Influence of weather and time of deposition on sheep faeces colonization
by nematophagous fungi in the Mata region of Minas Gerais State, Brazil
C.A. Saumell
∗, T. Padilha
Embrapa-Centro Nacional de Pesquisa de Gado de Leite, Rua Eugênio do Nascimento 610, Dom Bosco, Juiz de Fora, MG 36038-330, Brazil
Received 1 October 1998; received in revised form 20 September 1999; accepted 20 September 1999
Abstract
The influence of climate and time of deposition on the colonization of sheep faeces by nematophagous fungi was examined in the Mata Region of Minas Gerais State, Brazil. Sheep faeces were collected from the rectums of animals and deposited on pastures ofBrachiaria decumbensin the months of July and October 1995 and January and April 1996. Samples of the deposited faeces were recovered 3, 7 and 14 days after deposition. Sub-samples of faeces (2 g) were placed in the centre of a 2% water–agar petri dish. Free-living nematodes were added as bait, the plates incubated at room temperature and examined for 3 weeks. Isolated fungi were further cultured on corn-meal agar plates for identification. A total of 123 fungi was isolated from the 120 sheep faecal samples deposited on pastures including 22 in July, 39 in October, 41 in January and 21 in April. More than one-third of the fungi were recovered on the third day after deposition.Arthrobotrys oligosporaandMonacrosporium eudermatum, were the most common predatory fungi andHarposporium anguillulaethe most common endoparasite. Results showed that sheep faeces deposited on pastures ofB. decumbensare colonized rapidly by a variety of nematophagous fungi and seasonality affects colonization. ©2000 Elsevier Science B.V. All rights reserved.
Keywords:Biological control; Nematophagous fungi; Nematode parasites; Ruminants
1. Introduction
Trichostrongylid nematode infection is the main health constraint to sheep production, especially in the tropics. The control of internal parasites is funda-mental among pasture-grazed livestock (Herd, 1986) and any control failure leads to reduced production and consequently, profits. Ruminant parasite con-trol programs make use of the availability of a wide variety of anthelmintic products (Lanusse, 1996), all
∗Corresponding author. Present address: ´Area Parasitolog´ıa y
Enfermedades Parasitarias, Facultad de Ciencias Veterinarias, UNI-CEN, Paraje Arroyo Seco, Campus Universitario, 7000, Tandil, Argentina.
E-mail address:[email protected] (C.A. Saumell).
of which are highly effective against gastro-intestinal nematodes. However, none of them is efficient as the only means of control, it always being necessary to combine them with other measures such as, sharing of pastures by more than one animal species, or use of the pasture by both young and adult animals (Niezen et al., 1996).
Anthelmintics provide prompt but transitory solu-tions to internal parasites as they do not diminish the risk of re-infection among animals if they are kept on contaminated pastures (Steffan and Fiel, 1994). Other limitations related to anthelmintic use are resis-tance (Echevarria, 1996, Echevarria et al., 1996), drug residues in food (Padilha, 1996) and eco-toxic effects (Wall and Strong, 1987, Madsen et al., 1990, Herd, 1995). The various available control measures should
64 C.A. Saumell, T. Padilha / Applied Soil Ecology 14 (2000) 63–70
be organised into an integrated program to ensure maximum efficiency (Waller, 1998) with minimum use of the available compounds.
Recently, there has been growing interest in de-veloping alternatives to anthelmintics, including those stimulated by the difficulties associated with chemical controls mentioned above. Biological control seems likely to become a reality in the not-too-distant future (Larsen, 1999) and offers an effective and safe alter-native in the reduction of infective larva populations among gastro-intestinal nematodes in pastures. Bio-logical control is based on the use of nematophagous fungi against pre-parasitic forms of trichostrongylid (Larsen, 1999).
Nematophagous fungi are natural inhabitants of the soil (Barron, 1977) and can also be isolated from an-imal faeces (Juniper, 1953, 1954, 1957; Duddington, 1951, 1955; Gray, 1983; Larsen et al., 1994; Mahoney and Strongman, 1994; Hay et al., 1997). The influence of the weather on the behaviour of nematophagous fungi in soil samples collected from pastures used by sheep and the speed of colonization by nematophagous fungi, was observed. Peloille (1981) demonstrated that favourable temperature conditions and the presence of rain were important factors in the isolation of preda-tory fungi. It is also known that the state of decompo-sition of the faeces may influence the biology of the fungi (Mahoney and Strongman, 1994), there existing a definite relation with seasonal variations. Recently, Hay et al. (1998) observed that ovine faeces deposited on pasture, were colonized by nematophagous fungi 1 day after deposition and that dung was colonized by a greater number of species and at a faster rate in autumn compared to summer.
The aim of this work was to study the seasonal fluctuation as well as the speed of colonization of nematophagous fungi in ovine faeces deposited on Brachiaria decumbenspastures in the Mata Region of Gerais State, Brazil.
2. Materials and methods
The experiment was carried out at the Coronel Pacheco Experimental Farm (CECP) of the National Dairy Cattle Research Centre (Embrapa-Gado de
Leite), in Coronel Pacheco, Minas Gerais (21◦33′22′′S
e 43◦06′′W) at an altitude of 426 m above sea level.
The annual rainfall (1600 mm) is concentrated in the spring and summer, with air humidity ranging be-tween 70 and 85% throughout the year. The climate of the region is of the Cwa-type (mesotermic), after Köppen, rainy in the summer and with a dry win-ter from June to September (Embrapa, 1980). The
average annual temperature is 22.5◦C (average in the
period from 1985 to 1994). Air humidity ranged from 84 to 89% throughout the duration of the study. Rain-fall varied from 7.5 to 237.4 mm during the months of July 1995 and January 1996, respectively (Fig. 1). The soil at the site of the experiment was classified as Red–Yellow Latossolol (oxisol), with a pH reading on the surface and to a depth of 18 cm, of 4.6.
The faeces of three clinically healthy donor sheep were used. These animals were kept in total confine-ment and fed with a diet based on corn silage and a feed supplement containing corn grain, cottonseed, urea, mineral salt and dolomite calcareum. At the time of faeces collection, bags were fitted to the animals for a few hours to ensure the collection of a suffi-cient quantity of faeces for deposition on the plots. To ensure the absence of nematophagous fungi in the fresh faeces, faeces were homogenised and 2 g sam-ples (10 samsam-ples for each sample month) were crushed and placed on the centre of petri dishes containing a
2% water–agar and 1500–2000 Panagrellus sp. The
dishes were incubated and observed microscopically for nematophagous fungi. Collected faeces were ho-mogenised and divided into 30 samples of 25
pel-lets each and placed in 30 individual plots (1.5 m2)
ofB. decumbens. Faeces were deposited in July and October 1995 and January and April 1996 and placed on different parts of the plot on each occasion. Plots were randomly divided into three groups of 10 plots, which were sampled at 3, 7 and 14 days after depo-sition. Plots were separated by tracks and the grass on each plot was cut every 15 days and kept at a height of between 35–40 cm throughout the experi-ment. Faeces were collected from 10 plots on each of 3, 7 and 14 days after deposition. Faeces were broken up and homogenised manually. 2 g from each sample were placed in the centre of petri dishes containing
2% water agar. 1500–2000 nematodes of the
incu-Fig. 1. Number of species of nematophagous fungi isolated from ovine faeces of 3, 7 and 14 days after deposition at different times on Brachiaria decumbenspastures.
bated at room temperature (21–26◦C) for 3 weeks
and observed with the aid of a microscope. The ne-matophagous fungi isolated in the dishes seeded with faeces were re-divided into other dishes containing
wa-ter agar with 1500–2000 Panagrellus sp. nematodes
added. The conidia isolated in these were transferred to dishes of corn meal agar (2%) (Corn Meal Difco) in order to obtain pure cultures. Fungal identification was based on the keys of Subramanian (1963), Cooke and Godfrey (1964), Cooke and Dickinson (1965), Haard (1968), Schenck et al. (1977), van Oorschot (1985), de Hoog and van Oorschot (1985), Gams (1988), Liu and Zhang (1994) and Rubner (1996) and on the orig-inal descriptions of each species. Whenever necessary the fungi were stained with cotton blue at 0.05%. Af-ter identification, the isolated fungi were maintained
in corn meal agar (2%) cultures at 4◦C in the
Labora-tory of Helminthology at Embrapa, Gado de Leite. The pH of the fresh faeces and faeces collected 3, 7 and 14 days after deposition, was determined imme-diately after collection. The homogenised faeces were diluted in distilled water in the ratio of 1 : 2.5. The re-maining faeces were used for dry matter analysis by
means of drying at 65◦C for 72 h (AOAC, 1970).
Monthly data on rainfall, humidity and temperature were gathered at a weather station situated 200 m from the site of the experiment.
The data were analysed using PROC CAT-MOD/SAS (SAS, 1989) procedure.
3. Results
Nematophagous fungi were present in sheep faeces
deposited on B. decumbens throughout each month.
From a total of 120 samples collected during the 4 months, 123 fungi were isolated. Nematophagous fungi were not detected in fresh faeces, prior to de-position.
Predacious fungi were more abundant than en-doparasites fungi. Between predacious fungi, adhe-sive net-forming fungi prevailed. In this group, in the sampled month, no significant differences in the
num-ber of isolations were observed (p> 0.05). Among the
endoparasites, those species that infect nematodes by means of ingestible spores predominated with supe-rior percentages up to 66% and significant differences
in the sampled months were not observed (p> 0.05)
66 C.A. Saumell, T. Padilha / Applied Soil Ecology 14 (2000) 63–70
Table 1
Frequency of species of predatory fungi and endoparasites isolated in ovine faeces deposited onBrachiaria decumbensin the months of July and October of 1995 and January and April of 1996
Predatory fungi July/95 October January/96 April Total
Arthrobotrys brochopaga 1 1
A. haptospora 1 1 2
A. musiformis 2 2 2 6
A. oligospora 6 6 2 3 17
A. robusta 1 1
Monacrosporium aphrobrochum 2 2 4
M. eudermatum 5 6 2 6 19
M. gampsosporum 1 1
M. gephyropa gum 1 4 5
M. leptosporum 2 9 1 12
M. phymatopagum 1 1
Monacrosporiumsp.
Adhesive branches 1 1
Adhesive nets 2 2 4
Non identified fungi
Adhesive nets 1 1 2
Aseptate adhesive hyphae 1 1
Non-constricting rings 3 7 1 11
Sub-total 14 27 31 16 88
Endoparasites
Harposporium anguillulae 6 8 8 3 25
H. bysmatosporum 1 1
H. lilliputanum 1 1
Myzocytiumsp. 1 1 2 1 5
Nematoctonus robustus 1 1
Verticillium balanoides 1 1 2
Sub-total 8 12 10 5 35
Total 123
Sixteen species of nematophagous fungi were iso-lated in ovine faeces. The majority of the species were
fungi with predatory characteristics.Monacrosporium
eudermatumandArthrobotrys oligospora, which both form adhesive nets, were observed throughout all months. This group was the most frequently isolated (Table 1, Fig. 1) especially in the months of July, Oc-tober and April which had low or moderate rainfall (Fig. 2). Non-constricting ring-forming fungi made up the second most abundant group among the
preda-tory fungi andM. leptosporumwas the most common
species recovered (Table 1, Fig. 1). Among the
en-doparasites,H. anguillulae, which infects nematodes
by means of ingestible spores, was the most impor-tant species and isolated in every month of sampling
(Table 1). Myzocytium sp., whose infection is based
on mobile zoospores, was isolated less frequently,
but was also observed in every month of sampling (Table 1).
The season influenced in the number of isolated of
nematophagous fungi (p<0.05). In the rainy months,
a great diversity of nematophagous fungi species was observed (Table 1, Figs. 1 and 2). Non-constricting ring-forming fungi were the most significant species
in January (p<0.05) (Fig. 1). However, in the
month of January, there was a greater prevalence of non-constricting ring-forming fungi than in the months
of October and April (p<0.05) and they were not
isolated in July. Adhesive net-forming species were significantly more abundant than other mechanisms in the dry month of July, and October when the rainy
period began (p<0.05) (Fig. 1). There was a period
Fig. 2. Average maximum and minimum temperatures and total rainfall recorded during the experiment.
and consequently, the colonization of the faeces (Table 3). During this period, nematophagous fungi were not observed in the samples collected on the seventh day after deposition. Rainfall prior to the sam-pling on the fourteenth day, dampened the faeces again (Table 2), increasing colonization by nematophagous fungi, but differences between the frequency that the mechanisms of action of species of predacious and
endoparasites fungi were not detected (p> 0.05).
The distribution of the fungi, which were isolated on the three sampling days showed no significant
dif-ferences (p> 0.05). The percentage of isolated fungi
for 3, 7 and 14 days after deposition was 36, 25 and 39%, respectively (Table 3).
Table 2
Percentage dry matter of ovine faeces, collected in the months of July and October of 1995 and January and April of 1996
Month Fresh faeces Day after deposition
3 7 14
July/95 38.5 46.0 47.8 49.1
October 34.4 41.6 42.5 46.9
January/96 39.8 41.9 40.7 43.2
April 44.7 72.6 76.4 26.8
The pH values for fresh faeces and faeces aged in
the environment for 3, 7 and 14 days were 8.17±0.5,
8.32±0.4, 9.03±0.2 and 9.7±0.1, respectively.
Table 3
Frequency of species of predatory nematophagous fungi and en-doparasites isolated in ovine faeces on the third, seventh and four-teenth day after deposition on B. decumbens pastures, grouped according to the nematophagous mechanism
Predatory fungi Day after deposition
Mechanism of action 3 7 14 Total
Adhesive nets 19 11 20 50
Adhesive knobs 2 1 3
Non constricting rings 8 9 6 23
Constricting rings 2 2 1 5
Adhesive branches 1 2 3 6
Aseptate adhesive hyphae 1 1
Total 32 24 32 88
Endoparasites Mode of infection
Adhesive spores 1 1 1 3
Ingestible spores 8 9 10 27
Mobile zoospores 1 4 5
68 C.A. Saumell, T. Padilha / Applied Soil Ecology 14 (2000) 63–70
4. Discussion
The frequency and temporal distribution of ne-matophagous fungi in ovine faeces appeared to be influenced by this weather. The largest number of isolates were retrieved from faeces in the month of October (the beginning of the rainy season) and Jan-uary (a month of high registered rainfall). During this period of the year, the average minimum tem-perature increased, as well as the average maximum temperature. Under these conditions non-constricting
ring-forming fungi such as M. leptosporum became
abundant, particularly in the month of January. In the rainy months, the invasion of the faeces by an abun-dance of free-living nematodes would facilitate the colonization by non-constricting ring-forming fungi. When nematodes are infected by non-constricting ring forming fungi, they maintain their motility for some time thus facilitating dispersal. The presence of a larger number of isolates of non-constricting ring-forming fungi in rainy months, was also reported by Peloille (1981) in the organic matter of the soil, where sheep pastured.
Fungi, which form adhesive nets, were predominant during the dry season and decreased in numbers in the month of January when compared with other fungi with different nematophagous mechanisms (Fig. 2).
However,M. eudermatum andA. oligospora, among
the predatory fungi, andH. anguillulaeamong the
en-doparasites, were isolated in every month sampled, demonstrating their abundance in the environment and their capacity to colonize faeces and develop in both
the wet and dry months. A. oligospora and M.
eu-dermatum, which were the most abundant predatory species in the dry months, may resist drying out, as was observed by Gray and Bailey (1985) in adhesive net-forming fungi.
The ovine faeces were rapidly colonized by ne-matophagous fungi. Among the 123 isolates recov-ered, 42 colonized the faeces within 3 days of depo-sition. Hay et al. (1997), in New Zealand also found colonization of sheep faeces to be rapid. In the 3 days after deposition, they reported 83 and 58% of faecal samples contained nematophagous fungi in February and April, respectively.
The occurrence of nematophagous fungi in faeces is often reported to increase with the time of deposition (Duddington, 1955, Juniper, 1957, Soprunov, 1958,
Larsen et al., 1994). Nevertheless, in this study, faeces which remained for as long as 14 days in the pasture did not shown a markedly superior degree of coloniza-tion. Similar findings were observed in New Zealand where ovine faeces where kept in the environment for 3, 7, 14 and 28 days and the colonization level did not increase over the time the faeces remained on the soil (Hay et al., 1997).
Apparently, fungal colonization is favoured by the natural moisture content of the faecal pellets and sub-sequently by climatic factors, particularly rainfall. It is noteworthy that the number of isolates recovered after 7 days was influenced by a dry spell in the month of April 1996. It is possible that in this month, fae-ces sampled on the third day maintained sufficient moisture to facilitate fungal colonization while fae-ces remaining until seventh day, due to environmental dryness, were unable to support the growth of fungi which reached faeces in the first few days or allowed colonization when natural moisture was not present.
The ideal temperature range for the growth of
vari-ous species of fungi is situated between 15 and 30◦C
(Cooke, 1963, Pandey, 1973, Grønvold et al., 1985). Temperatures recorded over the experiment were close to or within the ideal temperature range, thus facilitat-ing fungal growth. This might explain why no marked difference was observed in relation to the number of isolates and time spent in the environment.
The colonization of sheep faeces by nematophagous fungi appears not to be influenced by the pH within the range studied here. A number of studies have demon-strated that the growth of a nematophagous fungus may be altered when the pH of the environment is modified (Grønvold et al., 1985, Mitsui, 1985). How-ever, in this experiment, in spite of the soil being acid and fresh faeces as well as faeces which remained for different intervals on the pastures, having an alkaline pH, the fungi rapidly colonized the faeces, showing their ability to develop within a wide pH range, as demonstrated previously by Soprunov (1958).
in the natural control of these parasites. Studies aimed at increasing the understanding of the association be-tween fungi and free-living stages of these nematodes are encouraged.
Acknowledgements
The authors express their gratitude to Clóvis de Paula Santos, for his technical assistance and to Edgardo Rodr´ıguez and Margarita West for their sta-tistical comments on the manuscript. This work was partially supported by CNPq and FAPEMIG.
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