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Nematophagous fungi in sheep faeces in Minas Gerais, Brazil
Mycol. Res. 104 (8) : 1005–1008 (August 2000). Printed in the United Kingdom.
1005
Nematophagous fungi in sheep faeces in Minas Gerais, Brazil
C. A. SAUMELL†, T. PADILHA* and C. DE P. SANTOS Embrapa-Centro Nacional de Pesquisa de Gado de Leite, Rua EugeV nio do Nascimento 610, Dom Bosco, Juiz de Fora, MG 36038-330, Brazil E-mail: tpadilha!Ipsi.barc.usda.gov Received 5 October 1998 ; accepted 10 October 1999.
Few studies have examined faeces as a substratum for nematophagous fungi, and no information is available about colonisation of faeces in tropical regions. Sheep faeces were deposited on a Brachiaria decumbens pasture in the Mata Region of Minas Gerais, Brazil. Up to 2 wk after deposition, samples were collected and cultivated on water agar (2 %) with free-living nematodes as bait. From 150 samples, 130 isolates were recovered after 3 wk. More than 70 % were predators and Arthrobotrys and Monacrosporium spp. predominated. Endoparasitic species of Drechmeria, Harposporium, Myzocytium, Nematoctonus and Verticillium were also isolated.
I N T R O D U C T I ON Gastrointestinal infections with nematode parasites are one of the major causes of reduced ruminant performance throughout the world (Parkins & Holmes 1989). Traditionally, the control of infections by nematodes in ruminants has combined the use of anthelminthic drugs, to eliminate the adult population of parasites within the animal and where possible, with pasture management, to avoid ingestion of infective larvae. Increasing levels of resistance to anthelminthic drugs in several helminth species (Waller et al. 1996), however, and growing concern about the presence of drug residues in ruminant products (Padilha 1996), and the potential toxicity of active drug metabolites for non-target organisms (Herd 1995), has prompted the need for alternative control methods. In the last decade, laboratory and field trials have shown that nematophagous fungi present in livestock faeces can reduce the number of infective larvae in the pasture (Larsen 1999), thus supporting the concept that specific fungal products may be used in the biological control of the freeliving stages of nematodes parasites in ruminants (Waller 1998). Selection of potential candidates amongst the local species requires that nematophagous fungi colonising ruminant faeces are first identified and their nematophagous activity quantified. The objective of the present study was to isolate and identify nematophagous fungi that naturally colonise sheep faeces
* Corresponding author : USDA-ARS, Embrapa-Labex Program, LPSI, IDRL, Barc-East, Building 1040, Room 105, Beltsville, MD 20705, USA. † Present Address : Area de Parasitologia y Enfermedades Parasitarias, Departamento de Sanidad Animal y Medicina Preventiva, Facultad de Ciencias Veterinarias, Universidad Nacional del Centro, Campus Universitario, (7000) Tandil, Argentina.
deposited on a Brachiaria decumbens pasture in the Mata Region, Minas Gerais, Brazil. M A T E R I A L S A N D M E T H O DS The study was carried out at the Coronel Pacheco Experimental Station of the National Dairy Cattle Research Centre, a branch of the Brazilian Agricultural Research Corporation. The Station is located at 21m 33h 22d S and 43m 06d W, 426 m a.s.l. In the region, annual rainfall (1600 mm) is concentrated in spring and summer (October–March) and the mean humidity ranges from 70–85 %. The climate is classified as rainy temperate in the summer and dry in the winter. The mean annual temperature is 19n5 mC. The soil is a red-yellow latosol (Oxisol) with pH 4n6. In June, July and October 1995 and January and April 1996, 150 samples of sheep faeces deposited on a pasture of Brachiaria decumbens were recovered at varying intervals not exceeding 14 d after deposition. Samples were cultivated for 3 wk on water-agar (2 %) using Panagrellus sp. as baits. Nematophagous fungi present on the plates were isolated and cultivated in pure culture for identification. Three sheep were used as donors of the faeces deposited on the pastures. These animals were kept in total confinement and fed a diet based on maize silage and a feed supplement containing maize grain, cotton seed, urea, mineral salt and dolomite lime. At the time of faeces collection, bags were fitted to the animals for a few hours to ensure the collection of a sufficient amount of faeces for deposition on the plots. In every month, collected faeces were homogenised and 10 samples of 2 g each taken, crushed and placed on the centre of Petri dishes containing 2 % water agar and Panagrellus sp. to detect a possible presence of nematophagous fungi in fresh
Nematophagous fungi in sheep faeces
1006
faeces. Faeces collected in faecal bags fixed on the sheep were mixed thoroughly and 25 pellets were deposited on each of thirty 1n5 m# plots of Brachiaria decumbens pasture. Two weeks later samples were carefully collected, and 2 g from each sample were placed in the centre of a Petri dish containing water agar (2 %) and free-living Panagrellus nematodes added as bait. Petri dishes were then sealed and kept at room temperature (21–26m) for 3 wk and observed under the microscope weekly for fungal growth. Nematophagous fungi were transferred to clean water agar plates and free-living nematodes were added. Conidia were transferred to corn meal (2 %) agar plates and net formation stimulated by addition of free-living nematodes to pure cultures. The identification of nematophagous fungi was based on the keys of Subramanian (1963), Cooke & Godfrey (1964), Cooke & Dickinson (1965), Haard (1968), Schenck, Kendrick & Pramer (1977), van Oorschot (1985), de Hoog & van Oorschot (1985), Gams (1988), Liu & Zhang (1994) and Rubner (1996). Original descriptions were consulted whenever needed. RESULTS AND DISCUSSION Nematophagous fungi were abundant in the sheep faeces both with respect to quantity and diversity of isolates (Tables 1 and 2). From 150 samples collected on the pastures, 130 fungal isolates were recovered. More than 70 % of the isolates were predacious fungi, and 14 species belong to Arthrobotrys and Monacrosporium (Table 1). Six species and five genera of endoparasitic fungi were identified : Drechmeria, Harposporium, Myzocytium, Nematoctonus and Verticillium (Table 2). Five isolates were identified as Monacrosporium sp. and 15 were classified only as hyphomycetes since they did not produce the necessary structures for identification. Table 1. Species of predatory nematophagous fungi isolated from sheep faeces, their trapping mechanism and frequency of occurrence out of 50 samples.
Arthrobotrys brochopaga A. dactyloides A. haptospora A. musiformis A. oligospora A. robusta A. scaphoides Monacrosporium aphrobrochum M. eudermatum M. gampsosporum M. gephyropagum M. leptosporum M. megalosporum M. phymatopagum Monacrosporium sp. 1 Monacrosporium sp. 2 Unidentified hyphae Unidentified hyphae Unidentified hyphae (Zoopagales) Total
Trapping mechanism
Frequency
Constricting rings Constricting rings Adhesive knobs Adhesive nets Adhesive nets Adhesive nets Adhesive nets Constricting rings Adhesive nets Adhesive nets Adhesive branches Non-constricting rings Adhesive nets Adhesive knobs Adhesive nets Adhesive branches Adhesive nets Non-constricting rings Aseptate adhesive hyphae
2 1 2 6 18 1 1 4 17 1 5 12 1 1 4 1 2 12 1 92
Table 2. Species of endoparasitic nematophagous fungi isolated from sheep faeces their trapping mechanism and frequency of occurrence out of 150 samples. Mode of infection Verticillium balanoides Drechmeria coniospora Harposporium anguillulae H. bysmatosporum H. lilliputanum Myzocytium sp. Nematoctonus robustus Total
Frequency
Adhesive conidia 2 Adhesive conidia 1 Ingested conidia 27 Ingested conidia 1 Ingested conidia 1 Encystment of motile zoospores 5 Adhesive conidia 1 38
Several traps were observed amongst the predacious fungi. Adhesive nets predominated, followed by isolates with constricting rings (Table 1). Arthrobotrys oligospora and Monacrosporium eudermatum predominated among the species that produce adhesive nets, while M. leptosporum was the only species identified that produced non-constricting rings. Previous studies in the Brazilian soil have identified A. musiformis as the prevalent species in different ecosystems (Naves & Campos 1991, Santos, Ferraz & Muchovej 1991, Dalla Pria, Ferraz & Muchovej 1991 ; Dias et al. 1995), and it was the fourth most abundant found in our survey. This result could be due to the fact that we stopped examination after 3 wk. As A. musiformis is slower growing than A. oligospora and M. eudermatum, it has less chance of being found within the three weeks observation period. Some taxonomic differences were encountered in the predator species recovered and generally concerned the size of the conidia. For example, isolates identified as A. musiformis had longer conidia than previous descriptions, and conidia of M. eudermatum were narrower than in the original description. Two isolates, which did not produce chlamydospores, were identified as M. eudermatum. They had two- or three-septate conidia of similar size to those described for M. cytosporum and the number of septa coincided with M. rutgeriense. The conidia, however, were larger than in the latter. As the shape of the conidia matched M. eudermatum, they were identified as this species. In a soil survey in Ecuador, Rubner (1994) found two isolates, one of which had conidia the length of these of our isolates, and the other was similar in width to those of our isolates. It is important to note that when fungi are grown in pure culture the conidia can vary in appearance, size and number of septa (Soprunov 1958), and the apical and basal segments can generally be smaller (Drechsler 1950), thus making identification difficult. One isolate could not be identified. This isolate grew fast in the presence of nematodes but did not grow alone on corn meal agar. It formed long aseptate adhesive hyphae, grew abundantly in the presence of Panagrellus and colonies had a slightly wrinkled surface. The hyphae were 4–5n3 µm wide and at the end point of contact with the nematode, an adhesive substance forming an irregular cushion could be observed. The hyphae that grew on the body of the nematodes were the same width, and some appeared to have a septum. The tips of the growing hyphae were rounded, 7 µm wide. No differentiated nets, conidia or chlamydospores
C. A. Saumell and others were observed. Fungi with similar morphological characteristics were isolated from soil by Duddington (1954), Norton (1963) and Estey & Olthof (1965). Such sterile hyphae are considered as belonging to the Zoopagales and cannot be grown axenically. In a study conducted by Mahoney & Strongman (1994), a fungus with similar characteristics isolated from decomposing faeces was classified as Cystopage sp. Twelve isolates produced non-constricting rings and adhesive knobs and had structures and measurements similar to M. leptosporum. They did not, however, produce conidia in pure or nematode-baited culture and it was not possible to identify them. These isolates were able to grow and prey from a ring in the presence of nematodes. Among the endoparasitic fungi, species with adhesive conidia or ingested conidia, were the most prevalent. Isolates with ingested conidia represented 22 % of the nematophagous fungi isolated, and were all Harposporium pp. (Table 2). Harposporium anguillulae was the most common endoparasite (20 % of the isolated fungi). The characteristics of our isolates coincide with the original description. The average size of the conidia (8n7i1n5 µm), were within the range described by Karling (1938) as 5–14i1n5–2n5 µm. H. anguillulae is a common endoparasite found in soil and faeces of animals. In a study of the occurrence of endoparasites in soil of different ecosystems in Brazil, Silva (1990) observed that H. anguillulae represented more than 40 % of the endoparasitic fungi recovered from infected nematodes. Duddington (1951) found that two out of 10 fungi isolated from faeces were H. anguillulae. Gray (1983) found it in five out of 16 fungi in decomposed dungs and dungs overgrown by plants collected from sites throughout Ireland. H. anguillulae represented the most frequently recorded endoparasitic fungus isolated from cattle faeces in different stages of decomposition in a survey in Nova Scotia, Canada (Mahoney & Strongman 1994). H. lilliputanum and H. bysmatosporum were isolated in smaller proportions (Table 2). The isolation frequency of these fungi was low in previous studies of soil collected in Brazil (3 isolates of H. bysmatosporum out of 22 endoparasites isolated ; Silva, 1990) and El Salvador (1 isolate of H. lilliputanum out of 57 nematophagous fungi isolated ; Bu! caro, 1983). Myzocytium spp., the second commonest among the endoparasites, were not identified to species level. The differences among species of this genus are small and differentiation difficult (Gray 1983). Hay (1995) also found Myzocytium sp. in sheep faeces deposited on Festuca arundinacea. Gray (1983) obtained Myzocytium four times from old faeces among a total of 16 other fungi. This survey demonstrated that sheep faeces deposited on Brachiaria decumbens pastures in the Mata Region of Minas Gerais State are colonised by a number of different species of nematophagous fungi. Further studies are needed to understand their ecology and assess possible effects on the freeliving stages of trichostrongylid nematodes of ruminants.
REFERENCES Bu! caro, R. D. (1983) Hongos nemato! fagos de El Salvador. Revista de BiologıT a Tropical 31 : 25–28. Cooke, R. C. & Dickinson, C. H. (1965) Nematode-trapping species of
1007 Dactylella and Monacrosporium. Transactions of the British Mycological Society 48 : 621–629. Cooke, R. C. & Godfrey, B. E. S. (1964) A key to the nematode-destroying fungi. Transactions of the British Mycological Society 47 : 61–74. Dalla Pria, M., Ferraz, S. & Muchovej, J. J. (1991) Isolamento e identificaça4 o de fungos nemato! fagos de amostras de solo de diversas regio4 es do Brasil. Nematologia Brasileira 15 : 170–176. Dias, W. P., Ferraz, S. & Muchovej, J. J. (1995) Detecça4 o, isolamento e identificaça4 o de fungos predadores de nemato! ides em amostras de solo de diferentes regio4 es do Brasil. Revista Ceres 42 : 615–620. Drechsler, C. (1950) Several species of Dactylella and Dactylaria that capture free-living nematodes. Mycologia 42 : 1–79. Duddington, C. L. (1951) The ecology of predacious fungi. 1. Preliminary survey. Transactions of the British Mycological Society 34 : 322–331. Duddington, C. L. (1954) Nematode-destroying fungi in agricultural soils. Nature 173 : 500–501. Estey, R. H. & Olthof, Th. H. A. (1965) The occurrence of nematophagous fungi in Que! bec. Phytoprotection 46 : 14–17. Gams, W. (1988) A contribution to the knowledge of nematophagous species of Verticillium. Netherlands Journal of Plant Pathology 94 : 123–148. Gray, N. F. (1983) Ecology of nematophagous fungi : distribution and habitat. Annals of Applied Biology 102 : 501–509. Haard, K. (1968) Taxonomic studies on the genus Arthrobotrys Corda. Mycologia 60 : 1140–1159. Hay, F. S. (1995) Endoparasites infecting nematodes in New Zealand. New Zealand Journal of Botany 33 : 401–407. Herd, R. (1995) Endectocidal Drugs : Ecological Risks and Counter-Measures. International Journal for Parasitology 25 : 875–885. Hoog, G. S. de & Oorschot, C. A. N. van (1985) Taxonomy of the Dactylaria complex. VI. Key to the genera and check-list of epithets. Studies in Mycology 26 : 97–122. Karling, J. S. (1938) Harposporium anguillulae. Mycologia 30 : 512–519. Larsen, M. (1999) Biological Control of Helminths. International Journal for Parasitology, 29 : 139–146. Liu, X. Z. & Zhang, K. Q. (1994) Nematode-trapping species of Monacrosporium with special reference to two new species. Mycological Research 98 : 862–868. Mahoney, C. J. & Strongman, D. B. (1994) Nematophagous fungi from cattle manure in four states of decomposition at three sites in Nova Scotia, Canada. Mycologia 86 : 371–375. Naves, R. L. & Campos, V. P. (1991) Ocorre# ncia de fungos predadores de nemato! ides no sul de Minas Gerais e estudo da capacidade predato! ria e crescimento in vitro de alguns de seus isolados. Nematologia Brasileira 15 : 152–162. Norton, Don C. (1963) Iowa fungi parasitic on nematodes. Proceedings of the Academy of Science 69 : 108–117. Oorschot, C. A. N. van (1985) Taxonomy of the Dactylaria complex. V. A review of Arthrobotrys and allied genera. Studies in Mycology 26 : 61–95. Padilha, T. (1996) Resı! duos de anti-helmı! nticos na carne e leite. In Controle da verminose em ruminantes. (T. Padilha, ed.) : 77–93. EMBRAPA, CNPGL, Coronel Pacheco, Brasil. Parkins, J. & Holmes, P. (1989) Effects of gastrointestinal helminth parasites on ruminant nutrition. Nutrition Research Reviews 2 : 227–246. Rubner, A. (1994) Predacious fungi from Ecuador. Mycotaxon 51 : 143–151. Rubner, A. (1996) Revision of predacious Hyphomycetes in the DactylellaMonacrosporium complex. Studies in Mycology 39 : 1–134. Santos, M. A., Ferraz, S. & Muchovej, J. J. (1991) Detection and ecology of nematophagous fungi from Brazilian soils. Nematologia Brasileira 15 : 121–134. Schenck, S., Kendrick, W. B. & Pramer, D. (1977) New nematode-trapping hyphomycete and a reevaluation of Dactylaria and Arthrobotrys. Canadian Journal of Botany 55 : 977–985. Silva, J. F. V. (1990) Fungus endoparasitas de nematoides na regiag o de Lavras e Sag o Sebastiag o do paraiso : ocorrencia, caracterizaçag o e potencial para o controle bioloT gico. MSc thesis, Escola Superior de Agricultura de Lavras, Lavras, MG, Brasil. Soprunov, F. F. (1958) Predacious hyphomycetes and their application in the control of pathogenic nematodes. Academy of Sciences of the Turkmen SSR, Ashkhabad : 1–365. [English Translation. 1966.] Israel Program for Scientific Translation.
Nematophagous fungi in sheep faeces Subramanian, C. V. (1963) Dactylella, Monacrosporium and Dactylina. Journal of the Indian Botanical Society 42 : 291–300. Waller, P. (1998) Parasite epidemiology, resistance and the prospects for implementation of alternative control programs. Proceedings of a workshop organized by FAO and the Danish Centre for Experimental Parasitology, Ipoh, Malaysia, 5–12 October 1997 : 1–10.
1008 Waller, P., Eddi, C., Maciel, S., Nari, A. & Hansen, J. W. (1996) The prevalence of anthelmintic resistance in nematode parasites of sheep in Southern Latin America – general overview. Veterinary Parasitology 62 : 181–187.
Corresponding Editor : B. Senn-Irlet
1005
Nematophagous fungi in sheep faeces in Minas Gerais, Brazil
C. A. SAUMELL†, T. PADILHA* and C. DE P. SANTOS Embrapa-Centro Nacional de Pesquisa de Gado de Leite, Rua EugeV nio do Nascimento 610, Dom Bosco, Juiz de Fora, MG 36038-330, Brazil E-mail: tpadilha!Ipsi.barc.usda.gov Received 5 October 1998 ; accepted 10 October 1999.
Few studies have examined faeces as a substratum for nematophagous fungi, and no information is available about colonisation of faeces in tropical regions. Sheep faeces were deposited on a Brachiaria decumbens pasture in the Mata Region of Minas Gerais, Brazil. Up to 2 wk after deposition, samples were collected and cultivated on water agar (2 %) with free-living nematodes as bait. From 150 samples, 130 isolates were recovered after 3 wk. More than 70 % were predators and Arthrobotrys and Monacrosporium spp. predominated. Endoparasitic species of Drechmeria, Harposporium, Myzocytium, Nematoctonus and Verticillium were also isolated.
I N T R O D U C T I ON Gastrointestinal infections with nematode parasites are one of the major causes of reduced ruminant performance throughout the world (Parkins & Holmes 1989). Traditionally, the control of infections by nematodes in ruminants has combined the use of anthelminthic drugs, to eliminate the adult population of parasites within the animal and where possible, with pasture management, to avoid ingestion of infective larvae. Increasing levels of resistance to anthelminthic drugs in several helminth species (Waller et al. 1996), however, and growing concern about the presence of drug residues in ruminant products (Padilha 1996), and the potential toxicity of active drug metabolites for non-target organisms (Herd 1995), has prompted the need for alternative control methods. In the last decade, laboratory and field trials have shown that nematophagous fungi present in livestock faeces can reduce the number of infective larvae in the pasture (Larsen 1999), thus supporting the concept that specific fungal products may be used in the biological control of the freeliving stages of nematodes parasites in ruminants (Waller 1998). Selection of potential candidates amongst the local species requires that nematophagous fungi colonising ruminant faeces are first identified and their nematophagous activity quantified. The objective of the present study was to isolate and identify nematophagous fungi that naturally colonise sheep faeces
* Corresponding author : USDA-ARS, Embrapa-Labex Program, LPSI, IDRL, Barc-East, Building 1040, Room 105, Beltsville, MD 20705, USA. † Present Address : Area de Parasitologia y Enfermedades Parasitarias, Departamento de Sanidad Animal y Medicina Preventiva, Facultad de Ciencias Veterinarias, Universidad Nacional del Centro, Campus Universitario, (7000) Tandil, Argentina.
deposited on a Brachiaria decumbens pasture in the Mata Region, Minas Gerais, Brazil. M A T E R I A L S A N D M E T H O DS The study was carried out at the Coronel Pacheco Experimental Station of the National Dairy Cattle Research Centre, a branch of the Brazilian Agricultural Research Corporation. The Station is located at 21m 33h 22d S and 43m 06d W, 426 m a.s.l. In the region, annual rainfall (1600 mm) is concentrated in spring and summer (October–March) and the mean humidity ranges from 70–85 %. The climate is classified as rainy temperate in the summer and dry in the winter. The mean annual temperature is 19n5 mC. The soil is a red-yellow latosol (Oxisol) with pH 4n6. In June, July and October 1995 and January and April 1996, 150 samples of sheep faeces deposited on a pasture of Brachiaria decumbens were recovered at varying intervals not exceeding 14 d after deposition. Samples were cultivated for 3 wk on water-agar (2 %) using Panagrellus sp. as baits. Nematophagous fungi present on the plates were isolated and cultivated in pure culture for identification. Three sheep were used as donors of the faeces deposited on the pastures. These animals were kept in total confinement and fed a diet based on maize silage and a feed supplement containing maize grain, cotton seed, urea, mineral salt and dolomite lime. At the time of faeces collection, bags were fitted to the animals for a few hours to ensure the collection of a sufficient amount of faeces for deposition on the plots. In every month, collected faeces were homogenised and 10 samples of 2 g each taken, crushed and placed on the centre of Petri dishes containing 2 % water agar and Panagrellus sp. to detect a possible presence of nematophagous fungi in fresh
Nematophagous fungi in sheep faeces
1006
faeces. Faeces collected in faecal bags fixed on the sheep were mixed thoroughly and 25 pellets were deposited on each of thirty 1n5 m# plots of Brachiaria decumbens pasture. Two weeks later samples were carefully collected, and 2 g from each sample were placed in the centre of a Petri dish containing water agar (2 %) and free-living Panagrellus nematodes added as bait. Petri dishes were then sealed and kept at room temperature (21–26m) for 3 wk and observed under the microscope weekly for fungal growth. Nematophagous fungi were transferred to clean water agar plates and free-living nematodes were added. Conidia were transferred to corn meal (2 %) agar plates and net formation stimulated by addition of free-living nematodes to pure cultures. The identification of nematophagous fungi was based on the keys of Subramanian (1963), Cooke & Godfrey (1964), Cooke & Dickinson (1965), Haard (1968), Schenck, Kendrick & Pramer (1977), van Oorschot (1985), de Hoog & van Oorschot (1985), Gams (1988), Liu & Zhang (1994) and Rubner (1996). Original descriptions were consulted whenever needed. RESULTS AND DISCUSSION Nematophagous fungi were abundant in the sheep faeces both with respect to quantity and diversity of isolates (Tables 1 and 2). From 150 samples collected on the pastures, 130 fungal isolates were recovered. More than 70 % of the isolates were predacious fungi, and 14 species belong to Arthrobotrys and Monacrosporium (Table 1). Six species and five genera of endoparasitic fungi were identified : Drechmeria, Harposporium, Myzocytium, Nematoctonus and Verticillium (Table 2). Five isolates were identified as Monacrosporium sp. and 15 were classified only as hyphomycetes since they did not produce the necessary structures for identification. Table 1. Species of predatory nematophagous fungi isolated from sheep faeces, their trapping mechanism and frequency of occurrence out of 50 samples.
Arthrobotrys brochopaga A. dactyloides A. haptospora A. musiformis A. oligospora A. robusta A. scaphoides Monacrosporium aphrobrochum M. eudermatum M. gampsosporum M. gephyropagum M. leptosporum M. megalosporum M. phymatopagum Monacrosporium sp. 1 Monacrosporium sp. 2 Unidentified hyphae Unidentified hyphae Unidentified hyphae (Zoopagales) Total
Trapping mechanism
Frequency
Constricting rings Constricting rings Adhesive knobs Adhesive nets Adhesive nets Adhesive nets Adhesive nets Constricting rings Adhesive nets Adhesive nets Adhesive branches Non-constricting rings Adhesive nets Adhesive knobs Adhesive nets Adhesive branches Adhesive nets Non-constricting rings Aseptate adhesive hyphae
2 1 2 6 18 1 1 4 17 1 5 12 1 1 4 1 2 12 1 92
Table 2. Species of endoparasitic nematophagous fungi isolated from sheep faeces their trapping mechanism and frequency of occurrence out of 150 samples. Mode of infection Verticillium balanoides Drechmeria coniospora Harposporium anguillulae H. bysmatosporum H. lilliputanum Myzocytium sp. Nematoctonus robustus Total
Frequency
Adhesive conidia 2 Adhesive conidia 1 Ingested conidia 27 Ingested conidia 1 Ingested conidia 1 Encystment of motile zoospores 5 Adhesive conidia 1 38
Several traps were observed amongst the predacious fungi. Adhesive nets predominated, followed by isolates with constricting rings (Table 1). Arthrobotrys oligospora and Monacrosporium eudermatum predominated among the species that produce adhesive nets, while M. leptosporum was the only species identified that produced non-constricting rings. Previous studies in the Brazilian soil have identified A. musiformis as the prevalent species in different ecosystems (Naves & Campos 1991, Santos, Ferraz & Muchovej 1991, Dalla Pria, Ferraz & Muchovej 1991 ; Dias et al. 1995), and it was the fourth most abundant found in our survey. This result could be due to the fact that we stopped examination after 3 wk. As A. musiformis is slower growing than A. oligospora and M. eudermatum, it has less chance of being found within the three weeks observation period. Some taxonomic differences were encountered in the predator species recovered and generally concerned the size of the conidia. For example, isolates identified as A. musiformis had longer conidia than previous descriptions, and conidia of M. eudermatum were narrower than in the original description. Two isolates, which did not produce chlamydospores, were identified as M. eudermatum. They had two- or three-septate conidia of similar size to those described for M. cytosporum and the number of septa coincided with M. rutgeriense. The conidia, however, were larger than in the latter. As the shape of the conidia matched M. eudermatum, they were identified as this species. In a soil survey in Ecuador, Rubner (1994) found two isolates, one of which had conidia the length of these of our isolates, and the other was similar in width to those of our isolates. It is important to note that when fungi are grown in pure culture the conidia can vary in appearance, size and number of septa (Soprunov 1958), and the apical and basal segments can generally be smaller (Drechsler 1950), thus making identification difficult. One isolate could not be identified. This isolate grew fast in the presence of nematodes but did not grow alone on corn meal agar. It formed long aseptate adhesive hyphae, grew abundantly in the presence of Panagrellus and colonies had a slightly wrinkled surface. The hyphae were 4–5n3 µm wide and at the end point of contact with the nematode, an adhesive substance forming an irregular cushion could be observed. The hyphae that grew on the body of the nematodes were the same width, and some appeared to have a septum. The tips of the growing hyphae were rounded, 7 µm wide. No differentiated nets, conidia or chlamydospores
C. A. Saumell and others were observed. Fungi with similar morphological characteristics were isolated from soil by Duddington (1954), Norton (1963) and Estey & Olthof (1965). Such sterile hyphae are considered as belonging to the Zoopagales and cannot be grown axenically. In a study conducted by Mahoney & Strongman (1994), a fungus with similar characteristics isolated from decomposing faeces was classified as Cystopage sp. Twelve isolates produced non-constricting rings and adhesive knobs and had structures and measurements similar to M. leptosporum. They did not, however, produce conidia in pure or nematode-baited culture and it was not possible to identify them. These isolates were able to grow and prey from a ring in the presence of nematodes. Among the endoparasitic fungi, species with adhesive conidia or ingested conidia, were the most prevalent. Isolates with ingested conidia represented 22 % of the nematophagous fungi isolated, and were all Harposporium pp. (Table 2). Harposporium anguillulae was the most common endoparasite (20 % of the isolated fungi). The characteristics of our isolates coincide with the original description. The average size of the conidia (8n7i1n5 µm), were within the range described by Karling (1938) as 5–14i1n5–2n5 µm. H. anguillulae is a common endoparasite found in soil and faeces of animals. In a study of the occurrence of endoparasites in soil of different ecosystems in Brazil, Silva (1990) observed that H. anguillulae represented more than 40 % of the endoparasitic fungi recovered from infected nematodes. Duddington (1951) found that two out of 10 fungi isolated from faeces were H. anguillulae. Gray (1983) found it in five out of 16 fungi in decomposed dungs and dungs overgrown by plants collected from sites throughout Ireland. H. anguillulae represented the most frequently recorded endoparasitic fungus isolated from cattle faeces in different stages of decomposition in a survey in Nova Scotia, Canada (Mahoney & Strongman 1994). H. lilliputanum and H. bysmatosporum were isolated in smaller proportions (Table 2). The isolation frequency of these fungi was low in previous studies of soil collected in Brazil (3 isolates of H. bysmatosporum out of 22 endoparasites isolated ; Silva, 1990) and El Salvador (1 isolate of H. lilliputanum out of 57 nematophagous fungi isolated ; Bu! caro, 1983). Myzocytium spp., the second commonest among the endoparasites, were not identified to species level. The differences among species of this genus are small and differentiation difficult (Gray 1983). Hay (1995) also found Myzocytium sp. in sheep faeces deposited on Festuca arundinacea. Gray (1983) obtained Myzocytium four times from old faeces among a total of 16 other fungi. This survey demonstrated that sheep faeces deposited on Brachiaria decumbens pastures in the Mata Region of Minas Gerais State are colonised by a number of different species of nematophagous fungi. Further studies are needed to understand their ecology and assess possible effects on the freeliving stages of trichostrongylid nematodes of ruminants.
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