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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)
Experimental Parasitology 128 (2011) 460–463
Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Research Brief
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) Fabio Ribeiro Braga a,⇑, Jackson Victor Araújo a,1, Juliana Milani Araujo a, Alexandre de Oliveira Tavela a, Sebastião Rodrigo Ferreira a, Filippe E. Freitas Soares b, Laércio dos Anjos Benjamin a, Luiza Neme Frassy a a b
Departamento de Veterinária, Universidade Federal de Viçosa, Viçosa-MG, CEP 36570-000 Vicosa, Brazil Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Viçosa, Viçosa-MG, CEP 3657-000 Vicosa, Brazil
a r t i c l e
i n f o
Article history: Received 30 September 2010 Received in revised form 12 May 2011 Accepted 16 May 2011 Available online 24 May 2011 Keywords: Nematophagous fungi Duddingtonia flagrans Silica-gel Cyathostomins
a b s t r a c t 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. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Horses host a wide variety of helminths, and cyathostomins are one of the most important (Love and Duncan, 1992). These helminths are present in pastures throughout the year and can cause episodes of diarrhea in adult animals (Braga et al., 2009). Anthelminthic drugs are usually used to control these gastrointestinal parasites. However, Bird and Herd (1995) report that no drug has efficacy against encysted larval stages of these nematodes. Biological control with predatory nematophagous fungi, especially Duddingtonia flagrans are considered as a viable alternative (Braga et al., 2009). One of the principal advantages is the ability to survive for long periods under laboratory conditions; however, some isolates may lose their predatory activity (Stirling, 1991). In this context, the use of silica-gel allows the storage of isolated spores for prolonged periods. According to Ryan et al. (2000) this method has been considered as one of the most ⇑ Corresponding author. Fax: +55 31 3899 1464. 1
E-mail address: [email protected] (F.R. Braga). CNPq scholarship.
0014-4894/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2011.05.013
effective, preserving the fungi for long periods. The main advantages are: (1) reduced chances contamination due to low humidity. (2) The financial cost is low and no specific equipment is required. (3) Many cultures of the same isolate may be recovered from a single sample storage. However, there is a lack of studies to assess whether the duration of preservation in silica-gel can interfere with the predatory activity. The current study evaluates the predatory activity of the fungus D. flagrans (isolates AC001 and CG722) stored for 7 years in silica-gel. The fungal isolates predatory activity was tested against cyathostomin infective larvae (L3). 2. Materials and methods 2.1. Fungi Two isolates of nematophagous fungi D. flagrans (AC001 and CG722) were used. The isolates were stored in test tubes containing 2% corn-meal-agar (2% CMA), in the dark, at 4 °C for 10 days. These isolates were obtained from Brazilian agricultural soil, in Viçosa city, Zona da Mata region of Minas Gerais state. They were
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F.R. Braga et al. / Experimental Parasitology 128 (2011) 460–463
collected using the soil-sprinkling method of Duddington (1955), modified by Santos et al. (1991). 2.2. Maintenance of fungal cultures in vials containing silica-gel Samples of the isolates AC001 and CG722 removed from test tubes containing 2% CMA, at 25 °C, were inoculated in sporulation medium AELA (yeast extract, 4 g; K2HPO4, 1 g; MgSO47H2O, 0.5 g; soluble starch, 20 g; distilled water, 1 l) as described by Mota et al. (2002). After 7 days, a sterile solution of 5% skimmed milk and rice grains was added to the surface of the plates. Then the grains were soaked in the suspension of fungal material and transferred to five vials containing grains of sterile silica-gel. The samples were stored at 4 °C in the absence of light and humidity. After 7 years, the cultures were recovered by transferring a grain of rice containing the fungus D. flagrans (AC001 and CG722) from the bottle with silica-
Reduction% ¼
thostomin L3 and 500 conidia of isolates AC001 or CG722 in 2% WA. The control group contained only 500 L3 on dishes in 2% WA. For 7 days, every 24 h, 10 random 4 mm diameter fields on each plate of treated and control groups were observed under an optical microscope with a 10 objective, counting the number of non-predated L3 on each. At the end of 7 days, the non-predated L3 were recovered from the Petri dishes using the Baermann apparatus with water at 42 °C. 2.6. Statistical analysis Data obtained were examined by analysis of variance at 1% and 5% probability levels using the BioEstat 3.0 software (Ayres et al., 2003). The efficiency predation activity was evaluated using Tukey’s test at 1% probability level. The percent reduction in the mean larval recovery was calculated by the following equation:
ðMean of L3 recovered from control mean of L3 recovered from treatmentÞ 100= Mean of L3 recovered from control
gel to the surface of 9.0 cm Petri dishes containing 20 ml of potato-dextrose agar 2% (2% PDA) and kept at 25 °C, in the dark, for 10 days. 2.3. Conidia collection After growth of the isolates in 2% PDA, new culture disks 4 mm in diameter were transferred to 9.0 cm Petri dishes containing 20 ml of 2% water-agar (2% WA) and 1 ml of distilled water containing 1000 larvae of Panagrellus sp., were added daily for 21 days to induce fungal conidia formation. When complete fungal development was observed, 5 ml of distilled water were added to each Petri dish, and the conidial and mycelial fragments were removed using technique described by Araújo et al. (1993). The suspension present in the plates was screened through a sieve coupled to a plastic container to remove the mycelium fragments.
3. Results Cultures of D. flagrans (isolates AC001 and CG722) preserved with silica-gel for 7 years were viable and produced conidia able to predate cyathostomin larvae. The presence of cyathostomin L3 in Petri dishes containing 2% WA was essential for the trap formation by fungal isolates, since this medium is poor in nutrients. A difference (p < 0.01) was observed between the means number of non-predated cyathostomin L3 and the means in the groups treated with the fungus D. flagrans (AC001 and CG722) throughout the experiment (Table 1). Evidence of predation was observed by means of cyathostomin L3 recovered on the seventh day using the Baermann method, at the end of the experiment. In addition, there were typical fungal structures (conidia and traps) in the treated groups during the test (Fig. 1A–E). At the end of the experiment, a mean cyathostomin L3 reduction of 88.6% (AC001) and 78.4% (CG722) was observed (Fig. 2).
2.4. Obtaining cyathostomin infective larvae (L3) 4. Discussion 2.4.1. Fecal samples About 1 kg of fresh feces were collected directly from the rectum of horses (Equus caballus) naturally infected with cyathostomins. These animals were from the Veterinary Department, Federal University of Viçosa, Minas Gerais, Brazil. After that, a count of eggs per gram of feces (EPG) was made according to Gordon and Whitlock (1939), in order to find positive animals. Then coprocultures using fragmented industrial vermiculite (autoclaved) and water were performed. The coprocultures were maintained at 26 °C in the dark for 8 days. At the end of this period cyathostomin (L3) were obtained by the Baermann method, identified and quantified according to the criteria described by Bevilaqua et al. (1993) using an optical microscope with a 10 objective.
Eysker et al. (1989) mention that adults cyathostomin can be controlled by many different broad spectrum anthelminthics. However, there are some disadvantages related to the administration of these drugs including the lack of efficacy against cyathostomins during encasement and the emergence of benzimidazole
Table 1 Daily means and standard deviations of non-predated third stage larvae (L3) of cyathostomins per 4 mm diameter field in 2% water-agar during 7 days in treatments with the fungus Duddingtonia flagrans (AC001 and CG722) and control without fungus. Time (Days)
Treatments (means of non-predated L3) AC001
CG722
Control
1 2 3 4 5 6 7
4.2a ± 2.5 2.7a ± 2.6 2.9a ± 2.7 2.0a ± 1.9 1.3a ± 1.5 1.6a ± 1.7 0.9a ± 1.2
5.2a ± 3.8 3.1a ± 3.5 3.9a ± 2.8 2.3a ± 2.9 2.1a ± 2.2 1.7a ± 2.1 0.8a ± 1.1
26.3b ± 18.7 14.1b ± 24.7 10.6b ± 10.2 6.5b ± 7.1 5.9b ± 5.4 4.7b ± 4.4 3.8b ± 3.6
2.5. Experimental assay The predation test was conducted on the surface of Petri dishes according to a modified technique described by Mota et al. (2002). Three groups were formed on 9.0 cm diameter Petri dishes containing 20 ml of 2% WA; two treated groups (AC001 and CG722) and one control group (without fungus). Six replicates were made for each group. Petri dishes were previously marked in 4 mm diameter fields. In the treated groups each Petri dish contained 500 cya-
Means followed by the same small letter in the lines were not statistically different (p > 0.01).
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Fig. 1. (A) infective larvae (L3) of cyathostomins (white arrow) and hyphae of the fungus Duddingtonia flagrans (AC001 or CG722) (black arrow). (B) Cyathostomin L3 (white arrow), formation of traps (black Arrow) by the fungal isolates (AC001 or CG722). (C and D) Cyathostomin L3 (white arrow) and trap formation and chlamydospores by the fungal isolates of D. flagrans (black arrow). (E) cyathostomin L3 (white arrow) and formation of traps (constrictor ring) by the fungal isolates of D. flagrans (black arrow) in Petri dishes containing 2% water-agar (2% WA).
resistance. In this study, the predatory activity of D. flagrans was effective, even after prolonged storage. This is interesting because, according to Braga et al. (2009, 2010b), this fungus can be used in natural conditions for controlling cyathostomins of horses. According to Fontenot et al. (2003), D. flagrans has an optimal growth rate of 15–60 mm per week at temperatures between 20 °C and 30 °C. In the presence of nematodes, the fungus can produce 700–800 traps per cm2 in a short period of time. These results are in accordance with the present work; once the temperature was kept constant around 25 °C, there was trap formation and a reduction in the number of cyathostomin L3 by D. flagrans (AC001 and CG722) at the end of experimental essay (Fig. 1A–E). Larsen et al. (1992) mention that the most practical utilization of this and other nematophagous fungi is the oral administration of the fungal material as mycelium, conidia and chlamydospores. Moreover, the literature has argued that the use of different isolates of the same fungus at different concentrations may have different results (Araújo et al., 1993). Larsen et al. (1995) reported the effectiveness of an isolate of D. flagrans on cyathostomins recovered from coprocultures after administration of different concen-
Fig. 2. Means and standard deviation (bars) of infective non-predated cyathostomin larvae recovered from 2% water-agar plates by the Baermann method on the seventh day of treatment with the following fungal isolates: Duddingtonia flagrans (AC001 and CG722) and control (without fungus). Asterisk denotes significant difference (p < 0.01) between the fungus-treated group and the control – Tukey’s test at a 1% probability level.
trations of the chlamydospores in horses. The reduction of cyathostomin L3 in feces of horses was also obtained through the administration of conidia of D. flagrans under different climatic
F.R. Braga et al. / Experimental Parasitology 128 (2011) 460–463
conditions (Baudena et al., 2000). In a recent study, Braga et al. (2010b) reported that D. flagrans isolate AC001 was effective in the destruction and reduction of cyathostomin L3 under laboratory conditions. In that study, 1000 conidia were used and, at the end of the experiment, a 93.64% reduction in the cyathostomin population was observed. The isolate used had been stored at 4 °C for 10 days. This is in accordance with the present work, where a reduction of 88.6% in cyathostomin L3 recovery was observed after treatment. These AC001 and CG722 isolates had been stored in silica-gel for 7 years. In relation to preservation time, Mota et al. (2002) reported that maintaining the samples in silica-gel after eighteen months had a negative influence on the predatory ability of the fungi Arthrobotrys robusta and Monacrosporium thaumasium, but they were still able to reduce the population of Haemonchus contortus L3 in relation to the control group. Braga et al. (in press) reported that A. robusta (I-31) was effective against H. contortus L3 (73.8% reduction) after 7 years preserved in silica-gel. The results of this study are similar; fungal cultures preserved with silica-gel for 7 years, and recovered in 2% PDA produced conidia and are able to trap cyathostomin larvae. The current work also observed spore production in groups treated with AC001 and CG722 (Fig. 1A–E). In this study, there was no statistically significant difference in the predatory ability of the two isolates of D. flagrans tested. However, differences in the inter and intra specific activity of predatory nematophagous fungi are common and have been observed in experiments with other fungal isolates (Araújo et al., 1993). Few reports have mentioned the predatory activity of nematophagous fungi in vitro after different conditions of storage on larvae of parasitic nematodes in domestic animals (Mota et al., 2002). Sharma and Smith (1999) demonstrated the possibility of recovering Ascomycota samples preserved in silica-gel after 25 years. Therefore this approach should not be discarded, not only because it is easy to implement, but also because it allows samples to remain ready for use for extended periods. Mota et al. (2002), mention that, despite all the advances already made in research on the use of nematophagous fungi for biological control of gastrointestinal parasites of domestic animals, a lot more has to be studied so this technique can be implemented. The preservation of microorganism cultures used in programs of biological control is essential for a large number of industrial processes and research. In this study, the fungus D. flagrans (AC001 and CG722) was effective in capturing, destroying and reducing cyathostomin L3, suggesting that storage time did not interfere with their predatory activity. This is the first study on the interaction of D. flagrans (AC001 and CG722) with cyathostomin L3 after 7 years of storage in silica-gel, showing that it resisted storage and that it maintained its predatory activity on cyathostomin L3. Acknowledgments The authors would like to thank CNPq, Capes, Fapemig and Capes/Finep for the financial support and grant concession, and
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to the Núcleo de Microscopia Eletrônica e Microanálise – NMM of the Federal University. References Araújo, J.V., Santos, M.A., Ferraz, S., Maia, A.S., 1993. Antagonistic effect of predacious Arthrobotrys fungi on infective Haemonchus placei larvae. Journal of Helminthology 67, 136–138. Ayres, M., Ayres, J.R.M., Ayres, D.L., Santos, A.S. 2003. Aplicações estatísticas nas áreas de ciências biológicas, CNPq, Brasília, p. 290. Baudena, M.A., Chapman, M.R., Larsen, M., Klei, R.R., 2000. Efficacy of the nematophagous fungus Duddingtonia flagrans in reducing equine cyathostome larvae on pasture in south Lousiana. Veterinary Parasitology 89, 219–230. Bevilaqua, C.M.L., Rodrigues, M.L., Cocordet, D., 1993. Identification of infective larvae of some common Eqüinos strongylids of horses. Revue de Médecine Vétérinaire 144, 989–995. Bird, J., Herd, R.P., 1995. In vitro assessment of two species of nematophagous fungi (Arthrobotrys oligospora and Arthrobotrys flagrans) to control the development of infective cyathostome larvae from naturally infected horses. Veterinary Parasitology 56, 181–187. Braga, F.R., Araújo, J.V., Silva, A.R., Araujo, J.M., Carvalho, R.O., Tavela, A.O., Campos, A.K., Carvalho, G.R., 2009. Biological control of horse cyathostomin (Nematoda: Cyathostominae) using the nematophagous fungus Duddingtonia flagrans in tropical southeastern Brazil. Veterinary Parasitology 163, 335–340. Braga, F.R., Araújo, J.V., Araujo, J.M., Silva, A.R., Carvalho, R.O., Ferreira, S.R., Benjamin, L.A., 2010b. Predatory activity of the nematophagous fungus Duddingtonia flagrans on horse cyathostomin infective larvae. Tropical Animal Health and Production 42, 1161–1163. Braga, F.R., Carvalho, R.O., Silva, A.R., Araújo, J.V., Frassy, L.N., Lafisca, A., Soares, F.E.F., in press. Predatory capability of the nematophagous fungus Arthrobotrys robusta preserved in silica-gel on infecting larvae of Haemonchus contortus. Tropical Animal Health and Production. Duddington, C.L., 1955. Notes on the technique of handling predaceous fungi. Transactions of the British Mycological Society 38, 97–103. Eysker, M., Boersema, J.H., Kooyman, F.N.J., 1989. Emergence from inhibited development of cyathostome larvae in ponies following failure to remove them by repeated treatments with benzimidazole compounds. Veterinary Parasitology 34, 87–93. Fontenot, M.E., Miller, J.E., Peña, M.T., Larsen, M., Gillespie, A., 2003. Efficiency of feeding Duddingtonia flagrans chlamydospores to grazing ewes on reducing availability of parasitic nematode larvae on pasture. Veterinary Parasitology 118, 203–213. Gordon, H.M., Whitlock, H.V., 1939. A new technique for counting nematode eggs in sheep faeces. Journal of the Council for Scientific and Industrial Research 12, 50–52. Larsen, M., Wolstrup, J., Henriksen, S.A., Gronvold, J., Nansen, P., 1992. In vivo passage through calves of nematophagous fungi selected for biocontrol of parasitic nematodes. Journal of Helminthology 66, 137–141. Larsen, M., Nansen, P., Wolstrup, J., Gronvold, J., Henriksen, S., Zorn, A., 1995. Biological control of trichostrongyles in calves by the fungus Duddingtonia flagrans fed to animals under natural grazing conditions. Veterinary Parasitology 60, 321–330. Love, S., Duncan, J.L., 1992. The development 1 of naturally acquired cyathostome infection in ponies. Veterinary Parasitology 44, 127–142. Mota, M.A., Campos, A.K., Araújo, J.V., 2002. Avaliação da capacidade predatória dos fungos Arthrobotrys robusta e Monacrosporium thaumasium sobre nematóides gastrintestinais parasitos de bovinos, submetidos a diferentes métodos de preservação. Revista Brasileira de Parasitologia Veterinária 11, 13–17. Ryan, M.J., Smith, D., Jeffries, P., 2000. A decision-based key to determine the most appropriate protocol for the preservation of fungi. World Journal of Microbiology and Biotechnology 16, 183–186. Santos, M., Ferraz, S., Muchovej, J., 1991. Detection and ecology of nematophagous fungi from Brazil soils. Nematologia Brasileira 15, 121–134. Sharma, B., Smith, D., 1999. Recovery of fungi after storage for over a quarter of a century. World Journal of Microbiology and Biotechnology 15, 517–519. Stirling, G.R., 1991. Biological control of plant parasitic nematodes. CAB International, Wallingford, United Kingdon, pp. 282.
Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Research Brief
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) Fabio Ribeiro Braga a,⇑, Jackson Victor Araújo a,1, Juliana Milani Araujo a, Alexandre de Oliveira Tavela a, Sebastião Rodrigo Ferreira a, Filippe E. Freitas Soares b, Laércio dos Anjos Benjamin a, Luiza Neme Frassy a a b
Departamento de Veterinária, Universidade Federal de Viçosa, Viçosa-MG, CEP 36570-000 Vicosa, Brazil Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Viçosa, Viçosa-MG, CEP 3657-000 Vicosa, Brazil
a r t i c l e
i n f o
Article history: Received 30 September 2010 Received in revised form 12 May 2011 Accepted 16 May 2011 Available online 24 May 2011 Keywords: Nematophagous fungi Duddingtonia flagrans Silica-gel Cyathostomins
a b s t r a c t 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. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Horses host a wide variety of helminths, and cyathostomins are one of the most important (Love and Duncan, 1992). These helminths are present in pastures throughout the year and can cause episodes of diarrhea in adult animals (Braga et al., 2009). Anthelminthic drugs are usually used to control these gastrointestinal parasites. However, Bird and Herd (1995) report that no drug has efficacy against encysted larval stages of these nematodes. Biological control with predatory nematophagous fungi, especially Duddingtonia flagrans are considered as a viable alternative (Braga et al., 2009). One of the principal advantages is the ability to survive for long periods under laboratory conditions; however, some isolates may lose their predatory activity (Stirling, 1991). In this context, the use of silica-gel allows the storage of isolated spores for prolonged periods. According to Ryan et al. (2000) this method has been considered as one of the most ⇑ Corresponding author. Fax: +55 31 3899 1464. 1
E-mail address: [email protected] (F.R. Braga). CNPq scholarship.
0014-4894/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2011.05.013
effective, preserving the fungi for long periods. The main advantages are: (1) reduced chances contamination due to low humidity. (2) The financial cost is low and no specific equipment is required. (3) Many cultures of the same isolate may be recovered from a single sample storage. However, there is a lack of studies to assess whether the duration of preservation in silica-gel can interfere with the predatory activity. The current study evaluates the predatory activity of the fungus D. flagrans (isolates AC001 and CG722) stored for 7 years in silica-gel. The fungal isolates predatory activity was tested against cyathostomin infective larvae (L3). 2. Materials and methods 2.1. Fungi Two isolates of nematophagous fungi D. flagrans (AC001 and CG722) were used. The isolates were stored in test tubes containing 2% corn-meal-agar (2% CMA), in the dark, at 4 °C for 10 days. These isolates were obtained from Brazilian agricultural soil, in Viçosa city, Zona da Mata region of Minas Gerais state. They were
461
F.R. Braga et al. / Experimental Parasitology 128 (2011) 460–463
collected using the soil-sprinkling method of Duddington (1955), modified by Santos et al. (1991). 2.2. Maintenance of fungal cultures in vials containing silica-gel Samples of the isolates AC001 and CG722 removed from test tubes containing 2% CMA, at 25 °C, were inoculated in sporulation medium AELA (yeast extract, 4 g; K2HPO4, 1 g; MgSO47H2O, 0.5 g; soluble starch, 20 g; distilled water, 1 l) as described by Mota et al. (2002). After 7 days, a sterile solution of 5% skimmed milk and rice grains was added to the surface of the plates. Then the grains were soaked in the suspension of fungal material and transferred to five vials containing grains of sterile silica-gel. The samples were stored at 4 °C in the absence of light and humidity. After 7 years, the cultures were recovered by transferring a grain of rice containing the fungus D. flagrans (AC001 and CG722) from the bottle with silica-
Reduction% ¼
thostomin L3 and 500 conidia of isolates AC001 or CG722 in 2% WA. The control group contained only 500 L3 on dishes in 2% WA. For 7 days, every 24 h, 10 random 4 mm diameter fields on each plate of treated and control groups were observed under an optical microscope with a 10 objective, counting the number of non-predated L3 on each. At the end of 7 days, the non-predated L3 were recovered from the Petri dishes using the Baermann apparatus with water at 42 °C. 2.6. Statistical analysis Data obtained were examined by analysis of variance at 1% and 5% probability levels using the BioEstat 3.0 software (Ayres et al., 2003). The efficiency predation activity was evaluated using Tukey’s test at 1% probability level. The percent reduction in the mean larval recovery was calculated by the following equation:
ðMean of L3 recovered from control mean of L3 recovered from treatmentÞ 100= Mean of L3 recovered from control
gel to the surface of 9.0 cm Petri dishes containing 20 ml of potato-dextrose agar 2% (2% PDA) and kept at 25 °C, in the dark, for 10 days. 2.3. Conidia collection After growth of the isolates in 2% PDA, new culture disks 4 mm in diameter were transferred to 9.0 cm Petri dishes containing 20 ml of 2% water-agar (2% WA) and 1 ml of distilled water containing 1000 larvae of Panagrellus sp., were added daily for 21 days to induce fungal conidia formation. When complete fungal development was observed, 5 ml of distilled water were added to each Petri dish, and the conidial and mycelial fragments were removed using technique described by Araújo et al. (1993). The suspension present in the plates was screened through a sieve coupled to a plastic container to remove the mycelium fragments.
3. Results Cultures of D. flagrans (isolates AC001 and CG722) preserved with silica-gel for 7 years were viable and produced conidia able to predate cyathostomin larvae. The presence of cyathostomin L3 in Petri dishes containing 2% WA was essential for the trap formation by fungal isolates, since this medium is poor in nutrients. A difference (p < 0.01) was observed between the means number of non-predated cyathostomin L3 and the means in the groups treated with the fungus D. flagrans (AC001 and CG722) throughout the experiment (Table 1). Evidence of predation was observed by means of cyathostomin L3 recovered on the seventh day using the Baermann method, at the end of the experiment. In addition, there were typical fungal structures (conidia and traps) in the treated groups during the test (Fig. 1A–E). At the end of the experiment, a mean cyathostomin L3 reduction of 88.6% (AC001) and 78.4% (CG722) was observed (Fig. 2).
2.4. Obtaining cyathostomin infective larvae (L3) 4. Discussion 2.4.1. Fecal samples About 1 kg of fresh feces were collected directly from the rectum of horses (Equus caballus) naturally infected with cyathostomins. These animals were from the Veterinary Department, Federal University of Viçosa, Minas Gerais, Brazil. After that, a count of eggs per gram of feces (EPG) was made according to Gordon and Whitlock (1939), in order to find positive animals. Then coprocultures using fragmented industrial vermiculite (autoclaved) and water were performed. The coprocultures were maintained at 26 °C in the dark for 8 days. At the end of this period cyathostomin (L3) were obtained by the Baermann method, identified and quantified according to the criteria described by Bevilaqua et al. (1993) using an optical microscope with a 10 objective.
Eysker et al. (1989) mention that adults cyathostomin can be controlled by many different broad spectrum anthelminthics. However, there are some disadvantages related to the administration of these drugs including the lack of efficacy against cyathostomins during encasement and the emergence of benzimidazole
Table 1 Daily means and standard deviations of non-predated third stage larvae (L3) of cyathostomins per 4 mm diameter field in 2% water-agar during 7 days in treatments with the fungus Duddingtonia flagrans (AC001 and CG722) and control without fungus. Time (Days)
Treatments (means of non-predated L3) AC001
CG722
Control
1 2 3 4 5 6 7
4.2a ± 2.5 2.7a ± 2.6 2.9a ± 2.7 2.0a ± 1.9 1.3a ± 1.5 1.6a ± 1.7 0.9a ± 1.2
5.2a ± 3.8 3.1a ± 3.5 3.9a ± 2.8 2.3a ± 2.9 2.1a ± 2.2 1.7a ± 2.1 0.8a ± 1.1
26.3b ± 18.7 14.1b ± 24.7 10.6b ± 10.2 6.5b ± 7.1 5.9b ± 5.4 4.7b ± 4.4 3.8b ± 3.6
2.5. Experimental assay The predation test was conducted on the surface of Petri dishes according to a modified technique described by Mota et al. (2002). Three groups were formed on 9.0 cm diameter Petri dishes containing 20 ml of 2% WA; two treated groups (AC001 and CG722) and one control group (without fungus). Six replicates were made for each group. Petri dishes were previously marked in 4 mm diameter fields. In the treated groups each Petri dish contained 500 cya-
Means followed by the same small letter in the lines were not statistically different (p > 0.01).
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Fig. 1. (A) infective larvae (L3) of cyathostomins (white arrow) and hyphae of the fungus Duddingtonia flagrans (AC001 or CG722) (black arrow). (B) Cyathostomin L3 (white arrow), formation of traps (black Arrow) by the fungal isolates (AC001 or CG722). (C and D) Cyathostomin L3 (white arrow) and trap formation and chlamydospores by the fungal isolates of D. flagrans (black arrow). (E) cyathostomin L3 (white arrow) and formation of traps (constrictor ring) by the fungal isolates of D. flagrans (black arrow) in Petri dishes containing 2% water-agar (2% WA).
resistance. In this study, the predatory activity of D. flagrans was effective, even after prolonged storage. This is interesting because, according to Braga et al. (2009, 2010b), this fungus can be used in natural conditions for controlling cyathostomins of horses. According to Fontenot et al. (2003), D. flagrans has an optimal growth rate of 15–60 mm per week at temperatures between 20 °C and 30 °C. In the presence of nematodes, the fungus can produce 700–800 traps per cm2 in a short period of time. These results are in accordance with the present work; once the temperature was kept constant around 25 °C, there was trap formation and a reduction in the number of cyathostomin L3 by D. flagrans (AC001 and CG722) at the end of experimental essay (Fig. 1A–E). Larsen et al. (1992) mention that the most practical utilization of this and other nematophagous fungi is the oral administration of the fungal material as mycelium, conidia and chlamydospores. Moreover, the literature has argued that the use of different isolates of the same fungus at different concentrations may have different results (Araújo et al., 1993). Larsen et al. (1995) reported the effectiveness of an isolate of D. flagrans on cyathostomins recovered from coprocultures after administration of different concen-
Fig. 2. Means and standard deviation (bars) of infective non-predated cyathostomin larvae recovered from 2% water-agar plates by the Baermann method on the seventh day of treatment with the following fungal isolates: Duddingtonia flagrans (AC001 and CG722) and control (without fungus). Asterisk denotes significant difference (p < 0.01) between the fungus-treated group and the control – Tukey’s test at a 1% probability level.
trations of the chlamydospores in horses. The reduction of cyathostomin L3 in feces of horses was also obtained through the administration of conidia of D. flagrans under different climatic
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conditions (Baudena et al., 2000). In a recent study, Braga et al. (2010b) reported that D. flagrans isolate AC001 was effective in the destruction and reduction of cyathostomin L3 under laboratory conditions. In that study, 1000 conidia were used and, at the end of the experiment, a 93.64% reduction in the cyathostomin population was observed. The isolate used had been stored at 4 °C for 10 days. This is in accordance with the present work, where a reduction of 88.6% in cyathostomin L3 recovery was observed after treatment. These AC001 and CG722 isolates had been stored in silica-gel for 7 years. In relation to preservation time, Mota et al. (2002) reported that maintaining the samples in silica-gel after eighteen months had a negative influence on the predatory ability of the fungi Arthrobotrys robusta and Monacrosporium thaumasium, but they were still able to reduce the population of Haemonchus contortus L3 in relation to the control group. Braga et al. (in press) reported that A. robusta (I-31) was effective against H. contortus L3 (73.8% reduction) after 7 years preserved in silica-gel. The results of this study are similar; fungal cultures preserved with silica-gel for 7 years, and recovered in 2% PDA produced conidia and are able to trap cyathostomin larvae. The current work also observed spore production in groups treated with AC001 and CG722 (Fig. 1A–E). In this study, there was no statistically significant difference in the predatory ability of the two isolates of D. flagrans tested. However, differences in the inter and intra specific activity of predatory nematophagous fungi are common and have been observed in experiments with other fungal isolates (Araújo et al., 1993). Few reports have mentioned the predatory activity of nematophagous fungi in vitro after different conditions of storage on larvae of parasitic nematodes in domestic animals (Mota et al., 2002). Sharma and Smith (1999) demonstrated the possibility of recovering Ascomycota samples preserved in silica-gel after 25 years. Therefore this approach should not be discarded, not only because it is easy to implement, but also because it allows samples to remain ready for use for extended periods. Mota et al. (2002), mention that, despite all the advances already made in research on the use of nematophagous fungi for biological control of gastrointestinal parasites of domestic animals, a lot more has to be studied so this technique can be implemented. The preservation of microorganism cultures used in programs of biological control is essential for a large number of industrial processes and research. In this study, the fungus D. flagrans (AC001 and CG722) was effective in capturing, destroying and reducing cyathostomin L3, suggesting that storage time did not interfere with their predatory activity. This is the first study on the interaction of D. flagrans (AC001 and CG722) with cyathostomin L3 after 7 years of storage in silica-gel, showing that it resisted storage and that it maintained its predatory activity on cyathostomin L3. Acknowledgments The authors would like to thank CNPq, Capes, Fapemig and Capes/Finep for the financial support and grant concession, and
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