ABSTRACT

In vivo experiments were performed to test six isolates of nematode-trapping fungi: two of Arthrobotrys robusta, one of A. conoides, one of Monacrosporium ellypsosporum, one of M. thaumasium and one of M. sinense for passage through the gastrointestinal tract of calves without losing the ability to entrap infective Haemonchus placei larvae. Following administration of conidia and mycelium of fungi isolates to the calves, two isolates of A. robusta, one isolate of A. conoides and one isolate of M. thaumasium met that goal when harvested from faecal samples and was later able to entrap the infective H. placei larvae. There was no evidence that the other isolates (M. ellypsosporum and M. sinense) passed through the gastrointestinal tract of the calves. Isolation from the faeces occurred from between 15 and 110 h following oral administration of fungi, with more isolation, mainly at the 15^th hour.

Key words: biological control, nematode-trapping fungi, Arthrobotrys, Monacrosporium, nematodes, calves

Introduction

The fungi antagonistic to nematodes consist of a wide variety of organisms, including nematode-trapping or predacious fungi, endoparasitic fungi, parasites of nematode eggs and cysts, and those producing nematotoxic metabolites. It is remarkable that fungi belonging to highly divergent orders and families occur in each of the above groups (MANKAU, 1980).

The predacious group, which includes the genus Arthrobotrys and Monacrosporium, produces an extensive system of hyphae in the environment. Along the hyphae there are organs able to capture living nematodes (BARRON, 1977).

The fungi inhabit a wide variety of substrates including soil, moulds, animal dung, decaying plant material, root plants and others. The most practical use of nematophagous fungi in the control of animal parasitic nematodes is by means of the oral administration of fungal material. After this material passes through the gastrointestinal tract of animals and is eliminated, together with the faeces, into the environment, faecal material is colonised by these fungi and a close contact between the recently hatched larvae and the fungi takes place, promoting the production of traps and further capture and death of nematodes. However, most nematode-trapping fungi are extremely susceptible to destruction by the severe conditions met in the gastrointestinal tract.

The present study was designed to select nematophagous fungi of the genera Arthrobotrys and Monacrosporium which are able to pass through the gastrointestinal tract of calves without loss of viability to prey on nematodes.

Materials and methods

Six isolates of nematode-trapping fungi, two isolates of Arthrobotrys robusta (A and B), one isolate of A. conoides (C), one isolate of Monacrosporium ellypsosporum (D), one isolate of M. thaumasium (E) and one of M. sinense (F) were obtained from Brazilian soil and kept in small flasks containing 2% Potato-Dextrose-Agar (2% PDA) at 4 °C.

Free-living nematodes (Panagrellus spp) originating from Brazilian soil were cultivated at room temperature in Petri dishes containing dampened and mashed oat flakes. The nematodes were extracted from surrounding material using the Baermann funnel.

Two Holstein calves, 100 kg of live weight each and free of helminth infection, were infected with 1000 infective Haemonchus placei larvae per kg of body mass. After 30 days of parasitic infection, and for a period of one month thereafter, daily faecal samples from the animals were obtained and, along with sterile charcoal, were kept at 26 °C for two weeks. Larvae were recovered in a Baermann apparatus, with water at 42 °C and washed three times with 0.9% NaCl physiological solution, and were then stored at 4 °C for one to two months. In order to obtain H. placei and Panagrellus spp free from bacteria and fungi, the nematodes were washed ten times in distilled water by centrifugation at 1000 rpm for five minutes. The nematodes were stored at 4 °C for one week in a solution containing 0.05% of streptomycin sulfate, 0.01% of chloramphenicol and 0.05% of amphotericin B. Washing twice with distilled water was repeated. Before their use, nematodes were maintained at 25 °C in the light for six hours and their viability was checked by stereoscopic microscope.

Mycelium grown of the fungi was performed in liquid medium of KADO and HESKET (1970) after 7 days of incubation at 25 °C in the dark. A part of the liquid medium was supplemented with 0.05 mg of L-Phenilalanine and 0.05 mg of L-Valine per ml of medium in order to induce the trap formation in the isolates according to NORDBRING-HERTZ (1973). Conidia of the isolates were obtained from the stock (2% PDA) and cultures were transferred to Petri dishes containing calf faecal agar made with 50 g macerated faeces, filtered through a 61 µm sieve. This filtrate was adjusted to 500 ml, added to 2% agar-agar and then autoclaved for 15 min. at 120 °C. To these dishes was added 1 ml suspension with 1000 Panagrellus over a period of 2 days. On the 5^th day the dishes became completely overgrown by the various fungal isolates. Two ml of distilled water was added to each Petri dish, conidia removed with a delicate brush and the isolated fungal material stored in 50 ml Erlenmeyer flasks.

Twenty-four, six- to eight-month-old housed male Holstein × Zebu calves were randomly separated into six (four animals in the test of each isolate) and four groups of one animal each: groups 1, 2, 3 and 4.

Group 1 - Animals received 20×10⁶ conidia; orally.

Group 2 - Animals received 100 g mycelium with trap, orally.

Group 3 - Each animal received 100 g mycelium without trap, orally.

Group 4 - Each calf received 100 ml water.

Five days before and five days following the administration, each animal was fed daily an autoclaved special ration of 1 kg of crushed corn and 3 kg of Pennisetum purpureum grass to control other nematodes and fungi.

Faecal samples were collected from each animal at 15, 18, 21, 24, 48, 72, 96 and 110 h after administration. Two grammes of faeces were removed from these samples and individually added to 9-cm Petri dishes containing 2% water agar at 25 °C, in the dark. The dishes were baited with 1000 infective H. placei larvae. The amount of conidiophore growth was observed daily. A count was performed at 24-h intervals from the time when the material was placed on the Petri dishes, during a period of 10 days for the Arthrobotrys isolates and during a period of 15 days for the Monacrosporium isolates. These assays were repeated three times.

Results and discussion

Figures 1, 2, 3 and 4 show the number of conidiophores in the faecal samples after administration of the A (A. robusta), B (A. robusta), C (A. conoides) and E (M. thaumasium) isolates, respectively. No fungi were found in the other faecal samples belonging to the calves after administration of the D (M. ellypsosporum) and F (M. sinense) isolates. The A and C isolates presented a greater production of conidiophores than the B and E isolates. In all the isolates that passed through the gastrointestinal tract of the calves in group 1 (animals received 20×10⁶ conidia, orally) the growth curve was higher than in the other groups. There was no fungal growth in the dishes containing faeces of the calves used as control (group 4). Ten days after administration of the infective H. placei larvae on isolates that passed through the gastrointestinal tract of the calves, no infective larvae free from fungi trapping were observed.

Fig. 1. Amount of the conidiophores of A (Arthrobotrys robusta) isolate, which appeared on 2% wateragar after the administration conidia (G1); mycelium with trap (G2) and mycelium without trap (G3)

Fig. 2. Amount of the conidiophores of B (Arthrobotrys robusta) isolate, which appeared on 2% wateragar after the administration conidia (G1); mycelium with trap (G2) and mycelium without trap (G3)

Fig. 3. Amount of the conidiophores of C (Arthrobotrys conoides) isolate, which appeared on 2% wateragar after the administration conidia (G1); mycelium with trap (G2) and mycelium without trap (G3)

Fig. 4. Amount of the conidiophores of E (Monacrosporium thaumasium) isolate, which appeared on 2% wateragar after the administration
conidia (G1) and mycelium without trap (G3)

According to LARSEN et al. (1991) the major problem in the use of nematophagous fungi as bio-control agents is the deposition of the fungal material in dung pats where the entrapment of the parasite larvae should take place. The most obvious possibility would be to add nematophagous fungi to the alimentary tract without loss of viability. The present study was designed to select trapping fungi of the genus Arthrobotrys and Monacrosporium, according to the above explanation. In this experiment, the A, B, C and E isolates were successful in passing through the gastrointestinal tract of calves, subsequently demonstrating its viability to prey on infective H. placei larvae. The D and F isolates did not perform as well, which could be due to their possible destruction while passing through the gastrointestinal tract of the calves.

These results demonstrate the importance of screening nematode-trapping fungi according to their ability to pass through the gastrointestinal tract before administering them in bio-control. HASHMI and CONNAN (1989) were first to mention this passage after performing oral administration of A. oligospora conidia on calves. Further, LARSEN et al. (1992) achieved this goal using isolates of A. oligospora, A. superba and Duddingtonia flagrans, as well as GRONVOLD et al. (1993), WOLSTRUP et al. (1994), NANSEN et al. (1995) using D. flagrans and ARAÚJO et al. (1996) using A. robusta.

WALLER et al. (1994) demonstrated through an in vivo experiment that the average time spent in the passage of the conidia through the digestive system of bovines after oral administration is about 24 h. In the present study, this time varied from 15 to 110 h after oral administration of fungi in calves, with more isolation mainly at the 15^th h.

It is very important to screen nematophagous fungi in each location as they can be effective, or even be found, in certain ecological niches. Future experiments will demonstrate whether our fungi isolates can be used for the biological control of gastrointestinal nematodes of grazing calves under natural conditions. The final challenge is to develop an efficient, low cost means of scaling up production of fungal material to satisfy industrial needs for the commercial exploitation of this technology.

References

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Received: 10 August 1998
Accepted: 5 May 1999

Araújo, J. V., M. A. Stephano, W. M. Sampaio: Prolazenje nematofagnih gljivica kroz probavnu cijev teladi. Vet. arhiv 69, 69-78, 1999.