-stage strongylid larvae on pasture was significantly lower on Sim plots compared to the remaining plot types for both years at all deposition times (P

The ability of a nematode-trapping fungus to establish in field soil is an important characteristic when considering its use as a biological control agent. The outgrowth of the nematode-trapping fungus Arthrobotrys superba from wood was recorded by labelling the fungus with [(14)C]3-O-methylglucose and [(32)P]orthophosphoric acid and by using the soil sprinkling method. The fungus reached a distance of 7-8 cm during 25 days in heat-treated (60 degrees C) soil, detected by either radioactive tracing or the soil sprinkling technique. The two labelled compounds were co-distributed at all sampling times (r(2)=0.946) which indicates that the glucose pool (as methylglucose) and phosphorus content were correlated throughout the mycelium. In natural, non-heat-treated soil the fungus reached a distance of 1.5 cm from one disc of birch wood after 30 days, while it reached 3.2 cm during the same period when the food base was a pile of five inoculated discs. The experiments showed, for the first time, that a nematophagous fungus, A. superba, can grow out into soil from a piece of wood and supported by nutrients translocated from the resource base to the edge of the mycelium.

Biological control is a broad concept which encompasses a range of control strategies including cultural practices, host plant resistance and the introduction or encouragement of antagonistic organisms. Baker and Cook (1974) defined biological control of nematodes as reduction in nematode damage by organisms antagonistic to nematodes through the regulation of nematode populations and/or a reduction in the capacity of nematodes to cause damage, which occurs naturally or is accomplished through the manipulation of the environment or by the mass introduction of antagonists.

Summary: It was recently shown that the nematode-infecting fungus Arthrobotrys oligospora contains a saline-soluble lectin (designated AOL) that is a member of a novel family of fungal lectins sharing similar primary sequences and binding specificities. During saprophytic growth in liquid cultures, levels of AOL and AOL mRNA were found to vary depending on the growth phase of the mycelium and the carbon/nitrogen (C/N) ratio of the medium. AOL was not detected in young mycelium. In older mycelium (stationary growth phase) grown in media with low C/N ratios (1 or 6), AOL comprised 5-20% of the total amount of saline-soluble proteins present in the mycelium. Neither the lectin nor its transcript was detected in mycelia grown in medium with higher C/N ratios (≥150). Under conditions of nitrogen starvation, AOL was preferentially degraded in relation to the total amount of saline-soluble proteins present in the mycelium. During the infection of nematodes, the level of AOL protein and AOL mRNA increased significantly once the nematodes had been penetrated and digested. Large amounts of AOL accumulated in the trophic hyphae growing inside the nematode as visualized by immunofluorescence microscopy. Later, AOL labelling was detected outside the digested nematodes, preferentially in strands of aggregated hyphae and in newly developed trap cells. Electron microscopy showed that AOL was localized to the cytoplasm and the nucleus of both vegetative mycelium and trap cells, and in the trophic hyphae growing inside the infected nematodes. These results indicate that AOL functions as a storage protein during both saprophytic and parasitic growth.

The fungal genus Arthrobotrys Corda contains fortyeight various nematode-trapping species. Within the genus fungi that produce traps of the adhesive network type as well as a few species that form constricting rings are found.

Nematodes are common soil inhabitants. Some feed on microorganisms and others are parasites of plants and animals (Stirling, 1991). Nematophagous fungi are parasites of nematodes and are found in almost every soil. They can be divided into three groups: 1) The nematode-trapping fungi, which capture nematodes by means of hyphal traps. These fungi live to varying degrees as saprophytes. 2) The endoparasitic fungi, which parasitize nematodes using minute conidia or zoospores and are obligate parasites (Barron, 1977; Gray, 1988). 3) Egg and female parasites which use their hyphal tips to infect the nematodes. These fungi live also as saprophytes (Morgan-Jones and Rodriguez Kabana, 1988).

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 mm2. 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.

Individuals of the free-living nematode Panagrellus redivivus were infected with conidia of the endoparasitic nematophagous fungus Drechmeria coniospora. At different stages of infection the nematodes were added to an agar-plate culture of the predatory fungus Arthrobotrys oligospora which contained traps. Initially, nematodes infected with D. coniospora were invariably captured, penetrated and colonized by A. oligospora. Then both fungi used the nematode contents for development of hyphal biomass outside the nematode. But inside nematodes in which infection by D. coniospora was advanced development of A. oligospora was markedly hampered. In dual-infected nematodes (colonized from opposite ends), the hyphae of A. oligospora were often observed to be dead or degenerate when in close proximity to living mycelium of D. coniospora, indicating that the latter acts as an antagonist against A. oligospora under these conditions. The interaction between trophic hyphae of D. coniospora and A. oligospora involved thickening of the cell wall of A. oligospora at the sites of contact and degradation of the hyphal compartments and shrivelling of the cell wall of A. oligospora. The observed interactions are discussed in relation to mycoparasitism and possible ecological function. © 1994, British Mycological Society. All rights reserved.

A new fungal parasite of the reniform nematode has been observed parasitizing nematode populations that have increased on cotton in a sandy loam field soil in the greenhouse. When enumerated, 46% of the stock reniform nematode population was colonized by this fungus. Egg, vermiform, and adult stages of the reniform nematode were observed with light microscopy and scanning electron micrography (SEM). The nematophagous fungus, Catenaria auxiliaries, was identified morphologically. There are no sequences on the GenBank to achieve a molecular identification. This nematophagous fungus has been previously reported on the beet cyst nematode in Europe (1,2); however, to our knowledge there are no reports of this fungus parasitizing the reniform nematode. In vermiform life stages of the nematode, rhizomycelium is observed in the initial phase of infection and is characterized by ovoid cells, 9.5 to 13.5 × 17.0 to 24.5 μm in diameter, separated by septa. Usually 10 to 15 ovoid cells lacking intercellular hyphal filaments are produced within each vermiform body. Rhizoids 3.5 to 4.0 μm wide develop from the rhizomycelium. Mature swollen cells produce precursor sporangia that may mature into resting spores or zoosporangia. Resting spores are yellow-to-cream, 20 to 40 μm in diameter with a reticulate appearance, and are common in the vermiform nematode life stages. Zoosporangia are ovoid, 9.5 to 13.5 × 17.0 to 24.5 μm, and will erupt from the cuticle of the vermiform nematode releasing zoospores via papillae. Zoospores are 2.9 to 4.9 μm with visible globules in the anterior region and single flagella that are 9 to 11 μm long. The zoospores swim short distances, maneuvering in the direction of the flagellum. Adult reniform females observed through SEM exhibit zoospores encysted in the metacorpus region of the nematode. Parasitized eggs are internally colonized with zoosporangia that are 20 to 25 μm in diameter. In advanced stages of infection, the eggs darken in color and zoosporangia erupt through the cuticle of the egg. Reniform nematodes visibly colonized with zoosporangia and resting spores were placed on corn meal, water, and potato dextrose agars. None of these media supported growth of the fungus, supporting our theory that this organism appears to be an obligate parasite of the nematode. Koch's postulates was completed when eggs colonized with rhizomycelium and resting spores or zoosporangia were added to cotton plants in sterile soil previously inoculated with 2,000 healthy vermiform reniform life stages. Plants were allowed to grow for 30 days in the greenhouse after which the next generation of vermiform nematodes were extracted from the soil and examined under the microscope. Rhizomycelium, resting spores, and zoosporangia were present in 42% of the reniform vermiform life stages. Morphological comparisons of the rhizomycelium, resting spores or zoosporangia, and zoospores colonizing the reniform nematodes were similar to the initial observations. Thus to our knowledge, this is the first report of Catenaria auxiliaries parasitizing the reniform nematode. References: (1) B. Kerry. J. Nematol. 12:253, 1980. (2) H. T. Tribe. Trans. Br. Mycol. Soc. 69:367, 1977.

Conidia of the endoparasitic fungus Drechmeria coniospora were able to adhere to the nematode Acrobeloides buetschilii; approximately 70% of the nematodes were carrying spores 16 h after their addition to the fungal culture. Young nematodes were preferentially affected; adhesion mainly took place in the head region of the animal. Full infection, characterized by colonization of the nematode body and production of conidiophores and conidia, was observed, albeit at low frequencies. In case of failure, a second adhesive knob was often formed on the germinated spore. The low frequencies of infection are not related to a loss of virulence, due to prolonged in vitro culturing of the fungus. Fresh spores, obtained from cultures which were passed several times through the host, did not show higher infection frequencies.

We studied the population biology of the nematophagous fungus Hirsutella rhossiliensis to understand its potential as a biological control agent. Because the fungus is an infectious and transmissible parasite, we framed our study within an epidemiological context. Field observations, theory, and experiments demonstrated that (i) parasitism of nematodes by H. rhossiliensis is dependent on nematode density, (ii) local populations of the fungus will go extinct unless supplied with some minimum number of nematodes (the host threshold density), and (iii) natural epidemics of this fungus in populations of nematodes develop slowly and only after long periods of high host density. Additional in-depth research on population biology is needed to explain other biological control systems and to guide future research. The most effective research will combine field observation, theory, and experimentation. Key words: density-dependent parasitism, host-parasite dynamics, modeling, nematophagous fungi.