A suitable model for the utilization of Duddingtonia flagrans fungus in small-flock-size sheep farms

Effective alternatives to anthelmintic treatment of nematode parasite infections of sheep are required because of the high prevalence of drug resistance. Within this context, the nematode-trapping fungus Duddingtonia flagrans has become a valuable component of various integrated control strategies. Toward this objective, a small quantity of lyophilized D. flagrans chlamydospores (10⁶ spores per animal) was administered to sheep in a one-year plot study. Animals grazing on native pasture were divided into two homogeneous groups and were kept in 1-ha paddocks in the southern region of Brazil. The oral administration of chlamydospores led to a significant reduction (p < 0.05) in the number of nematode eggs per gram of feces and in the larval availability on herbage (difference of 37.6%) in comparison to the control group. Control animals needed to be dewormed three times during the experiment, whereas the fungus-treated animals maintained a low parasite load, independent of seasonal variation. Although D. flagrans cannot serve as a panacea for nematode parasite control of livestock, it represents a significant advance toward rationalizing the use of endoparasitic drugs in small animals.     

The STRIPAK component SipC is involved in morphology and cell-fate determination in the nematode-trapping fungus Duddingtonia flagrans

The striatin-interacting phosphatase and kinase (STRIPAK) complex is a highly conserved eukaryotic signaling hub involved in the regulation of many cellular processes. In filamentous fungi, STRIPAK controls multicellular development, hyphal fusion, septation, and pathogenicity. In this study, we analyzed the role of the STRIPAK complex in the nematode-trapping fungus Duddingtonia flagrans which forms three-dimensional, adhesive trapping networks to capture Caenorhabditis elegans. Trap networks consist of several hyphal loops which are morphologically and functionally different from vegetative hyphae. We show that lack of the STRIPAK component SipC (STRIP1/2/HAM-2/PRO22) results in incomplete loop formation and column-like trap structures with elongated compartments. The misshapen or incomplete traps lost their trap identity and continued growth as vegetative hyphae. The same effect was observed in the presence of the actin cytoskeleton drug cytochalasin A. These results could suggest a link between actin and STRIPAK complex functions.     

Influence of the preservation period in silica-gel on the predatory activity of the isolates of Duddingtonia flagrans on infective larvae of cyathostomins (Nematoda: Cyathostominae)

The continued maintenance of nematophagous fungi predatory activity under laboratory conditions is one of the basic requirements for a successful biological control. The purpose of this study was to evaluate the influence of time on the preservation of the fungus Duddingtonia flagrans (AC001 and CG722) stored in silica-gel for 7 years and their subsequent predatory activity on cyathostomin L₃ larvae in 2% water-agar medium (2% WA). Samples of the isolates AC001 and CG722, originating from vials containing grains of silica-gel sterilized and stored for 7 years, were used. After obtaining fungal conidia, the predation test was conducted over 7 days on the surface of 9.0 cm Petri dishes filled with 2% WA. In the treated groups each Petri dish contained 500 cyathostomin L₃ and conidia of fungal isolates in 2% WA. In the control group (without fungi) the plates contained 500 L₃ in 2% WA. The experimental results showed that isolated AC001 and CG722 were efficient in preying on cyathostomin L₃ (p < 0.01) compared to control (without fungus). However, no difference was observed (p > 0.01) in the predatory activity of the fungal isolates tested. Comparing the groups, there was a significant reductions of cyathostomin L₃ (p < 0.01) of 88.6% and 78.4% on average recovered from the groups treated with the isolates AC001 and CG722, respectively, after 7 days. The results of this test showed that the fungus D. flagrans (AC001 and CG722) stored in silica-gel for at least 7 years maintained its predatory activity on cyathostomin L₃.     

Duddingtonia flagrans: centrifugal flotation technique with magnesium sulphate for the quantification and qualification of chlamydospores in sheep faeces

Duddingtonia flagrans is a nematode-trapping fungus responsible for attacking larval stages of helminths in pasture, which has potential as a biological control method. The aim of this study was to test the magnesium sulphate centrifugal flotation technique for the quantification of D. flagrans chlamydospores in sheep faeces and to verify their morphological viability. In this experiment one sheep received an oral dose of 4.5 × 10⁶ chlamydospores/day during 20 days. Fecal samples were collected between days 15 and 20 and analyzed by the centrifugal flotation technique with magnesium sulphate. Densities of 1.23, 1.27 and 1.31 g mL⁻¹ recovered 1.45 × 10⁵, 3.87 × 10⁵ and 1.65 × 10⁵ chlamydospores from the faeces, respectively. Based upon the results it was concluded that this is an efficient technique for the chlamydospores quantification in ovine faeces. Moreover, it allowed more accurate visualization of chlamydospore morphology.     

An extracellular serine protease of an isolate of Duddingtonia flagrans nematophagous fungus

This study aimed to present a protease produced by Duddingtonia flagrans fungus (AC001), and to evaluate its activity in the biological control of cyathostomin infective larvae (L₃). The crude extract from D. flagrans grown in liquid medium was applied first to a DEAE-Sepharose? and later to a CM-Sepharose? ion exchange column. Protease activity was determined under different pHs and temperatures. Subsequently, the effects of metal ions and phenylmethylsulfonyl fluoride (PMSF) inhibitor on activity were evaluated. Next, the protease activity in the biological control of nematodes was tested. A new 38 kDa serine protease (Df1) was purified. Optimum activity was obtained at pH 8.0 and 60°C; CuSO₄, ZnSO₄ and PMSF strongly inhibited the activity. Df1 (AC001) showed an L₃ reduction rate of 58%. In conclusion, a serine protease produced by D. flagrans (AC001) has been isolated, which is effective in the in vitro destruction of cyathostomin L₃.     

捕食性真菌Duddingtonia flagrans原生质体获得与再生的荧光标记评价

为了更准确快捷地评价利用酶解法酶解捕食性真菌Duddingtonia flagrans菌丝产生的原生质体数量及菌丝细胞壁降解情况,采用活体荧光染料羧基荧光素乙酰乙酸(carboxyfluorescein diacetate succinimidyl ester,CFSE),对该捕食性真菌菌丝酶解后产生的原生质体进行荧光标记,分别考察了标记浓度、标记时间、孵育温度对原生质体标记效果的影响,并观察CFSE标记后的原生质体再生情况。结果表明CFSE终浓度为10μmol/L,标记时间为15min,孵育温度为36℃,是CFSE标记捕食性真菌原生质体的理想条件,该试验同时表明CFSE不影响原生质体的再生率。CFSE作为一种活细胞示踪荧光探针,可以快速高效地标记捕食性真菌原生质体,该方法为捕食性真菌原生质体制备质量的快速评价提供了新的思路。     

Culture medium characteristics on the predatory activity of the nematophagous fungus Duddingtonia flagrans

The influence of casein and pH on the activity of the nematophagous fungus Duddingtonia flagrans (AC001) on trichostrongylide larvae was evaluated. A ‘positive influence’ was observed contributing to the reduction of 63% in the average number of recovered L₃ in the media supplemented with casein and pH 7.0.     

Extracellular lipase production by a sapwood-staining fungus,Ophiostoma piceae

The extracellular lipase production of a sapwood-staining fungus, Ophiostoma piceae, grown in liquid media, was optimally active at pH 5.5 and 37°C. Although glucose, fructose, sucrose, starch and dextrin, as carbon sources for growth gave similar mycelial yields, which were higher than those obtained with arabinose, galactose or raffinose, the cells growing on those carbohydrates produced little extracellular lipase. However, both high biomass and lipase activity were obtained when plant oils (olive, soybean, corn, sunflower seed, sesame, cotton seed or peanut) were used as carbon sources. Among the nitrogen sources examined, Casamino acids gave the best growth, whereas (NH₄)₂SO₄ gave the best lipase production. The highest lipase productivity seen was obtained in a medium with olive oil as carbon source and a combination of (NH₄)₂SO₄and peptone as nitrogen source.The authors are with Forest Products Biotechnology, Department of Wood Science, Facully of Forestry, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada     

Trematodes enhance the development of the nematode-trapping fungus Arthrobotrys (Duddingtonia) flagrans

The capability of helminth (nematode and trematode) parasites in stimulating nematode trap and chlamydospore development of the nematophagous fungus Arthrobotrys (formerly Duddingtonia) flagrans was explored. Dead adult specimens of trematodes (the liver fluke Fasciola hepatica and the rumen fluke Calicophoron daubneyi) and nematodes (the ascarid Parascaris equorum and the strongylid Oesophagostomum spp.), as well as their secretory products, were placed onto corn meal agar plates concurrently inoculated with A. flagrans. Trapping organs were observed after 5 d and chlamydospores after 16 d, including in the control plates in the absence of parasitic stimulus. However, our data shows that both nematodes and trematodes increase trap and chlamydospore production compared with controls. We show for the first time that significantly higher numbers of traps and chlamydospores were observed in the cultures coinoculated with adult trematodes. We conclude that both the traps and chlamydospores formation are not only related to nematode-specific stimuli. The addition of secretory products of the trematode C. daubneyi to culture medium has potential for use in the large scale production of chlamydospores.     

Enzyme production in continuous cultivation by the thermophilic fungus, Thermomyces lanuginosus

The production of extracellular enzymes by the thermophilic fungus Thermomyces lanuginosus was studied in chemostat cultures at a dilution rate of 0.08 h^–1 in relation to variation in the ammonium concentration in the feed medium. Under steady state conditions, three growth regimes were recognised and the production of several extracellular enzymes from T. lanuginosus was recorded under different nutrient limitations ranging from nitrogen limitation to carbon/energy limitation. The range and the production of carbohydrate hydrolysing enzymes and lipase increased from Regime I (NH₄Cl 600 mg l^–1) to Regime III (NH₄CI 1200 mg l^–1), whereas production of protease was highest in Regime II (600 mg l^–1 < NH₄Cl <1200 mg l^–1).     

Application of a formulation of the nematophagous fungus <Emphasis Type="Italic">Duddingtonia</Emphasis><Emphasis Type="Italic">flagrans</Emphasis> in the control of cattle gastrointestinal nematodiosis

The viability of a formulation of Duddingtonia flagrans was assessed in the control of parasite gastrointestinal nematodes of cattle. Two groups (A and B) of eight crossbred Holstein × Zebu cattle, approximately one year old, were placed in Brachiaria decumbens pasture. Each animal in group B (treated) received orally 20 g sodium alginate pellets containing mycelial mass of the D. flagrans fungus, while the animals in the group A (control) received pellets without fungus for seven months, starting in March 2005. The egg per gram of feces counting the gastrointestinal nematodes showed a difference (P < 0.05) in the treated group in June, July and August, with reductions of 58% (June), 47% (July) and 51% (August) compared to the control group. The infective larvae recovered in the pastures collected up to 20 cm from distance of the fecal dung in group B differed (P < 0.01) from the larvae recovered in group A. At the end of the experimental period, the animals in group B presented a greater weight gain (P < 0.01) compared to the untreated group (A). The treatment of cattle with pellets containing the D. flagrans nematophagous fungus, at the dose and duration used was effective in controlling the infective larvae of gastrointestinal nematodes of cattle.     

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.     

Efficacy of the nematode-trapping fungus Duddingtonia flagrans against infections by Haemonchus and Trichostrongylus species in lambs at pasture

The efficacy of the nematode-trapping fungus Duddingtonia flagrans against infections by trichostrongyle nematodes in sheep was assessed throughout 6 months. Twenty Ile de France lambs were divided into two groups (control and treated groups), which were kept in separate pastures. Animals of the treated group were fed with D. flagrans twice a week (Tuesdays and Fridays). Pellets were prepared with the fungus mycelia in liquid culture medium and contained approximately 20% fungus. They were mixed with the animals' diet at a concentration of 1 g pellet per 10 kg live weight. Faecal egg counts (FEC), packed cell volume (PCV), total serum protein and the animals' body weight were determined fortnightly from 7 October 2005 to 24 March 2006. Comparison of such parameters between groups showed no significant differences (P > 0.05), except on 10 February 2006, when the control group presented a higher mean FEC than the treated group (P < 0.05). Feeding sheep with pellets containing D. flagrans had no benefit to the prophylaxis of nematode infections under the experimental conditions used in the present study.     

Interaction of the nematophagous fungus Duddingtonia flagrans on Amblyomma cajannense engorged females and enzymatic characterisation of its chitinase

The present work aimed to evaluate the production and the characterisation of a chitinase from nematophagous fungus Duddingtonia flagrans (AC001) and observe the interaction of this fungus on engorged females of Amblyomma cajennense under laboratory conditions. In assay A, the engorged females of A. cajennense were separated and immersed for 5 seconds in a fungal suspension of 10⁶ conidia/ml of the fungus D. flagrans and placed in Petri dishes, in the dark. In assay B, wheat bran supplemented with 1% chitin and liquid minimal medium was used [K₂HPO₄ (5.0 g/l), MgSO₄ (0.10 g/l), ZnSO₄ (0.0050 g/l), FeSO₄ (0.001 g/l) e CuSO₄ (0.50 mg/l)], as a substrate for chitinase production. To demonstrate the presence of chitinase in the crude extract obtained after the enzymatic extraction, a purification process was developed using a specific adsorption technique. The results from assay A demonstrated the interaction of the D. flagrans conidia produced from chitin-agar on engorged females of A. cajennense. In the assay B, D. flagrans produced a chitinase successfully, with a high value for enzyme activity. The molecular mass of semi-purified enzyme was estimated at approximately 34 kDa. It was concluded that the fungus produced a chitinase and has some entomopathogenic activity, as demonstrated here for the first time; however, it is strongly suggested that further studies are needed to elucidate the molecular mechanism of infection of target organisms by this fungus.     

Production of neutral and alkaline extracellular proteases by the thermophilic fungus, Scytalidium thermophilum, grown on microcrystalline cellulose

Extracellular proteases produced by Scytalidium thermophilum, grown on microcrystalline cellulose, were most active at pH 6.5–8 and 37–45 °C when incubated for 60 min. Highest protease activity was at day 3 where endoglucanase activity was low. Protease activity measurements with and without the protease inhibitors, p-chloromercuribenzoate, PMSF, antipain, E-64, EDTA and pepstatin A, suggest production of thiol-containing serine protease and serine proteases. Endoglucanase and Avicel-adsorbable endoglucanase activity in culture medium was not significantly affected by protease inhibitors.     

Effects of chelators (EGTA, EDTA) and calcium ions on nematode capture by the nematode-trapping fungusArthrobotrys ellipsospora

The nematode-trapping fungusArthrobotrys ellipsospora developed an adhesive knob and trapped nematodes when cultured on a low-nutrient medium. It also trapped polystyrene beads in the same way. The adhesive knob produced mucus that was stained with alcian blue, while mycelium of the fungus was stained with periodic acid/Schiff (PAS). The amount of mucus increased with in days after culturing in the low-nutrient media. The fungus completely lost its ability to trap nematodes when treated with EDTA and EGTA, but it recovered the ability after incubation in the presence of a low concentration of Ca (10⁻⁶–10⁻⁷ M) for 1 h. Calmodulin inhibitor W-7 also inhibited the trapping ability of the fungus, and there was a significant (p<0.05) difference between the effects of W-7 and W-5. Ca-binding protein was also detected in the fungus.     

Interspecific competition between the nematode-trapping fungus, Duddingtonia flagrans, and selected microorganisms and the effect of spore concentration on the efficacy of nematode trapping

The fungus, Duddingtonia flagrans, is able to trap and kill free-living nematode larvae of the cattle parasite Cooperia oncophora when chlamydospores are mixed in cattle faeces. Isolates of Bacillus subtilis (two isolates), Pseudomonas spp. (three isolates) and single isolates of the fungal genera Alternaria, Cladosporium, Fusarium, Trichoderma and Verticillium were isolated from cattle faeces and shown to reduce D. flagrans growth on agar plates. When these isolates were added to cattle faeces containing D. flagrans and nematode larvae of C. oncophora, developing from eggs, none of the isolates reduced nematode mortality attributed to D. flagrans. Similarly, the coprophilic fungus Pilobolus kleinii, which cannot be cultivated on agar, also failed to suppress the ability of D. flagrans to trap and kill developing larvae of C. oncophora. Increasing chlamydospore doses of D. flagrans in faecal cultures resulted in higher nematode mortality. Thus, no evidence of interspecific or intraspecific competition was observed. The consequences of these findings are discussed.     

Detection and production condition of acetylxylan esterase from a wood-rotting fungus,Coriolus versicolor

Acetyl esterase production was detected in a wood-rotting fungus,Coriolus versicolor, by the formation of a clear zone on a double layer agar plate containing glucose β-d-pentaacetate. Two polysaccharide acetates, carboxymethyl cellulose acetate and xylan acetate, also served as detectable substrates in place of glucose acetate to form clear zone. In an esterase assay, this fungal esterase showed a higher specificity to acetylxylan than did porcine liver esterase, indicating that it is an acetylxylan esterase.     

Acid phosphatase activity during the interaction of the nematophagous fungus Duddingtonia flagrans with the nematode Panagrellus sp

The use of Duddingtonia flagrans, a nematode-trapping fungus, has been investigated as a biological control method against free living larvae of gastrointestinal nematodes of livestock animals. This fungus captures and infects the nematode by cuticle penetration, immobilization and digestion of the internal contents. It has been suggested that this sequence of events occurs by a combination of physical and enzymatical activities. This report characterizes the acid phosphatase activity during the interaction of D. flagrans with the free-living nematode Panagrellus sp. The optimum pH for the hydrolysis of the acid phosphatase substrate p-nitrophenyl phosphate was 2.2, 2.8 and 5.4 from D. flagrans alone and 2.2 and 5.4 for Panagrellus sp alone, fungus-nematode interaction in liquid medium and fungus-nematode interaction in solid medium. Different acid phosphatase activity bands were detected by SDS-PAGE. Maximum acid phosphatase activity of the fungus or nematode alone and of the fungus-nematode interaction occurred within 70 min of incubation in the presence of the substrate 4-methylumbelliferyl phosphate. The activity of this enzyme was significantly higher for the fungus-nematode interaction when compared to the organisms alone, indicating a synergistic response. Furthermore, structures appeared in the hyphae after 30 min, nematodes were observed adhered after 40 min and many were captured by the typical fungus traps after 70 min of interaction. The participation of acid phosphatase activity and its importance during the interaction of the fungus with the nematode were discussed.     

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.