- Email: [email protected]
Animal-crop rotation system: A hurdle for the use of the nematophagous fungus Duddingtonia flagrans
Biological Control 62 (2012) 82–85
Contents lists available at SciVerse ScienceDirect
Biological Control journal homepage: www.elsevier.com/locate/ybcon
Animal-crop rotation system: A hurdle for the use of the nematophagous fungus Duddingtonia flagrans Deise L. Mahl a, Régis A. Zanette a, Francielli P.K. Jesus b, Anelise B. Friedriczewski c, Sydney H. Alves a, Janio M. Santurio a,⇑ a b c
Programa de Pós-Graduação em Farmacologia, Universidade Federal de Santa Maria, Santa Maria, Brazil Programa de Pós-Graduação em Ciências Veterinárias, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil Laboratório de Pesquisas Micológicas, Universidade Federal de Santa Maria, Santa Maria, Brazil
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
" The activity of 14 agricultural fungicides against Duddingtonia flagrans was evaluated in vitro. " Minimum inhibitory concentrations of the fungicides were obtained by the standard broth microdilution M38-A2 method. " Agar diffusion test showed that D. flagrans is resistant to only two out of the 14 fungicides at the concentrations assayed. " Efficient biological control of nematodes using fungal agents may not be possible in animal-crop rotation systems.
Duddingtonia flagrans growth in potato dextrose agar supplemented with water/DMSO (control) and several agricultural fungicides at the concentration of their maximum residue limit.
a r t i c l e
a b s t r a c t
i n f o
Article history: Received 12 May 2011 Accepted 25 March 2012 Available online 2 April 2012 Keywords: Agricultural fungicides Susceptibility test Duddingtonia flagrans Biological control
The fallow period can represent an opportunity to farmers if the field can be trimmed to receive finishing cattle. Nematode control remains important at this stage, and the use of biological agents such as nematophagous fungi is desirable. Nonetheless, the indiscriminate use of fungicides on agricultural crops may affect the development and survival of fungal propagules. Therefore, we evaluated the in vitro activity of 14 agricultural fungicides against the Duddingtonia flagrans strain ARSEF 5701. The assays were based on CLSI M38-A2 broth microdilution and the agar diffusion method. The fungicides azoxystrobin, cyproconazole, cyprodinil and tebuconazole showed minimum inhibitory concentrations for D. flagrans below their respective maximum residue limits. Although complete radial growth inhibition was only observed for carbendazim at the maximum concentration tested, D. flagrans proved to be resistant to only two fungicides, cyprodinil and iprodione. The present study indicates that D. flagrans is sensitive to most of the common agricultural fungicides, and resistance depends upon the structure, i.e., hypha or chlamydospore. On the basis of these in vitro results, we concluded that the use of agricultural fungicides could seriously hamper the survival of the fungus D. flagrans in amended soils. Ó 2012 Elsevier Inc. All rights reserved.
⇑ Corresponding author. Address: Campus UFSM, Prédio 20, Sala 4139, Santa Maria, RS 16 97105-900, Brazil. Fax: +55 55 3220 8906. E-mail address: [email protected] (J.M. Santurio). 1049-9644/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.biocontrol.2012.03.009
83
D.L. Mahl et al. / Biological Control 62 (2012) 82–85
1. Introduction Animal-crop rotation systems have been fostering the interest of farmers searching for diversification and increased profitability. In the summer season, for example, soybean and corn fields assume economic importance in Southern Brazil, but most of the harvested area becomes unutilised in the winter (Lopes et al., 2008). This situation opens space for the introduction of feeder cattle or sheep that can be fattened prior to the next summer season. The effective control of gastrointestinal parasites is essential to optimizing production in grazing cattle. Strategic worm control programs include pasture rotation management, the identification of susceptible animals (Larsson et al., 2007) and the utilization of nematophagous fungi, notably, Duddingtonia flagrans, which bears the stamp of being the fungus with the most promising field results (Jobim et al., 2008; Santurio et al., 2011). This fungus produce diffuse thick-walled chlamydospores, the stage responsible for its survival during passage through the gut of ruminants following oral administration. Once reaching the feces, the chlamydospores form specialized, three-dimensional networks that trap gastrointestinal parasite larvae (Sanyal, 2000), which recently dislodged from the eggs. Therefore, D. flagrans is commonly isolated from fresh fecal samples of grazing livestock, although it has been successfully detected by the polymerase chain reaction technique from decaying plant parts and soil specimens (Kelly et al., 2008). The larvae, if not killed, develop in the environment to attain the infective stage, which are ingested when the animals feed the grass. The intensive and indiscriminate use of fungicides on agricultural crops, coupled with their low biodegradability, presents a constraint problem for nematophagous fungi, as observed in vitro and substantiated in vivo by Sanyal et al. (2004). Luz et al. (2007) showed that the susceptibility of fungi pathogenic to insects and other invertebrates can be both species-specific and dependent on the individual fungicide compound. Therefore, we report on the in vitro susceptibility of D. flagrans to 14 agricultural fungicides using an agar dilution method and a standardized broth microdilution method for testing filamentous fungi. 2. Materials and methods The D. flagrans isolate, strain ARSEF 5701 (Collection of Entomopathogenic Fungal Cultures), was used. The fungus was cultivated on potato dextrose agar tubes for three weeks at 28 °C. Thereafter, a sterile aqueous solution containing 0.85% saline and 0.05% Triton X-100 was added to the tubes, and the chlamydospores were carefully rinsed from the agar surface by gentle agitation. Microscopic analysis of the suspension showed a large quantity of chlamydospores and few broken hyphae. The suspension was read in a spectrophotometer at 530 nm, and the transmittance was adjusted to 68–70%. The inoculum was diluted in RPMI 1640 broth containing L-glutamine and was buffered to pH 7 with 0.165 morpholinepropanesulfonic acid, yielding a final concentration of 104 chlamydospores/ml. The agricultural fungicides azoxystrobin, carbendazim, cyproconazole, cyprodinil, difenoconazole, fludioxonil, iprodione, mancozeb, metalaxyl, pencycuron, tebuconazole, thiabendazole, triadimenol and trifloxystrobin were obtained from Sigma–Aldrich (St. Louis, MO). The broth microdilution test was performed in accordance with the Clinical and Laboratory Standards Institute (CLSI) document M38-A2 (CLSI, 2008). The fungicides were diluted 1:5 in RPMI 1640 broth, and 100 ll aliquots were sequentially dispensed into the microdilution plates. The maximum residue limit (MRL) for each fungicide, according to Brazilian National Health Vigilance Agency (ANVISA, Brazilian authority), was used as the central dilution. Thereafter, five additional dilutions were performed using the RPMI 1640 medium above and below the MRL,
totaling 10 dilutions for each chemical. Then, 100 ll of inoculum was added to each well. The tests were performed in triplicate, and the MIC readings were visually assessed for the presence (i.e., growth) or absence of hyphae compared with the positive control (hyphae under optimal growth conditions) after 48 h of incubation at 28 °C, as preconized by CLSI document for nondermatophyte fungal species. Potato dextrose agar was used for the agar diffusion method according to Alves and Cury (1992), with minor modifications. After the medium was autoclaved, it was allowed to cool to 50 °C before antifungal compounds diluted in DMSO were added in the volumes necessary to obtain the desired final concentrations. The agar and the compounds were mixed and poured into plates (20 ml/plate). The agar from the control plates only had water and DMSO corresponding to the volume of the fungicide dilution used. For each assay, mycelial plugs (9 mm in diameter) were cut from the margin of actively growing colonies of 7-d-old stock cultures and placed in the center of each test plate (at room temperature) with the mycelium in contact with the agar. Additional dilutions were performed using the RPMI 1640 medium above and below the MRL of the fungicide assayed, totaling six dilutions for each chemical. Each antifungal concentration and control sample was plated in triplicate, and the plates were incubated at at 28 °C for 7 d. The mean colony diameter was found to have a normal distribution (D0 Agostino-Pearson test). When a significant (analysis of variance; P < 0.05) effect of concentration was detected, comparisons were made with the results for the water-DMSO control samples by using Dunnett’s post hoc test. 3. Results and discussion Chlamydospore growth in control wells containing RPMI 1640 broth after 48 h at 28 °C was excelent, allowing for the visual observation of hyphae in the wells. For all test plates in the agar diffusion method, colony growth appeared uniform and radially symmetric. Maximum growth of the D. flagrans fungus in control plates (9 cm) was attained after 7 d. The azoxystrobin, cyproconazole, cyprodinil and tebuconazole showed MICs for D. flagrans below their respective MRLs (Table 1). However, considering the fungicide’s ability to accumulate and adsorb to soil particles and that their intensive application increases the harvest indexes (Ludwig et al., 2010), the content of fungicides in soils is likely to be much greater than the residue limits remaining on the food product samples. Komárek et al. (2010) can be consulted for an extensive review of the environmental and toxicological Table 1 Agricultural fungicides, crops, maximum residue limit (MRL) for each crop and minimum inhibitory concentrations (MICs) against the Duddingtonia flagrans strain ARSEF 5701. Agricultural fungicide
Crop
MRL (mg kg1)
MIC (mg l1)
Tebuconazole Cyproconazole Cyprodinil Difenoconazole Azoxystrobin Iprodione Metalaxyl Pencycuron Trifloxystrobin Fludioxonil Triadimenol Thiabendazole Carbendazim Mancozeb
Soybean Soybean Potato Soybean Soybean Potato Soybean Potato Soybean Soybean Oat Soybean Soybean Wheat
0.1 0.05 0.05 0.05 0.5 0.05 0.05 0.1 0.05 0.05 0.2 0.2 0.5 0.5
0.0125 0.025 0.025 0.1 0.25 0.4 0.4 0.4 0.4 0.8 0.8 1.6 4 >16
The tests were carried out in triplicate; MICs that were lower than the MRL for each fungicide are shown in bold.
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D.L. Mahl et al. / Biological Control 62 (2012) 82–85
Table 2 Mean ± SD radial growth of Duddingtonia flagrans cultured in potato dextrose agar supplemented with water/DMSO or various concentrations of agricultural fungicides. Agricultural fungicide
Cyproconazole Cyprodinil Difenoconazole Fludioxonil Iprodione Metalaxyl Trifloxystrobin Tebuconazole Pencycuron – Thiabendazole Triadimenol – Azoxystrobin Carbendazim Mancozeb
Concentration (mg/l) Water/DMSO
0.8
0.2
0.1
0.05
0.0125
0.003125
9 9 9 9 9 9 9 9 9
2.4 ± 0.1a 9 3.7 ± 0.3a 2.9 ± 0.1a 9 6 ± 0.5a 4.6 ± 0.3a 2.8 ± 0.3a 5.5 ± 0.5a 1.6 3.6 ± 0.16a 2.4 ± 1.1a 8 3.5 ± 0.4a 0a 3.8 ± 0.5a
3.9 ± 0.4a 9 2.7 ± 0.1a 9 9 5.4 ± 0.1a 5 ± 0.5a 2.9 ± 0.4a 5.8 ± 0.9a 0.4 4.9 ± 0.7a 4.3 ± 0.6a 4 3.1 ± 0.4a 1.2 ± 0.15a 1.5 ± 0.3a
4.2 ± 0.3a 9 4.2 ± 0.5a 9 9 7.6 ± 1.3a 5 ± 0.3a 4.1 ± 0.2a 6.5 ± 0.25a 0.2 5.4 ± 1.8a 5.5 ± 0.3a 2 4.3 ± 0.4a 3 ± 1.3a 2.2 ± 0.4a
4.7 ± 0.4a 9 2.7 ± 0.1a 9 9 8.3 ± 0.8 5.4 ± 0.6a 4.5 ± 0.5a 5.7 ± 0.4a 0.025 4.9 ± 0.7a 5.8 ± 0a 0.5 4.6 ± 1.5a 3.7 ± 0.1a 6.3 ± 0.4a
4.4 ± 0.3a 9 5.3 ± 0.5a 9 9 9 4.5 ± 0.3a 4.6 ± 0.1a 7.1 ± 0.1a 0.0125 3.5 ± 0.1a 6.1 ± 0.2a 0.125 4.6 ± 0.4a 3.8 ± 0.1a 5.6 ± 1.5a
6.8 ± 0.1a 9 6.2 ± 0.8a 9 9 9 6.7 ± 0.3a 4.6 ± 0.1a 6.4 ± 0.1a 0.00625 3.3 ± 0.4a 6.2 ± 0.1a 0.03125 5.4 ± 0.2a 4.2 ± 0.6a 5.7 ± 0.5a
9 9 9 9 9
Isolates were cultured in triplicate; values reported represent mean colony diameter in millimeters. a Within a row, the value differs significantly (P < 0.001; Dunnett’s test) from the value for the water/DMSO control sample. Bold numbers represent maximum residue limits for each fungicide assayed.
aspects of agricultural fungicides. The results for the agar dilution method can be observed in Table 2. D. flagrans proved to be resistant only against cyprodinil and iprodione at the concentrations tested. Resistance was also observed against fludioxonil up to the concentration of 0.2 g/l and to metalaxyl at concentrations below the MRL. The radial growth assay has been used extensively for the in vitro evaluation of fungicide resistance in plant-pathogenic oomycetes (Brown et al., 2008). This technique assesses growth inhibition by the direct measurement of colony diameter following the inoculation of isolates on agar supplemented with various concentrations of antifungal compounds. Differences in susceptibility were observed by comparing both in vitro techniques. For example, D. flagrans proved to be resistant to cyprodinil using the agar diffusion method at concentrations ranging from 0.003125 to 0.8 g/l, although the MIC for this fungicide was of 0.025 g/l. This discrepancy is probably due to the use of 9 mm culture disks as inocula, which supported initial fungal growth. In some soilborne fungi, chlamydospores have been documented to have a role as survival structures (Xiarong and Heitman, 2005; Campos et al., 2009). Conversely, the chlamydospores used in the microdilution method were more directly affected by the fungicides than the hyphae present in the agar diffusion test. Importantly, although chlamydospores are often formed within the agar in cultures grown on agar-solidified medium (Gardner et al., 2000), chlamydospore production is time sensitive (Campos et al., 2009); hence, none were observed in the 7-d-old cultures. The MIC observed for thiabendazole in our study was 1.6 g/l, which is in agreement with previous in vitro studies that show that D. flagrans is completely inhibited at concentrations of 10 g/l but develops well on 1 g/l of the fungicide (Paraud et al., 2004; Luz et al., 2007). According to Faedo et al. (2002) and Campos et al. (2009), a number of factors can potentially affect whether D. flagrans spreads into and establishes itself in the soil. These factors include climate, soil type, soil microorganisms and soil macrofauna. Moreover, these authors reported that this nematode-trapping fungus has minimal growth beyond the fecal environment into the surrounding soil when fungal chlamydospores were fed to sheep or cattle. However, their conclusions were based on the lack of a detectable impact of fungal predation on the nematode population in the surrounding soil. The emergence of adequate, direct techniques (Kelly et al., 2008; Ojeda-Robertos et al., 2008) allowed for work showing that animals reingest chlamydospores, which
spread from feces to the grass, giving continuity to the animalenvironment cycle (Santurio et al., 2009). The results of the study reported here support previous concerns about the inherent susceptibility of nematophagous-fungi against antimycotic agents. Only two out of the 14 agricultural fungicides, at the concentrations tested herein, did not inhibit growth of the D. flagrans strain used in our experiment. Such susceptibility could seriously hamper the prolonged chlamydospore viability in amended soils, which could improve nematode control in animal-crop systems. Acknowledgment The first author would like to thank CAPES (Brazilian Federal Agency for the Improvement of Higher Education) for the financial support. The authors 3, 4, 5 and 6 thank CNPq (Brazil) for grant support. References Alves, S.H., Cury, A.E., 1992. Candida from cancer patients: susceptibility ‘‘in vitro’’ to polyene antifungal agents. Revista do Instituto de Medicina Tropical de São Paulo 34, 251–254. Brown, T.A., Grooters, A.M., Hosgood, G.L., 2008. In vitro susceptibility of Pythium insidiosum and a Lagenidium sp. to itraconazole, posaconazole, voriconazole, terbinafine, caspofungin, and mefenoxam. American Journal of Veterinary Research 69, 1463–1468. Campos, A.K., Araújo, J.V., Guimarães, M.P., Dias, A.S., 2009. Resistance of different fungal structures of Duddingtonia flagrans to the digestive process and predatory ability on larvae of Haemonchus contortus and Strongyloides papillosus in goat feces. Parasitology Research 105, 913–919. CLSI, 2008. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi; approved standard. CLSI document M38–A2, second ed. Clinical Laboratory Standards Institute, Pennsylvania. Faedo, M., Larsen, M., Dimander, S.O., Yeates, G.W., Höglund, J., Waller, P.J., 2002. Growth of the fungus Duddingtonia flagrans in soil surrounding feces deposited by cattle or sheep fed the fungus to control nematode parasites. Biological Control 23, 64–70. Gardner, K., Wiebe, M.G., Gillespie, A.T., Trinci, A.P.J., 2000. Production of chlamydospores of the nematode-trapping Duddingtonia flagrans in shake flask culture. Mycological Research 104, 205–209. Jobim, M.B., Santurio, J.M., De La Rue, M., 2008. Duddingtonia flagrans: biological control of cattle nematodes in the field. Ciencia Rural 38, 2256–2263. Kelly, P., Good, B., Fitzpatrick, R., Hanrahan, J.P., De Wall, T.D.T., 2008. Development and application of a PCR diagnostic assay for the accurate and rapid identification of the nematophagous fungus Duddingtonia flagrans. Mycological Research 112, 1026–1030. Komárek, M., Cadková, E., Chrastny´, V., Bordas, F., Bollinger, J., 2010. Contamination of vineyard soils with fungicides: a review of environmental and toxicological aspects. Environment International 36, 138–151.
D.L. Mahl et al. / Biological Control 62 (2012) 82–85 Larsson, A., Dimander, S.O., Rydzik, A., Uggla, A., Waller, P.J., Höglund, J., 2007. A 3year field evaluation of pasture rotation and supplementary feeding to control parasite infection in first-season grazing cattle – dynamics of pasture infectivity. Veterinary Parasitology 145, 129–137. Lopes, M.L.T., Carvalho, P.C.F., Anghinoni, I., Santos, D.T., Kuss, F., Freitas, F.K., Flores, J.P.C., 2008. Crop-livestock integration system: performance and carcass quality of superprecoce beef steers finished in oat and ryegrass pasture managed under different heights. Ciencia Rural 38, 178–184. Ludwig, M.P., Dutra, L.M.C., Filho, O.A.L., Zabot, L., Uhry, D., Lisboa, J.I., 2010. Soybean grain yield in response to herbicide and fungicides. Ciencia Rural 40, 1516–1522. Luz, C., Netto, M.C.B., Rocha, L.F.N., 2007. In vitro susceptibility to fungicides by invertebrate-pathogenic and saprobic fungi. Mycopathologia 164, 39–47. Ojeda-Robertos, N.F., Torres-Acosta, J.F.J., Ayala-Burgos, A., Aguilar-Caballero, A.J., Cob-Galera, L.A., Mendoza-de-Gives, P., 2008. A technique for the quantification of Duddingtonia flagrans chlamydospores in sheep faeces. Veterinary Parasitology 152, 339–343. Paraud, C., Pors, I., Chartier, C., 2004. Activity of Duddingtonia flagrans on Trichostrongylus colubriformis larvae in goat feces and interaction with a benzimidazole treatment. Small Ruminant Research 55, 199–207.
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Santurio, J.M., Zanette, R.A., Da Silva, A.S., De La Rue, M., Monteiro, S.G., Alves, S.H., 2009. Improved method for Duddingtonia flagrans chlamydospores production for livestock use. Veterinary Parasitology 164, 344–346. Santurio, J.M., Zanette, R.A., Da Silva, A.S., Fanfa, V.R., Farret, M.H., Ragagnin, L., Hecktheuer, P.A., Monteiro, S.G., 2011. A suitable model for the utilization of Duddingtonia flagrans fungus in small-flock-size sheep farms. Experimental Parasitology 127, 727–731. Sanyal, P.K., 2000. Screening for Indian isolates of predacious fungi for use in biological control against nematode parasites of ruminants. Veterinary Research Communications 14, 119–122. Sanyal, P.K., Chauhan, J.B., Mukhopadhyaya, P.N., 2004. Implications of fungicidal effects of benzimidazole compounds on Duddingtonia flagrans in integrated nematode parasite management in livestock. Veterinary Research Communications 28, 375–385. Xiarong, L., Heitman, J., 2005. Chlamydospore formation during hyphal growth in Cryptococcus neoformans. Eukaryotic Cell 4, 1746–1754.
Contents lists available at SciVerse ScienceDirect
Biological Control journal homepage: www.elsevier.com/locate/ybcon
Animal-crop rotation system: A hurdle for the use of the nematophagous fungus Duddingtonia flagrans Deise L. Mahl a, Régis A. Zanette a, Francielli P.K. Jesus b, Anelise B. Friedriczewski c, Sydney H. Alves a, Janio M. Santurio a,⇑ a b c
Programa de Pós-Graduação em Farmacologia, Universidade Federal de Santa Maria, Santa Maria, Brazil Programa de Pós-Graduação em Ciências Veterinárias, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil Laboratório de Pesquisas Micológicas, Universidade Federal de Santa Maria, Santa Maria, Brazil
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
" The activity of 14 agricultural fungicides against Duddingtonia flagrans was evaluated in vitro. " Minimum inhibitory concentrations of the fungicides were obtained by the standard broth microdilution M38-A2 method. " Agar diffusion test showed that D. flagrans is resistant to only two out of the 14 fungicides at the concentrations assayed. " Efficient biological control of nematodes using fungal agents may not be possible in animal-crop rotation systems.
Duddingtonia flagrans growth in potato dextrose agar supplemented with water/DMSO (control) and several agricultural fungicides at the concentration of their maximum residue limit.
a r t i c l e
a b s t r a c t
i n f o
Article history: Received 12 May 2011 Accepted 25 March 2012 Available online 2 April 2012 Keywords: Agricultural fungicides Susceptibility test Duddingtonia flagrans Biological control
The fallow period can represent an opportunity to farmers if the field can be trimmed to receive finishing cattle. Nematode control remains important at this stage, and the use of biological agents such as nematophagous fungi is desirable. Nonetheless, the indiscriminate use of fungicides on agricultural crops may affect the development and survival of fungal propagules. Therefore, we evaluated the in vitro activity of 14 agricultural fungicides against the Duddingtonia flagrans strain ARSEF 5701. The assays were based on CLSI M38-A2 broth microdilution and the agar diffusion method. The fungicides azoxystrobin, cyproconazole, cyprodinil and tebuconazole showed minimum inhibitory concentrations for D. flagrans below their respective maximum residue limits. Although complete radial growth inhibition was only observed for carbendazim at the maximum concentration tested, D. flagrans proved to be resistant to only two fungicides, cyprodinil and iprodione. The present study indicates that D. flagrans is sensitive to most of the common agricultural fungicides, and resistance depends upon the structure, i.e., hypha or chlamydospore. On the basis of these in vitro results, we concluded that the use of agricultural fungicides could seriously hamper the survival of the fungus D. flagrans in amended soils. Ó 2012 Elsevier Inc. All rights reserved.
⇑ Corresponding author. Address: Campus UFSM, Prédio 20, Sala 4139, Santa Maria, RS 16 97105-900, Brazil. Fax: +55 55 3220 8906. E-mail address: [email protected] (J.M. Santurio). 1049-9644/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.biocontrol.2012.03.009
83
D.L. Mahl et al. / Biological Control 62 (2012) 82–85
1. Introduction Animal-crop rotation systems have been fostering the interest of farmers searching for diversification and increased profitability. In the summer season, for example, soybean and corn fields assume economic importance in Southern Brazil, but most of the harvested area becomes unutilised in the winter (Lopes et al., 2008). This situation opens space for the introduction of feeder cattle or sheep that can be fattened prior to the next summer season. The effective control of gastrointestinal parasites is essential to optimizing production in grazing cattle. Strategic worm control programs include pasture rotation management, the identification of susceptible animals (Larsson et al., 2007) and the utilization of nematophagous fungi, notably, Duddingtonia flagrans, which bears the stamp of being the fungus with the most promising field results (Jobim et al., 2008; Santurio et al., 2011). This fungus produce diffuse thick-walled chlamydospores, the stage responsible for its survival during passage through the gut of ruminants following oral administration. Once reaching the feces, the chlamydospores form specialized, three-dimensional networks that trap gastrointestinal parasite larvae (Sanyal, 2000), which recently dislodged from the eggs. Therefore, D. flagrans is commonly isolated from fresh fecal samples of grazing livestock, although it has been successfully detected by the polymerase chain reaction technique from decaying plant parts and soil specimens (Kelly et al., 2008). The larvae, if not killed, develop in the environment to attain the infective stage, which are ingested when the animals feed the grass. The intensive and indiscriminate use of fungicides on agricultural crops, coupled with their low biodegradability, presents a constraint problem for nematophagous fungi, as observed in vitro and substantiated in vivo by Sanyal et al. (2004). Luz et al. (2007) showed that the susceptibility of fungi pathogenic to insects and other invertebrates can be both species-specific and dependent on the individual fungicide compound. Therefore, we report on the in vitro susceptibility of D. flagrans to 14 agricultural fungicides using an agar dilution method and a standardized broth microdilution method for testing filamentous fungi. 2. Materials and methods The D. flagrans isolate, strain ARSEF 5701 (Collection of Entomopathogenic Fungal Cultures), was used. The fungus was cultivated on potato dextrose agar tubes for three weeks at 28 °C. Thereafter, a sterile aqueous solution containing 0.85% saline and 0.05% Triton X-100 was added to the tubes, and the chlamydospores were carefully rinsed from the agar surface by gentle agitation. Microscopic analysis of the suspension showed a large quantity of chlamydospores and few broken hyphae. The suspension was read in a spectrophotometer at 530 nm, and the transmittance was adjusted to 68–70%. The inoculum was diluted in RPMI 1640 broth containing L-glutamine and was buffered to pH 7 with 0.165 morpholinepropanesulfonic acid, yielding a final concentration of 104 chlamydospores/ml. The agricultural fungicides azoxystrobin, carbendazim, cyproconazole, cyprodinil, difenoconazole, fludioxonil, iprodione, mancozeb, metalaxyl, pencycuron, tebuconazole, thiabendazole, triadimenol and trifloxystrobin were obtained from Sigma–Aldrich (St. Louis, MO). The broth microdilution test was performed in accordance with the Clinical and Laboratory Standards Institute (CLSI) document M38-A2 (CLSI, 2008). The fungicides were diluted 1:5 in RPMI 1640 broth, and 100 ll aliquots were sequentially dispensed into the microdilution plates. The maximum residue limit (MRL) for each fungicide, according to Brazilian National Health Vigilance Agency (ANVISA, Brazilian authority), was used as the central dilution. Thereafter, five additional dilutions were performed using the RPMI 1640 medium above and below the MRL,
totaling 10 dilutions for each chemical. Then, 100 ll of inoculum was added to each well. The tests were performed in triplicate, and the MIC readings were visually assessed for the presence (i.e., growth) or absence of hyphae compared with the positive control (hyphae under optimal growth conditions) after 48 h of incubation at 28 °C, as preconized by CLSI document for nondermatophyte fungal species. Potato dextrose agar was used for the agar diffusion method according to Alves and Cury (1992), with minor modifications. After the medium was autoclaved, it was allowed to cool to 50 °C before antifungal compounds diluted in DMSO were added in the volumes necessary to obtain the desired final concentrations. The agar and the compounds were mixed and poured into plates (20 ml/plate). The agar from the control plates only had water and DMSO corresponding to the volume of the fungicide dilution used. For each assay, mycelial plugs (9 mm in diameter) were cut from the margin of actively growing colonies of 7-d-old stock cultures and placed in the center of each test plate (at room temperature) with the mycelium in contact with the agar. Additional dilutions were performed using the RPMI 1640 medium above and below the MRL of the fungicide assayed, totaling six dilutions for each chemical. Each antifungal concentration and control sample was plated in triplicate, and the plates were incubated at at 28 °C for 7 d. The mean colony diameter was found to have a normal distribution (D0 Agostino-Pearson test). When a significant (analysis of variance; P < 0.05) effect of concentration was detected, comparisons were made with the results for the water-DMSO control samples by using Dunnett’s post hoc test. 3. Results and discussion Chlamydospore growth in control wells containing RPMI 1640 broth after 48 h at 28 °C was excelent, allowing for the visual observation of hyphae in the wells. For all test plates in the agar diffusion method, colony growth appeared uniform and radially symmetric. Maximum growth of the D. flagrans fungus in control plates (9 cm) was attained after 7 d. The azoxystrobin, cyproconazole, cyprodinil and tebuconazole showed MICs for D. flagrans below their respective MRLs (Table 1). However, considering the fungicide’s ability to accumulate and adsorb to soil particles and that their intensive application increases the harvest indexes (Ludwig et al., 2010), the content of fungicides in soils is likely to be much greater than the residue limits remaining on the food product samples. Komárek et al. (2010) can be consulted for an extensive review of the environmental and toxicological Table 1 Agricultural fungicides, crops, maximum residue limit (MRL) for each crop and minimum inhibitory concentrations (MICs) against the Duddingtonia flagrans strain ARSEF 5701. Agricultural fungicide
Crop
MRL (mg kg1)
MIC (mg l1)
Tebuconazole Cyproconazole Cyprodinil Difenoconazole Azoxystrobin Iprodione Metalaxyl Pencycuron Trifloxystrobin Fludioxonil Triadimenol Thiabendazole Carbendazim Mancozeb
Soybean Soybean Potato Soybean Soybean Potato Soybean Potato Soybean Soybean Oat Soybean Soybean Wheat
0.1 0.05 0.05 0.05 0.5 0.05 0.05 0.1 0.05 0.05 0.2 0.2 0.5 0.5
0.0125 0.025 0.025 0.1 0.25 0.4 0.4 0.4 0.4 0.8 0.8 1.6 4 >16
The tests were carried out in triplicate; MICs that were lower than the MRL for each fungicide are shown in bold.
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Table 2 Mean ± SD radial growth of Duddingtonia flagrans cultured in potato dextrose agar supplemented with water/DMSO or various concentrations of agricultural fungicides. Agricultural fungicide
Cyproconazole Cyprodinil Difenoconazole Fludioxonil Iprodione Metalaxyl Trifloxystrobin Tebuconazole Pencycuron – Thiabendazole Triadimenol – Azoxystrobin Carbendazim Mancozeb
Concentration (mg/l) Water/DMSO
0.8
0.2
0.1
0.05
0.0125
0.003125
9 9 9 9 9 9 9 9 9
2.4 ± 0.1a 9 3.7 ± 0.3a 2.9 ± 0.1a 9 6 ± 0.5a 4.6 ± 0.3a 2.8 ± 0.3a 5.5 ± 0.5a 1.6 3.6 ± 0.16a 2.4 ± 1.1a 8 3.5 ± 0.4a 0a 3.8 ± 0.5a
3.9 ± 0.4a 9 2.7 ± 0.1a 9 9 5.4 ± 0.1a 5 ± 0.5a 2.9 ± 0.4a 5.8 ± 0.9a 0.4 4.9 ± 0.7a 4.3 ± 0.6a 4 3.1 ± 0.4a 1.2 ± 0.15a 1.5 ± 0.3a
4.2 ± 0.3a 9 4.2 ± 0.5a 9 9 7.6 ± 1.3a 5 ± 0.3a 4.1 ± 0.2a 6.5 ± 0.25a 0.2 5.4 ± 1.8a 5.5 ± 0.3a 2 4.3 ± 0.4a 3 ± 1.3a 2.2 ± 0.4a
4.7 ± 0.4a 9 2.7 ± 0.1a 9 9 8.3 ± 0.8 5.4 ± 0.6a 4.5 ± 0.5a 5.7 ± 0.4a 0.025 4.9 ± 0.7a 5.8 ± 0a 0.5 4.6 ± 1.5a 3.7 ± 0.1a 6.3 ± 0.4a
4.4 ± 0.3a 9 5.3 ± 0.5a 9 9 9 4.5 ± 0.3a 4.6 ± 0.1a 7.1 ± 0.1a 0.0125 3.5 ± 0.1a 6.1 ± 0.2a 0.125 4.6 ± 0.4a 3.8 ± 0.1a 5.6 ± 1.5a
6.8 ± 0.1a 9 6.2 ± 0.8a 9 9 9 6.7 ± 0.3a 4.6 ± 0.1a 6.4 ± 0.1a 0.00625 3.3 ± 0.4a 6.2 ± 0.1a 0.03125 5.4 ± 0.2a 4.2 ± 0.6a 5.7 ± 0.5a
9 9 9 9 9
Isolates were cultured in triplicate; values reported represent mean colony diameter in millimeters. a Within a row, the value differs significantly (P < 0.001; Dunnett’s test) from the value for the water/DMSO control sample. Bold numbers represent maximum residue limits for each fungicide assayed.
aspects of agricultural fungicides. The results for the agar dilution method can be observed in Table 2. D. flagrans proved to be resistant only against cyprodinil and iprodione at the concentrations tested. Resistance was also observed against fludioxonil up to the concentration of 0.2 g/l and to metalaxyl at concentrations below the MRL. The radial growth assay has been used extensively for the in vitro evaluation of fungicide resistance in plant-pathogenic oomycetes (Brown et al., 2008). This technique assesses growth inhibition by the direct measurement of colony diameter following the inoculation of isolates on agar supplemented with various concentrations of antifungal compounds. Differences in susceptibility were observed by comparing both in vitro techniques. For example, D. flagrans proved to be resistant to cyprodinil using the agar diffusion method at concentrations ranging from 0.003125 to 0.8 g/l, although the MIC for this fungicide was of 0.025 g/l. This discrepancy is probably due to the use of 9 mm culture disks as inocula, which supported initial fungal growth. In some soilborne fungi, chlamydospores have been documented to have a role as survival structures (Xiarong and Heitman, 2005; Campos et al., 2009). Conversely, the chlamydospores used in the microdilution method were more directly affected by the fungicides than the hyphae present in the agar diffusion test. Importantly, although chlamydospores are often formed within the agar in cultures grown on agar-solidified medium (Gardner et al., 2000), chlamydospore production is time sensitive (Campos et al., 2009); hence, none were observed in the 7-d-old cultures. The MIC observed for thiabendazole in our study was 1.6 g/l, which is in agreement with previous in vitro studies that show that D. flagrans is completely inhibited at concentrations of 10 g/l but develops well on 1 g/l of the fungicide (Paraud et al., 2004; Luz et al., 2007). According to Faedo et al. (2002) and Campos et al. (2009), a number of factors can potentially affect whether D. flagrans spreads into and establishes itself in the soil. These factors include climate, soil type, soil microorganisms and soil macrofauna. Moreover, these authors reported that this nematode-trapping fungus has minimal growth beyond the fecal environment into the surrounding soil when fungal chlamydospores were fed to sheep or cattle. However, their conclusions were based on the lack of a detectable impact of fungal predation on the nematode population in the surrounding soil. The emergence of adequate, direct techniques (Kelly et al., 2008; Ojeda-Robertos et al., 2008) allowed for work showing that animals reingest chlamydospores, which
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