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Evaluation of potential biocontrol agent for aflatoxin in Argentinean peanuts
International Journal of Food Microbiology 162 (2013) 220–225
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Evaluation of potential biocontrol agent for aflatoxin in Argentinean peanuts M.S. Alaniz Zanon, M.L. Chiotta, G. Giaj-Merlera, G. Barros, S. Chulze ⁎ Departamento de Microbiología e Inmunología, Facultad de Ciencias Exactas Físico Químicas y Naturales, Universidad Nacional de Río Cuarto, Ruta Nacional 36 km 601, Río Cuarto, Córdoba, Argentina
a r t i c l e
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Article history: Received 18 September 2012 Received in revised form 8 January 2013 Accepted 12 January 2013 Available online 31 January 2013 Keywords: Aflatoxin Aspergillus flavus Biological control Peanut Soil
a b s t r a c t Biocontrol by competitive exclusion has been developed as the most promising means of controlling aflatoxins in peanuts. A 2-year study was carried out to determine the efficacy of an Aspergillus flavus strain as biocontrol agent to reduce aflatoxin production in peanuts under field conditions in Argentina. The competitive strain used was a nontoxigenic A. flavus (AFCHG2) naturally occurring in peanut from Córdoba, Argentina. The inoculum was produced through solid-state fermentation on long grain rice and applied at rate of 50 kg inoculum/ha. The incidence of the released strain within the A. flavus communities in soil and peanuts was determined using the shift in the ratio toxigenic:nontoxigenic and VCG analysis. During the 2009/2010 growing season, treatments produced significant reductions in the incidence of toxigenic isolates of A. flavus/Aspergillus parasiticus in soil and peanuts. However, no preharvest aflatoxin contamination was observed. In the 2010/2011 growing season, plants were exposed to late season drought conditions that were optimal for aflatoxin contamination. Significant reductions in aflatoxin levels averaging 71% were detected in treated plots with different inoculation treatments. The results suggest that using the strategy of competitive exclusion A. flavus AFCHG2 can be applied to reduce aflatoxin contamination in Argentinean peanuts. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Aflatoxin contamination of peanuts results from growth in peanut kernels by toxigenic strains of Aspergillus flavus and Aspergillus parasiticus. Soil is the main source of inoculum for A. flavus/A. parasiticus and as peanut fruits develop underground, pods are in direct contact with the soil fungal populations (Horn and Dorner, 1998). Pre-harvest aflatoxin contamination of peanuts is associated with severe late-season drought stress. Contamination can also occur after peanuts are dug if they are not quickly harvested, dried and maintained at safe moisture level; or during storage when improper conditions of moisture and temperature exist (Cole et al., 1995). Lots of peanuts with excessive levels of contamination cannot be used for human consumption and therefore represent great economic losses for the peanut industry (Lamb and Sternitzke, 2001). Aflatoxins are carcinogens and genotoxins that directly alter the DNA structure (Williams et al., 2004). Government regulatory agencies have established very low tolerances for aflatoxins in food, including peanuts and peanut products. The upper limit for aflatoxins in peanuts is 2 ng/g for aflatoxin B1 and 4 ng/g for total aflatoxins (B1 + B2 + G1 + G2) in the European Union and 20 ng/g for total aflatoxins in the United States
⁎ Corresponding author at: Departamento de Microbiología e Inmunología, Facultad de Ciencias Exactas Físico Químicas y Naturales, Universidad Nacional de Río Cuarto, Ruta Nacional Nº 36 km 601 (5800) Río Cuarto, Córdoba, Argentina. Tel.: +54 358 4676429; fax: +54 358 4680280. E-mail address: [email protected] (S. Chulze). 0168-1605/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.01.017
(Wu, 2006) and Argentina according to Mercosur resolution 56/94 (FAO, 2004). Peanut (Arachis hypogaea L.) is an economically important crop in Argentina. Since 2006, exports of peanuts from Argentina have exceeded 400,000 t, making this country the largest peanut exporter in the world. Approximately 65% of these exports go to the European Union (mainly The Netherlands, Germany, UK, France, Greece and Poland) and also to the USA and Canada (Cámara Argentina del Maní, 2012). Aflatoxin control in peanuts relies on several approaches, both preharvest and postharvest, such as good cultural practices, irrigation, use of drought resistant cultivars and postharvest sorting by electronic devices and blanching (Dorner, 2008). However, these procedures are expensive and not always effective. One strategy that has been developed for reducing preharvest aflatoxin contamination of crops is biological control, which is achieved by applying competitive non-toxigenic strains of A. flavus and/or A. parasiticus to the soil of developing crops (Dorner and Cole, 2002). This approach is based on the premise that when high numbers of spores of the nontoxigenic strains are added to soil, they will compete with naturally occurring toxigenic strains for infection sites for growth on peanuts and for essential nutrients. Also, it has been demonstrated that soil inoculation with nontoxigenic strains has a carryover effect and may protects peanuts from contamination during storage (Dorner and Cole, 2002). Biological control using competitive exclusion of toxigenic strains by non-aflatoxigenic strains has been demonstrated under field conditions
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in peanut from USA and Australia (Dorner et al., 1992, 1998; Dorner and Cole, 2002; Horn et al., 2000; Horn and Dorner, 2009; Pitt and Hocking, 2006). In Aspergillus species the formation of a stable heterokaryon following hyphal anastomosis is controlled by a series of het loci (Leslie, 1993). Two individuals that have the same alleles at all het loci belong to the same vegetative compatibility group (VCG). Vegetative compatibility can be used to estimate genetic diversity, may limit the potential for heterokaryosis and asexual gene flow (Bayman and Cotty, 1991) and can be used for monitoring the incidence of biocontrol strains in field assays (Cotty, 1994). Aspergillus species from section Flavi have been isolated from soil and peanuts cultivated in the main peanut production area in Argentina and characterized in relation to their toxigenic profile and genetic diversity using VCG strategy and molecular markers (Barros et al., 2003, 2005, 2006a,b, 2007). Based on these studies an A. flavus strain was selected as potential biocontrol agent. Most studies on aflatoxin control in Argentinean peanuts have focused on postharvest strategies such as the use of synthetic and natural substances (Nesci et al., 2011; Passone et al., 2008, 2009). However, at present there are no data on biocontrol as preharvest strategy to reduce the entry of aflatoxins to the peanut food chain. The aim of this work was to evaluate the efficacy of a native non-aflatoxigenic A. flavus strain AFCHG2 to reduce aflatoxin production in peanuts under field conditions in Argentina. 2. Material and methods
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The planting dates were November 27, 2009 and October 12, 2010. The inoculum was added to fields with machines used to dispense fertiliser at rate of 50 kg inoculum/ha during two periods: at planting (Fig. 1A) and 50 days after planting (Fig. 1B). During the 2010/ 2011 growing season, in order to promote conducive conditions for aflatoxin production in peanuts, subplots of each treatment were covered during the last month prior to digging to impose drought stress. The drought condition was achieved by covering the plots with removable shelters (7 m × 6 m) constructed with a piece of polypropylene thermal long-term (LTD) of 150 μm thick. The shelters were mounted on structures built with polypropylene pipes of 1/2 inch in diameter (Fig. 1C). The control and treatment subplots were monitored with temperature sensor devices in order to register soil temperature at 5 cm depth (Fig. 1D). Another change made during the second season was the analysis of the different ways to inoculate the nontoxigenic A. flavus strain. Subplots comprised controls and treatments as follows: (1) uninoculated control without drought stress; (2) uninoculated control with drought stress during last month prior to digging; (3) inoculated at planting time without drought stress; (4) inoculated at planting time with drought stress during last month prior to digging; (5) inoculated 50 days after planting without drought stress; (6) inoculated 50 days after planting with drought stress during last month prior to digging; (7) inoculated at planting time and 50 days after planting without drought stress; and (8) inoculated at planting time and 50 days after planting with drought stress during last month prior to digging. At harvest, all treatments were dug and shelled by hand.
2.1. Strain selection 2.4. Soil fungal analysis The competitive strain used was an A. flavus strain known as AFCHG2, a naturally occurring isolate obtained from peanuts harvested in Córdoba, Argentina. This strain was characterized in previous studies and shown to produce neither aflatoxins nor cyclopiazonic acid, produce large sclerotia > 400 μm (L morphotype) and belongs to a vegetative compatibility group that includes only non-toxigenic strains (Barros et al., 2005, 2006a,b). The isolate was characterized morphologically using the methodology of Klich (2002) and maintained in glycerol 20% at −80 °C. 2.2. Inoculum preparation The A. flavus AFCHG2 inoculum was produced by solid-state fermentation on autoclaved long grain rice. The substrate (500 g) was conditioned in plastic bags; distilled water was added to attain 35– 40% moisture content in the rice. The bags were inoculated with 1 ml of a conidial suspension (10 7/ml) from malt extract agar slant and incubated at 30 °C for 4 days, and the bags were hand shaken daily to avoid clump production. At the end of incubation period, the substrate was dried in a forced air draft oven at 40 °C over night. The viable count (cfu/g) of A. flavus in the substrate was determined by homogenizing 10 g in 90 ml of peptone water 0.1% (wt/v). This mixture was then shaken and diluted to final concentrations of 10 −2 and 10−3. From each dilution, 0.1 ml was spread in triplicate on Dichloran Rose Bengal Chloramphenicol (DRBC) modified with 3% NaCl (Horn and Dorner, 1998). The Petri dishes were incubated in darkness for 5–7 days at 30 °C. 2.3. Field assays The field assays were done in commercial fields with previous history of peanut cultivation during the 2009/2010 and 2010/2011 growing seasons in Las Acequias, located within the peanut-growing region of Córdoba, Argentina. The experiments were established as split plot design; each plot consisted of 54 m × 7 m divided into three 18 m × 7 m subplots, with a buffer area among plots of ten rows. The peanut cultivar (Runner type) was planted in rows at 70 cm distance.
2.4.1. Soil sampling Ten soil samples were taken in two diagonal transects extending from opposing corners in each subplot immediately after planting and during maturation of the pods prior to digging to determine the A. flavus/A. parasiticus populations. Each soil sample (approximately 100 g) was a pool from 5 subsamples taken with a trowel from the top 5 cm of soil where peanuts would be or were forming. Sub-samples of each sample were combined in a paper bag and air-dried for 1–2 days at 25–30 °C. Samples were thoroughly mixed and passed through a testing sieve (2 mm mesh size). 2.4.2. Fungal isolation and identification From each soil sample 10 g was diluted with 90 ml of peptone water 0.1% (w/v). This mixture was shaken for 20 min and decimally diluted. A 0.1 ml aliquot of each dilution per sample was spread on the surface of two solid media: Dichloran Rose Bengal Chloramphenicol (DRBC) modified with 3% NaCl and Dichloran Glycerol 18% (DG18). The plates were incubated in darkness for 5–7 days at 30 °C. The results were expressed as colony forming units per gramme (cfu/g) of soil. Fungal colonies that resembled Aspergillus section Flavi were subcultured on malt extract agar medium (MEA) for further identification according to Klich (2002). 2.4.3. Toxigenic profile of A. flavus/A. parasiticus isolates A. flavus/A. parasiticus isolates were inoculated on malt extract agar slants at 28 °C for 7 days. At the end of the incubation period, 5 ml of spore suspension solution (sodium lauryl sulphate; 0.01% w/v) was added to the slants and the spores were harvested by vigorous agitation. The spore concentration was measured with a Neubauer chamber and adjusted to 10 5 spores/ml. This conidia concentration from each isolate (30 isolates × subplot) was used to inoculate 4-ml vials containing 1 ml of medium containing 150 g of sucrose, 20 g of yeast extract, 10 g of soytone, and 1 l of distilled water; the pH of the medium was adjusted to 5.9 with HCl. The cultures were incubated at 30 °C for 7 days in the dark (Horn and Dorner, 1999). Preliminary analysis of the extracts to screen for
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Fig. 1. Inoculum application in machines used to dispense fertiliser at planting (A) and 50 days after planting (B). Plot covered with a shelter that allowed drought conditions (C). Temperature sensor device used to register soil temperature at 5 cm deep (D).
aflatoxin production was carried out using thin-layer chromatography according to the methodology proposed by Geisen (1996). Aflatoxins were quantified by HPLC according to Horn et al. (1996).
incubated at 25 °C for 7 days. The incidence of toxigenic isolates of A. flavus/A. parasiticus in peanuts was determined by testing all the isolates for toxigenicity as described above for soil samples.
2.4.4. Vegetative compatibility group (VCG) analysis Nitrate non-utilizing mutants were obtained on plates of Czapek-Dox medium (Cz) containing 25 g/L potassium chlorate (Bayman and Cotty, 1991) with unadjusted pH. Five Cz–chlorate plates were inoculated with a conidial suspension of A. flavus in soft agar at four points. Cultures were incubated at 30 °C and the margins of the colonies with restricted growth were examined periodically for fast-growing sectors consisting of sparse mycelium. Hyphal tips from sectors arising from different colonies were transferred to Cz plates (nitrate medium) to confirm their inability to utilize nitrate. The nit mutant phenotypes were determined by growing the strains on several media in which the NaNO3 was replaced as the sole nitrogen source of either 700 mg/L NaNO2 (nitrite medium), 900 mg/L ammonium tartrate (ammonium medium) or 100 mg/L hypoxanthine (hypoxanthine medium) according to Papa (1986). Assignment of isolates to the VCG of applied strain AFCHG2 was made on the basis of complementation test between nia D − (deficient in the structural gene for nitrate reductase) of the field isolate to be assigned and a cnx (deficient in a molybdenum cofactor) tester mutant of AFCHG2.
2.5.2. Aflatoxin analysis The aflatoxin analysis was performed using the method of Trucksess et al. (1994). Aflatoxins were quantified by injecting 50 μL of extract from each vial into an HPLC system consisting of a Hewlett Packard model 1100 pump (Palo Alto, CA) connected to a Hewlett Packard model 1046A programmable fluorescence detector and a data module Hewlett Packard Kayak XA (HP ChemStation Rev. A.06.01). Chromatographic separations were performed on a stainless steel, C18 reversed-phase column (150 mm × 4.6 mm i.d., 5 μm particle size; Luna-Phenomenex, Torrance, CA, USA) connected to a precolumn Security Guard (20 mm × 4.6 mm i.d., 5 μm particle size, Phenomenex). The mobile phase was water:methanol:acetonitrile (4:1:1, v/v/v) at a flow rate of 1.5 ml/min and the limit of detection was 1 ng/ml of B1 and G1 and 0.8 ng/g of B2 and G2. Pure aflatoxin solutions were used as external standard (Sigma-Aldrich, St. Louis, MO, USA).
2.5. Peanut analysis 2.5.1. Mycobiota analysis from peanut samples From each subplot approximately 3 kg of kernels was used. This sample was mixed thoroughly and 100 kernels (2 replicates) were selected for fungal infection determination. The remaining sample was ground to obtain a subsample of 25 g (3 replicates) for aflatoxin analysis. Peanut kernels from each subplot were surface disinfected for 1 min in 1% sodium hypochlorite solution, rinsed three times in sterile distilled water and transferred to Petri dishes containing Dichloran Rose Bengal Chloramphenicol agar (DRBC) modified with 3% NaCl and Dichloran Glycerol 18% (DG18) (Pitt and Hocking, 1997). Plates were
2.6. Statistical analysis Data of fungal populations were log-transformed prior to analysis of variance (ANOVA). Means separation and comparison were made by Fisher's least significant difference (LSD) test at a probability level of p b 0.05. To compare the different treatments, aflatoxin concentrations were subjected to nonparametric Kruskal–Wallis test followed by Dunn's nonparametric multiple comparison test. The statistical analyses were performed using SigmaStat for windows version 2.03 (SPSS Inc., San Jose, CA). 3. Results and discussion 3.1. Field trial during 2009/2010 growing season Densities and toxigenicity of A. flavus and A. parasiticus soil populations were determined before planting in November and during
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pod maturation in February. The inoculum level and the incidence of toxigenic isolates of native A. flavus and A. parasiticus in soil at planting time were similar among plots and A. flavus was the dominant species from section Flavi, showing an isolation percentage nearly to 90% (Table 1). The remaining species identified were A. parasiticus (9%) and the nonaflatoxigenic species A. caelatus (1%). Data on incidence of the native population in the present study are consistent with those found by Barros et al. (2003, 2005) in different areas within the peanut-growing region of Córdoba. The soil samples at pod maturation showed a significant increase in the density of A. flavus between treated and control plots, while the viable count of A. parasiticus was not modified. Similar to planting time, A. flavus was the dominant species from section Flavi isolated from soil in the treated plots, but showing an isolation percentage slightly higher (>95%). As nontoxigenic A. flavus AFCHG2 colonies cannot be differentiated from wild-type strains, the efficacy of the biocontrol agent was monitored using the shift of the toxigenic/ nontoxigenic ratio in the treated plots in relation to control plots. At planting time, 72.5% and 94% of A. flavus and A. parasiticus isolates, respectively, were toxigenic. The analysis of A. flavus isolates recovered from soil at pod maturation showed that only 27% of isolates tested were toxigenic in treated plots compared with 80% in control plots. The nontoxigenic A. flavus AFCHG2 used in the present study was characterized previously as belong to vegetative compatibility group (VCG) which does not include toxigenic isolates (Barros et al., 2006a). Thus, VCG analysis was used as a second marker to monitor the presence of this biocontrol strain among the soil A. flavus population recovered at the pod maturation stage. Prior to application of the biocontrol agent and at pod maturation in control plots, only 3 of 90 soil isolates were vegetatively compatible with the AFCHG2. By contrast, 180 out of 256 A. flavus isolates recovered from treated soil at pod maturation belonged to the same VCG that the nontoxigenic A. flavus strain applied (Table 1). At harvest time, the percentages of peanuts infected by A. flavus and A. parasiticus ranged from 2 to 18% and were not significantly different (p > 0.05) between treated and control plots (Table 1). This result indicates that the rate of inoculum applied to soil had no effect on subsequent infection of peanut kernels. Similar results were obtained by Dorner and Cole (2002) and Dorner et al. (2003) when they used nontoxigenic A. flavus/A. parasiticus inoculum in peanut fields in USA. Among the Aspergillus section Flavi species identified, A. flavus was the most frequently recovered from peanuts, with isolation frequencies of 90 and 98% in control and treated plots, respectively. This result agrees with Horn et al. (1994) and Dorner and Horn (2007) who found that A. flavus was by far the predominant colonizer of peanuts, even in peanuts grown in soil treated with high levels of nontoxigenic A. parasiticus. This indicated that A. flavus was the more aggressive species and was responsible for
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most of the aflatoxin contamination of peanuts. Similar to those data found from soil samples at pod maturation, the toxigenic/ nontoxigenic ratio was nearly 3:7 in A. flavus strains isolated from peanuts in treated plots. With respect to the VCG analysis, only one of 30 peanut isolates recovered from the control plots was vegetatively compatible with the AFCHG2. A significant increase in the percentage of the VCG applied was observed in the peanuts recovered from the treated plots, where 16 of 30 isolates belonged to the atoxigenic VCG. Results obtained in both toxigenicity and VCG analyses demonstrated that the application of the biocontrol agent was efficient, showing competence within A. flavus and A. parasiticus community associated with the crop and resident in soil. This behaviour reduced both the average aflatoxin-producing potential of the native community and the risk of aflatoxin contamination. However, 2009/2010 growing season was characterized by abundant rainfall during the last month of crop development and the peanut samples showed no preharvest aflatoxin contamination. 3.2. Field trial during 2010/2011 growing season The density of native A. flavus/A. parasiticus soil population was similar among plots at pre-planting time, with viable count ranged from 500 to 1400 cfu/g with a mean of 815 cfu/g. Two aflatoxigenic species belonging to Aspergillus section Flavi were identified in the soil samples, A. flavus and A. parasiticus. However, A. flavus showed isolation frequencies higher than A. parasiticus in all soil samples evaluated accounting for nearly 85% of the isolates. In relation to the incidence of toxigenic native isolates at pre-planting time, a total of 85% of A. flavus isolates were toxigenic whereas all A. parasiticus isolates were toxigenic. The low percentage of atoxigenic strains in the native soil population found in this study is similar to those found among Aspergillus section Flavi populations isolated from agricultural soils in a range of geographic regions of the world (Barros et al., 2005; Horn and Dorner, 1999; Sweany et al., 2011). At pod maturation, densities of total A. flavus + A. parasiticus soil population were significantly higher in control and treated plots under drought stress compared with control and treated plots without drought stress. In all situations, A. flavus was the predominant species representing more than 90% of the isolates. For this reason, Table 2 shows only data on toxigenicity and percentages of isolates assigned to the applied VCG corresponding to A. flavus species. Regarding the toxigenicity of the A. flavus soil populations, significant displacement of toxigenic strains occurred in plots treated with nontoxigenic A. flavus AFCHG2 with different inoculation treatments in plots either under normal conditions or exposed to drought stress. Results obtained from the VCG analysis showed that only 4 of 60 strains soil isolates recovered from the control plots with and
Table 1 Density (cfu/g), toxigenic isolates (%) and isolates in applied VCG (%) of A. flavus and A. parasiticus in soil and peanuts and percentage of infection (%) and aflatoxin (μg/kg) in peanuts during 2009/2010 season. Treatment
Control A. flavus A. parasiticus Treated A. flavus A. parasiticus a b c d e f
Soil at planting
Soil at pod maturation
CFU/g
CFU/g
256 ac 230 a 23 b 215 ac 193 a 22 b
Toxigenic isolates (%)
75 a 98 a 70 a 92 a
225 abc 199 abe 26 a 647 bc 621 b 26 a
Peanuts
Toxigenic isolates (%)
Isolates in applied VCG (%)a
80 a 100 a
3b Ndf
27 b 98 a
70 a Ndf
Infection (%) 10 a 9a 1b 12 a 11 a 1b
Toxigenic isolates (%)b
Isolates in applied VCG (%)a
75 a 100 a
3b Nd
28 b 100 a
53a Nd
Percent isolates assigned to the applied vegetative compatibility group (VCG) to which AFCHG2 belongs on the basis of auxotroph complementation. For determination of toxigenic isolates (%) and isolates in applied VCG (%) a total of 401 isolates were evaluated. CFU/g: total count of Aspergillus section Flavi. ND = Not Detected b 1 μg/kg. Within a column, values not sharing a common letter are significantly different (p b 0.05). Nd = Not determined.
Mean aflatoxin NDd – – NDd – –
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Table 2 Density (cfu/g), toxigenic isolates (%) and isolates in applied VCG (%) of A. flavus in soil and peanuts and percentage of infection (%) and mean aflatoxin (μg/kg) in peanuts during 2010/2011 season. Treatment
Soil at pod maturation CFU/ga
Peanuts
Toxigenic isolates (%)
Isolates in applied VCG (%)b
Infection (%)
Toxigenic isolates (%)c
Isolates in applied VCG (%)b
Mean aflatoxind
Without drought stress Control Planting inoc. 50 days after planting inoc. Double inoc.
550 a 809 ab 1044 b 768 ab
68.6 38.2 32.3 45.2
a b b ab
3b 50 a 66 a 53 a
6.5 26.5 23.5 47.5
a abf ab bc
70 60 49 50
a ab ab ab
9b 35 a 30 ab 25 ab
N.De N.D N.D N.D
With drought stress Control Planting inoc. 50 days after planting inoc. Double inoc.
1965 cd 1260 bc 2480 c 2730 c
71.8 23.4 54.3 35.7
a b ab b
9b 56 a 50 a 50 a
57.0 75.7 78.5 99.5
bc cd cd d
67 57 50 51
a ab ab ab
5b 40 a 45 a 40 a
101.2 a 36.2 ab 25.4 b 28.1 b
a b c d e f
CFU/g: total count of Aspergillus section Flavi. Percent isolates assigned to the applied vegetative compatibility group (VCG) to which AFCHG2 belongs on the basis of auxotroph complementation. For determination of toxigenic isolates (%) and isolates in applied VCG (%) a total of 450 isolates were evaluated. Values are averages of three replicates. ND = Not Detected b 1 μg/kg. Within a column, values not sharing a common letter are significantly different (p b 0.05).
without drought stress were vegetatively compatible with the AFCHG2. After the application of the biocontrol agent, a higher incidence of isolates belong to the VCG applied was observed in all treatments both under normal conditions and exposed to drought stress (Table 2). At harvest the colonization of peanuts exhibited significant differences between control and treated plots in both conditions with and without drought stress; however a higher percentage of peanuts were infected in all inoculation treatments under drought condition, with A. flavus being responsible for this colonization. This result can be explained due to the capacity of A. flavus to infect peanuts under environmentally conducive conditions (Horn, 2003). Analysis of toxigenicity of the A. flavus isolates recovered from peanuts demonstrated that the rate of displacement of toxigenic strains was lower compare to those found in soil, with a ratio nontoxigenic to toxigenic near to 1:1. Similar results were obtained by Dorner and Horn (2007) who found relatively high incidence of toxigenic isolates in treated peanuts compared with the relatively low incidence in treated soil. These authors suggest that toxigenic isolates of A. flavus are more aggressive colonizers of peanuts than nontoxigenic strains. Analysis of aflatoxin contamination in peanuts showed no preharvest contamination in plots without drought stress. By comparison, the mean soil temperatures during the pod maturation period were close or above to 30 °C during several days in plots with drought stress, demonstrating an effective conducive condition for aflatoxin production. Significant reductions in aflatoxin levels averaging 71% were detected with all three treatments (Table 2). Although a low displacement of toxigenic isolates was observed in treated peanuts compare with treated soil, a significant increase in the percentage of the isolates in the VCG applied was observed in the treated peanuts (27 of 59 isolates) compared with the control (1 of 40 isolates). This result could explain the lower aflatoxin contamination observed in treated peanuts under conducive conditions. No statistically significant differences among inoculation 50 days after planting and plots inoculated twice were observed. According to this result, a single application preferably 50 days after planting will be adequate for future assays. The percentage reduction in aflatoxin levels obtained in this study is lower than the average reductions of 77% to 98% obtained in peanuts from USA and Australia (Dorner and Cole, 2002; Dorner and Lamb, 2006; Dorner and Horn, 2007; Pitt and Hocking, 2006). However, reductions around 70% obtained in the present study will be useful where there are conducive conditions for aflatoxin contamination, ensuring that levels are lower than those imposed by the regulations of the European Union, the major importer of Argentinean peanuts.
Regarding the inoculum application rate, the dosage used in this study was higher than other rates employed in peanuts from the USA (Dorner et al., 1998; Dorner and Cole, 2002; Dorner and Horn, 2007) but lower than other application rates evaluated in peanuts from Australia (Pitt and Hocking, 2006). More economical application rates should be tested in the future to determine the lowest effective rate. The present study demonstrated that nontoxigenic A. flavus AFCHG2 has the potential to become a management tool for the biocontrol of aflatoxin contamination of peanuts in Argentina.
Acknowledgements We are grateful to Aceitera General Deheza and Cotagro for kindly providing the commercial fields used in this study. The authors thank Ariel Bessone and Sebastian Rosso for technical assistance. This study was supported by grants from EC KBBE-2007-222690-2 MYCORED and Secretaría de Ciencia y Técnica, Universidad Nacional de Río Cuarto (SECyT-UNRC 2009–2010). Alaniz Zanon, M.S., Chiotta M.L. and Giaj-Merlera G. are fellows of CONICET and Barros, G. and Chulze, S. are members of the Research Career of CONICET.
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Horn, B.W., Greene, R.L., Dorner, J.W., 2000. Inhibition of aflatoxin B1 production by Aspergillus parasiticus using nonaflatoxigenic strains: role of vegetative compatibility. Biological Control 17, 147–154. Klich, M.A., 2002. Identification of Common Aspergillus Species. Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands. Lamb, M.C., Sternitzke, D.A., 2001. Cost of aflatoxin to the farmer, buying point, and sheller segments of the southeast United States peanut industry. Peanut Science 28, 58–63. Leslie, J.F., 1993. Fungal vegetative compatibility. Annual Review of Phytopathology 31, 127–150. Nesci, A.V., Montemarani, A., Passone, M.A., Etcheverry, M., 2011. Insecticidal activity of synthetic antioxidants, natural phytochemicals, and essential oils against an Aspergillus section Flavi vector (Oryzaephilus surinamensis L.) in microcosm. Journal of Pest Science 84, 107–115. Papa, K.E., 1986. Heterokaryon incompatibility in Aspergillus flavus. Mycologia 78, 98–101. Passone, M.A., Bluma, R., Nesci, A., Resnik, S., Etcheverry, M., 2008. Impact of food grade antioxidants on peanut pods and seeds mycoflora in storage system from Córdoba, Argentina. Journal of Food Science 28, 550–566. Passone, M., Doprado, M., Etcheverry, M., 2009. Food-grade antioxidants for control of Aspergillus section Flavi and interrelated mycoflora of stored peanuts with different water activities. World Mycotoxin Journal 9, 399–407. Pitt, J.W., Hocking, A.D., 1997. Fungi and Food Spoilage. Blackie Academic & Professional, London, United Kingdom. Pitt, J.I., Hocking, A.D., 2006. Mycotoxins in Australia: biocontrol of aflatoxin in peanuts. Mycopathologia 162, 233–243. Sweany, R.R., Damann, K.E., Kaller, M.D., 2011. Comparison of soil and corn kernel Aspergillus flavus populations: evidence for niche specialization. Phytopathology 101, 952–959. Trucksess, M.W., Stack, M.E., Nesheim, S., Albert, R.H., Romer, T.R., 1994. Multifunctional column coupled with liquid chromatography for determination of aflatoxins B1, B2, G1 and G2 in corn, almonds, Brazil nuts, peanuts, and pistachio nuts: collaborative study. Journal of AOAC International 77, 1512–1521. Williams, J.H., Phillips, T.D., Jolly, P.E., Stiles, J.K., Jolly, C.M., Aggarwal, D., 2004. Human aflatoxicosis in developing countries: a review of toxicology, exposure, potential health consequences, and interventions. American Journal of Clinical Nutrition 80, 1106–1122. Wu, F., 2006. Economic impact of fumonisin and aflatoxin regulations on global corn and peanut markets. In: Barug, D., Bhatnagar, D., van Egmond, H.P., van der Kamp, J.W., van Osenbruggen, W.A., Visconti, A. (Eds.), The Mycotoxin Factbook. Food and Feed Topics. Wageningen Academic Publishers, Wageningen, pp. 83–91.
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Evaluation of potential biocontrol agent for aflatoxin in Argentinean peanuts M.S. Alaniz Zanon, M.L. Chiotta, G. Giaj-Merlera, G. Barros, S. Chulze ⁎ Departamento de Microbiología e Inmunología, Facultad de Ciencias Exactas Físico Químicas y Naturales, Universidad Nacional de Río Cuarto, Ruta Nacional 36 km 601, Río Cuarto, Córdoba, Argentina
a r t i c l e
i n f o
Article history: Received 18 September 2012 Received in revised form 8 January 2013 Accepted 12 January 2013 Available online 31 January 2013 Keywords: Aflatoxin Aspergillus flavus Biological control Peanut Soil
a b s t r a c t Biocontrol by competitive exclusion has been developed as the most promising means of controlling aflatoxins in peanuts. A 2-year study was carried out to determine the efficacy of an Aspergillus flavus strain as biocontrol agent to reduce aflatoxin production in peanuts under field conditions in Argentina. The competitive strain used was a nontoxigenic A. flavus (AFCHG2) naturally occurring in peanut from Córdoba, Argentina. The inoculum was produced through solid-state fermentation on long grain rice and applied at rate of 50 kg inoculum/ha. The incidence of the released strain within the A. flavus communities in soil and peanuts was determined using the shift in the ratio toxigenic:nontoxigenic and VCG analysis. During the 2009/2010 growing season, treatments produced significant reductions in the incidence of toxigenic isolates of A. flavus/Aspergillus parasiticus in soil and peanuts. However, no preharvest aflatoxin contamination was observed. In the 2010/2011 growing season, plants were exposed to late season drought conditions that were optimal for aflatoxin contamination. Significant reductions in aflatoxin levels averaging 71% were detected in treated plots with different inoculation treatments. The results suggest that using the strategy of competitive exclusion A. flavus AFCHG2 can be applied to reduce aflatoxin contamination in Argentinean peanuts. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Aflatoxin contamination of peanuts results from growth in peanut kernels by toxigenic strains of Aspergillus flavus and Aspergillus parasiticus. Soil is the main source of inoculum for A. flavus/A. parasiticus and as peanut fruits develop underground, pods are in direct contact with the soil fungal populations (Horn and Dorner, 1998). Pre-harvest aflatoxin contamination of peanuts is associated with severe late-season drought stress. Contamination can also occur after peanuts are dug if they are not quickly harvested, dried and maintained at safe moisture level; or during storage when improper conditions of moisture and temperature exist (Cole et al., 1995). Lots of peanuts with excessive levels of contamination cannot be used for human consumption and therefore represent great economic losses for the peanut industry (Lamb and Sternitzke, 2001). Aflatoxins are carcinogens and genotoxins that directly alter the DNA structure (Williams et al., 2004). Government regulatory agencies have established very low tolerances for aflatoxins in food, including peanuts and peanut products. The upper limit for aflatoxins in peanuts is 2 ng/g for aflatoxin B1 and 4 ng/g for total aflatoxins (B1 + B2 + G1 + G2) in the European Union and 20 ng/g for total aflatoxins in the United States
⁎ Corresponding author at: Departamento de Microbiología e Inmunología, Facultad de Ciencias Exactas Físico Químicas y Naturales, Universidad Nacional de Río Cuarto, Ruta Nacional Nº 36 km 601 (5800) Río Cuarto, Córdoba, Argentina. Tel.: +54 358 4676429; fax: +54 358 4680280. E-mail address: [email protected] (S. Chulze). 0168-1605/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.01.017
(Wu, 2006) and Argentina according to Mercosur resolution 56/94 (FAO, 2004). Peanut (Arachis hypogaea L.) is an economically important crop in Argentina. Since 2006, exports of peanuts from Argentina have exceeded 400,000 t, making this country the largest peanut exporter in the world. Approximately 65% of these exports go to the European Union (mainly The Netherlands, Germany, UK, France, Greece and Poland) and also to the USA and Canada (Cámara Argentina del Maní, 2012). Aflatoxin control in peanuts relies on several approaches, both preharvest and postharvest, such as good cultural practices, irrigation, use of drought resistant cultivars and postharvest sorting by electronic devices and blanching (Dorner, 2008). However, these procedures are expensive and not always effective. One strategy that has been developed for reducing preharvest aflatoxin contamination of crops is biological control, which is achieved by applying competitive non-toxigenic strains of A. flavus and/or A. parasiticus to the soil of developing crops (Dorner and Cole, 2002). This approach is based on the premise that when high numbers of spores of the nontoxigenic strains are added to soil, they will compete with naturally occurring toxigenic strains for infection sites for growth on peanuts and for essential nutrients. Also, it has been demonstrated that soil inoculation with nontoxigenic strains has a carryover effect and may protects peanuts from contamination during storage (Dorner and Cole, 2002). Biological control using competitive exclusion of toxigenic strains by non-aflatoxigenic strains has been demonstrated under field conditions
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in peanut from USA and Australia (Dorner et al., 1992, 1998; Dorner and Cole, 2002; Horn et al., 2000; Horn and Dorner, 2009; Pitt and Hocking, 2006). In Aspergillus species the formation of a stable heterokaryon following hyphal anastomosis is controlled by a series of het loci (Leslie, 1993). Two individuals that have the same alleles at all het loci belong to the same vegetative compatibility group (VCG). Vegetative compatibility can be used to estimate genetic diversity, may limit the potential for heterokaryosis and asexual gene flow (Bayman and Cotty, 1991) and can be used for monitoring the incidence of biocontrol strains in field assays (Cotty, 1994). Aspergillus species from section Flavi have been isolated from soil and peanuts cultivated in the main peanut production area in Argentina and characterized in relation to their toxigenic profile and genetic diversity using VCG strategy and molecular markers (Barros et al., 2003, 2005, 2006a,b, 2007). Based on these studies an A. flavus strain was selected as potential biocontrol agent. Most studies on aflatoxin control in Argentinean peanuts have focused on postharvest strategies such as the use of synthetic and natural substances (Nesci et al., 2011; Passone et al., 2008, 2009). However, at present there are no data on biocontrol as preharvest strategy to reduce the entry of aflatoxins to the peanut food chain. The aim of this work was to evaluate the efficacy of a native non-aflatoxigenic A. flavus strain AFCHG2 to reduce aflatoxin production in peanuts under field conditions in Argentina. 2. Material and methods
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The planting dates were November 27, 2009 and October 12, 2010. The inoculum was added to fields with machines used to dispense fertiliser at rate of 50 kg inoculum/ha during two periods: at planting (Fig. 1A) and 50 days after planting (Fig. 1B). During the 2010/ 2011 growing season, in order to promote conducive conditions for aflatoxin production in peanuts, subplots of each treatment were covered during the last month prior to digging to impose drought stress. The drought condition was achieved by covering the plots with removable shelters (7 m × 6 m) constructed with a piece of polypropylene thermal long-term (LTD) of 150 μm thick. The shelters were mounted on structures built with polypropylene pipes of 1/2 inch in diameter (Fig. 1C). The control and treatment subplots were monitored with temperature sensor devices in order to register soil temperature at 5 cm depth (Fig. 1D). Another change made during the second season was the analysis of the different ways to inoculate the nontoxigenic A. flavus strain. Subplots comprised controls and treatments as follows: (1) uninoculated control without drought stress; (2) uninoculated control with drought stress during last month prior to digging; (3) inoculated at planting time without drought stress; (4) inoculated at planting time with drought stress during last month prior to digging; (5) inoculated 50 days after planting without drought stress; (6) inoculated 50 days after planting with drought stress during last month prior to digging; (7) inoculated at planting time and 50 days after planting without drought stress; and (8) inoculated at planting time and 50 days after planting with drought stress during last month prior to digging. At harvest, all treatments were dug and shelled by hand.
2.1. Strain selection 2.4. Soil fungal analysis The competitive strain used was an A. flavus strain known as AFCHG2, a naturally occurring isolate obtained from peanuts harvested in Córdoba, Argentina. This strain was characterized in previous studies and shown to produce neither aflatoxins nor cyclopiazonic acid, produce large sclerotia > 400 μm (L morphotype) and belongs to a vegetative compatibility group that includes only non-toxigenic strains (Barros et al., 2005, 2006a,b). The isolate was characterized morphologically using the methodology of Klich (2002) and maintained in glycerol 20% at −80 °C. 2.2. Inoculum preparation The A. flavus AFCHG2 inoculum was produced by solid-state fermentation on autoclaved long grain rice. The substrate (500 g) was conditioned in plastic bags; distilled water was added to attain 35– 40% moisture content in the rice. The bags were inoculated with 1 ml of a conidial suspension (10 7/ml) from malt extract agar slant and incubated at 30 °C for 4 days, and the bags were hand shaken daily to avoid clump production. At the end of incubation period, the substrate was dried in a forced air draft oven at 40 °C over night. The viable count (cfu/g) of A. flavus in the substrate was determined by homogenizing 10 g in 90 ml of peptone water 0.1% (wt/v). This mixture was then shaken and diluted to final concentrations of 10 −2 and 10−3. From each dilution, 0.1 ml was spread in triplicate on Dichloran Rose Bengal Chloramphenicol (DRBC) modified with 3% NaCl (Horn and Dorner, 1998). The Petri dishes were incubated in darkness for 5–7 days at 30 °C. 2.3. Field assays The field assays were done in commercial fields with previous history of peanut cultivation during the 2009/2010 and 2010/2011 growing seasons in Las Acequias, located within the peanut-growing region of Córdoba, Argentina. The experiments were established as split plot design; each plot consisted of 54 m × 7 m divided into three 18 m × 7 m subplots, with a buffer area among plots of ten rows. The peanut cultivar (Runner type) was planted in rows at 70 cm distance.
2.4.1. Soil sampling Ten soil samples were taken in two diagonal transects extending from opposing corners in each subplot immediately after planting and during maturation of the pods prior to digging to determine the A. flavus/A. parasiticus populations. Each soil sample (approximately 100 g) was a pool from 5 subsamples taken with a trowel from the top 5 cm of soil where peanuts would be or were forming. Sub-samples of each sample were combined in a paper bag and air-dried for 1–2 days at 25–30 °C. Samples were thoroughly mixed and passed through a testing sieve (2 mm mesh size). 2.4.2. Fungal isolation and identification From each soil sample 10 g was diluted with 90 ml of peptone water 0.1% (w/v). This mixture was shaken for 20 min and decimally diluted. A 0.1 ml aliquot of each dilution per sample was spread on the surface of two solid media: Dichloran Rose Bengal Chloramphenicol (DRBC) modified with 3% NaCl and Dichloran Glycerol 18% (DG18). The plates were incubated in darkness for 5–7 days at 30 °C. The results were expressed as colony forming units per gramme (cfu/g) of soil. Fungal colonies that resembled Aspergillus section Flavi were subcultured on malt extract agar medium (MEA) for further identification according to Klich (2002). 2.4.3. Toxigenic profile of A. flavus/A. parasiticus isolates A. flavus/A. parasiticus isolates were inoculated on malt extract agar slants at 28 °C for 7 days. At the end of the incubation period, 5 ml of spore suspension solution (sodium lauryl sulphate; 0.01% w/v) was added to the slants and the spores were harvested by vigorous agitation. The spore concentration was measured with a Neubauer chamber and adjusted to 10 5 spores/ml. This conidia concentration from each isolate (30 isolates × subplot) was used to inoculate 4-ml vials containing 1 ml of medium containing 150 g of sucrose, 20 g of yeast extract, 10 g of soytone, and 1 l of distilled water; the pH of the medium was adjusted to 5.9 with HCl. The cultures were incubated at 30 °C for 7 days in the dark (Horn and Dorner, 1999). Preliminary analysis of the extracts to screen for
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Fig. 1. Inoculum application in machines used to dispense fertiliser at planting (A) and 50 days after planting (B). Plot covered with a shelter that allowed drought conditions (C). Temperature sensor device used to register soil temperature at 5 cm deep (D).
aflatoxin production was carried out using thin-layer chromatography according to the methodology proposed by Geisen (1996). Aflatoxins were quantified by HPLC according to Horn et al. (1996).
incubated at 25 °C for 7 days. The incidence of toxigenic isolates of A. flavus/A. parasiticus in peanuts was determined by testing all the isolates for toxigenicity as described above for soil samples.
2.4.4. Vegetative compatibility group (VCG) analysis Nitrate non-utilizing mutants were obtained on plates of Czapek-Dox medium (Cz) containing 25 g/L potassium chlorate (Bayman and Cotty, 1991) with unadjusted pH. Five Cz–chlorate plates were inoculated with a conidial suspension of A. flavus in soft agar at four points. Cultures were incubated at 30 °C and the margins of the colonies with restricted growth were examined periodically for fast-growing sectors consisting of sparse mycelium. Hyphal tips from sectors arising from different colonies were transferred to Cz plates (nitrate medium) to confirm their inability to utilize nitrate. The nit mutant phenotypes were determined by growing the strains on several media in which the NaNO3 was replaced as the sole nitrogen source of either 700 mg/L NaNO2 (nitrite medium), 900 mg/L ammonium tartrate (ammonium medium) or 100 mg/L hypoxanthine (hypoxanthine medium) according to Papa (1986). Assignment of isolates to the VCG of applied strain AFCHG2 was made on the basis of complementation test between nia D − (deficient in the structural gene for nitrate reductase) of the field isolate to be assigned and a cnx (deficient in a molybdenum cofactor) tester mutant of AFCHG2.
2.5.2. Aflatoxin analysis The aflatoxin analysis was performed using the method of Trucksess et al. (1994). Aflatoxins were quantified by injecting 50 μL of extract from each vial into an HPLC system consisting of a Hewlett Packard model 1100 pump (Palo Alto, CA) connected to a Hewlett Packard model 1046A programmable fluorescence detector and a data module Hewlett Packard Kayak XA (HP ChemStation Rev. A.06.01). Chromatographic separations were performed on a stainless steel, C18 reversed-phase column (150 mm × 4.6 mm i.d., 5 μm particle size; Luna-Phenomenex, Torrance, CA, USA) connected to a precolumn Security Guard (20 mm × 4.6 mm i.d., 5 μm particle size, Phenomenex). The mobile phase was water:methanol:acetonitrile (4:1:1, v/v/v) at a flow rate of 1.5 ml/min and the limit of detection was 1 ng/ml of B1 and G1 and 0.8 ng/g of B2 and G2. Pure aflatoxin solutions were used as external standard (Sigma-Aldrich, St. Louis, MO, USA).
2.5. Peanut analysis 2.5.1. Mycobiota analysis from peanut samples From each subplot approximately 3 kg of kernels was used. This sample was mixed thoroughly and 100 kernels (2 replicates) were selected for fungal infection determination. The remaining sample was ground to obtain a subsample of 25 g (3 replicates) for aflatoxin analysis. Peanut kernels from each subplot were surface disinfected for 1 min in 1% sodium hypochlorite solution, rinsed three times in sterile distilled water and transferred to Petri dishes containing Dichloran Rose Bengal Chloramphenicol agar (DRBC) modified with 3% NaCl and Dichloran Glycerol 18% (DG18) (Pitt and Hocking, 1997). Plates were
2.6. Statistical analysis Data of fungal populations were log-transformed prior to analysis of variance (ANOVA). Means separation and comparison were made by Fisher's least significant difference (LSD) test at a probability level of p b 0.05. To compare the different treatments, aflatoxin concentrations were subjected to nonparametric Kruskal–Wallis test followed by Dunn's nonparametric multiple comparison test. The statistical analyses were performed using SigmaStat for windows version 2.03 (SPSS Inc., San Jose, CA). 3. Results and discussion 3.1. Field trial during 2009/2010 growing season Densities and toxigenicity of A. flavus and A. parasiticus soil populations were determined before planting in November and during
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pod maturation in February. The inoculum level and the incidence of toxigenic isolates of native A. flavus and A. parasiticus in soil at planting time were similar among plots and A. flavus was the dominant species from section Flavi, showing an isolation percentage nearly to 90% (Table 1). The remaining species identified were A. parasiticus (9%) and the nonaflatoxigenic species A. caelatus (1%). Data on incidence of the native population in the present study are consistent with those found by Barros et al. (2003, 2005) in different areas within the peanut-growing region of Córdoba. The soil samples at pod maturation showed a significant increase in the density of A. flavus between treated and control plots, while the viable count of A. parasiticus was not modified. Similar to planting time, A. flavus was the dominant species from section Flavi isolated from soil in the treated plots, but showing an isolation percentage slightly higher (>95%). As nontoxigenic A. flavus AFCHG2 colonies cannot be differentiated from wild-type strains, the efficacy of the biocontrol agent was monitored using the shift of the toxigenic/ nontoxigenic ratio in the treated plots in relation to control plots. At planting time, 72.5% and 94% of A. flavus and A. parasiticus isolates, respectively, were toxigenic. The analysis of A. flavus isolates recovered from soil at pod maturation showed that only 27% of isolates tested were toxigenic in treated plots compared with 80% in control plots. The nontoxigenic A. flavus AFCHG2 used in the present study was characterized previously as belong to vegetative compatibility group (VCG) which does not include toxigenic isolates (Barros et al., 2006a). Thus, VCG analysis was used as a second marker to monitor the presence of this biocontrol strain among the soil A. flavus population recovered at the pod maturation stage. Prior to application of the biocontrol agent and at pod maturation in control plots, only 3 of 90 soil isolates were vegetatively compatible with the AFCHG2. By contrast, 180 out of 256 A. flavus isolates recovered from treated soil at pod maturation belonged to the same VCG that the nontoxigenic A. flavus strain applied (Table 1). At harvest time, the percentages of peanuts infected by A. flavus and A. parasiticus ranged from 2 to 18% and were not significantly different (p > 0.05) between treated and control plots (Table 1). This result indicates that the rate of inoculum applied to soil had no effect on subsequent infection of peanut kernels. Similar results were obtained by Dorner and Cole (2002) and Dorner et al. (2003) when they used nontoxigenic A. flavus/A. parasiticus inoculum in peanut fields in USA. Among the Aspergillus section Flavi species identified, A. flavus was the most frequently recovered from peanuts, with isolation frequencies of 90 and 98% in control and treated plots, respectively. This result agrees with Horn et al. (1994) and Dorner and Horn (2007) who found that A. flavus was by far the predominant colonizer of peanuts, even in peanuts grown in soil treated with high levels of nontoxigenic A. parasiticus. This indicated that A. flavus was the more aggressive species and was responsible for
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most of the aflatoxin contamination of peanuts. Similar to those data found from soil samples at pod maturation, the toxigenic/ nontoxigenic ratio was nearly 3:7 in A. flavus strains isolated from peanuts in treated plots. With respect to the VCG analysis, only one of 30 peanut isolates recovered from the control plots was vegetatively compatible with the AFCHG2. A significant increase in the percentage of the VCG applied was observed in the peanuts recovered from the treated plots, where 16 of 30 isolates belonged to the atoxigenic VCG. Results obtained in both toxigenicity and VCG analyses demonstrated that the application of the biocontrol agent was efficient, showing competence within A. flavus and A. parasiticus community associated with the crop and resident in soil. This behaviour reduced both the average aflatoxin-producing potential of the native community and the risk of aflatoxin contamination. However, 2009/2010 growing season was characterized by abundant rainfall during the last month of crop development and the peanut samples showed no preharvest aflatoxin contamination. 3.2. Field trial during 2010/2011 growing season The density of native A. flavus/A. parasiticus soil population was similar among plots at pre-planting time, with viable count ranged from 500 to 1400 cfu/g with a mean of 815 cfu/g. Two aflatoxigenic species belonging to Aspergillus section Flavi were identified in the soil samples, A. flavus and A. parasiticus. However, A. flavus showed isolation frequencies higher than A. parasiticus in all soil samples evaluated accounting for nearly 85% of the isolates. In relation to the incidence of toxigenic native isolates at pre-planting time, a total of 85% of A. flavus isolates were toxigenic whereas all A. parasiticus isolates were toxigenic. The low percentage of atoxigenic strains in the native soil population found in this study is similar to those found among Aspergillus section Flavi populations isolated from agricultural soils in a range of geographic regions of the world (Barros et al., 2005; Horn and Dorner, 1999; Sweany et al., 2011). At pod maturation, densities of total A. flavus + A. parasiticus soil population were significantly higher in control and treated plots under drought stress compared with control and treated plots without drought stress. In all situations, A. flavus was the predominant species representing more than 90% of the isolates. For this reason, Table 2 shows only data on toxigenicity and percentages of isolates assigned to the applied VCG corresponding to A. flavus species. Regarding the toxigenicity of the A. flavus soil populations, significant displacement of toxigenic strains occurred in plots treated with nontoxigenic A. flavus AFCHG2 with different inoculation treatments in plots either under normal conditions or exposed to drought stress. Results obtained from the VCG analysis showed that only 4 of 60 strains soil isolates recovered from the control plots with and
Table 1 Density (cfu/g), toxigenic isolates (%) and isolates in applied VCG (%) of A. flavus and A. parasiticus in soil and peanuts and percentage of infection (%) and aflatoxin (μg/kg) in peanuts during 2009/2010 season. Treatment
Control A. flavus A. parasiticus Treated A. flavus A. parasiticus a b c d e f
Soil at planting
Soil at pod maturation
CFU/g
CFU/g
256 ac 230 a 23 b 215 ac 193 a 22 b
Toxigenic isolates (%)
75 a 98 a 70 a 92 a
225 abc 199 abe 26 a 647 bc 621 b 26 a
Peanuts
Toxigenic isolates (%)
Isolates in applied VCG (%)a
80 a 100 a
3b Ndf
27 b 98 a
70 a Ndf
Infection (%) 10 a 9a 1b 12 a 11 a 1b
Toxigenic isolates (%)b
Isolates in applied VCG (%)a
75 a 100 a
3b Nd
28 b 100 a
53a Nd
Percent isolates assigned to the applied vegetative compatibility group (VCG) to which AFCHG2 belongs on the basis of auxotroph complementation. For determination of toxigenic isolates (%) and isolates in applied VCG (%) a total of 401 isolates were evaluated. CFU/g: total count of Aspergillus section Flavi. ND = Not Detected b 1 μg/kg. Within a column, values not sharing a common letter are significantly different (p b 0.05). Nd = Not determined.
Mean aflatoxin NDd – – NDd – –
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Table 2 Density (cfu/g), toxigenic isolates (%) and isolates in applied VCG (%) of A. flavus in soil and peanuts and percentage of infection (%) and mean aflatoxin (μg/kg) in peanuts during 2010/2011 season. Treatment
Soil at pod maturation CFU/ga
Peanuts
Toxigenic isolates (%)
Isolates in applied VCG (%)b
Infection (%)
Toxigenic isolates (%)c
Isolates in applied VCG (%)b
Mean aflatoxind
Without drought stress Control Planting inoc. 50 days after planting inoc. Double inoc.
550 a 809 ab 1044 b 768 ab
68.6 38.2 32.3 45.2
a b b ab
3b 50 a 66 a 53 a
6.5 26.5 23.5 47.5
a abf ab bc
70 60 49 50
a ab ab ab
9b 35 a 30 ab 25 ab
N.De N.D N.D N.D
With drought stress Control Planting inoc. 50 days after planting inoc. Double inoc.
1965 cd 1260 bc 2480 c 2730 c
71.8 23.4 54.3 35.7
a b ab b
9b 56 a 50 a 50 a
57.0 75.7 78.5 99.5
bc cd cd d
67 57 50 51
a ab ab ab
5b 40 a 45 a 40 a
101.2 a 36.2 ab 25.4 b 28.1 b
a b c d e f
CFU/g: total count of Aspergillus section Flavi. Percent isolates assigned to the applied vegetative compatibility group (VCG) to which AFCHG2 belongs on the basis of auxotroph complementation. For determination of toxigenic isolates (%) and isolates in applied VCG (%) a total of 450 isolates were evaluated. Values are averages of three replicates. ND = Not Detected b 1 μg/kg. Within a column, values not sharing a common letter are significantly different (p b 0.05).
without drought stress were vegetatively compatible with the AFCHG2. After the application of the biocontrol agent, a higher incidence of isolates belong to the VCG applied was observed in all treatments both under normal conditions and exposed to drought stress (Table 2). At harvest the colonization of peanuts exhibited significant differences between control and treated plots in both conditions with and without drought stress; however a higher percentage of peanuts were infected in all inoculation treatments under drought condition, with A. flavus being responsible for this colonization. This result can be explained due to the capacity of A. flavus to infect peanuts under environmentally conducive conditions (Horn, 2003). Analysis of toxigenicity of the A. flavus isolates recovered from peanuts demonstrated that the rate of displacement of toxigenic strains was lower compare to those found in soil, with a ratio nontoxigenic to toxigenic near to 1:1. Similar results were obtained by Dorner and Horn (2007) who found relatively high incidence of toxigenic isolates in treated peanuts compared with the relatively low incidence in treated soil. These authors suggest that toxigenic isolates of A. flavus are more aggressive colonizers of peanuts than nontoxigenic strains. Analysis of aflatoxin contamination in peanuts showed no preharvest contamination in plots without drought stress. By comparison, the mean soil temperatures during the pod maturation period were close or above to 30 °C during several days in plots with drought stress, demonstrating an effective conducive condition for aflatoxin production. Significant reductions in aflatoxin levels averaging 71% were detected with all three treatments (Table 2). Although a low displacement of toxigenic isolates was observed in treated peanuts compare with treated soil, a significant increase in the percentage of the isolates in the VCG applied was observed in the treated peanuts (27 of 59 isolates) compared with the control (1 of 40 isolates). This result could explain the lower aflatoxin contamination observed in treated peanuts under conducive conditions. No statistically significant differences among inoculation 50 days after planting and plots inoculated twice were observed. According to this result, a single application preferably 50 days after planting will be adequate for future assays. The percentage reduction in aflatoxin levels obtained in this study is lower than the average reductions of 77% to 98% obtained in peanuts from USA and Australia (Dorner and Cole, 2002; Dorner and Lamb, 2006; Dorner and Horn, 2007; Pitt and Hocking, 2006). However, reductions around 70% obtained in the present study will be useful where there are conducive conditions for aflatoxin contamination, ensuring that levels are lower than those imposed by the regulations of the European Union, the major importer of Argentinean peanuts.
Regarding the inoculum application rate, the dosage used in this study was higher than other rates employed in peanuts from the USA (Dorner et al., 1998; Dorner and Cole, 2002; Dorner and Horn, 2007) but lower than other application rates evaluated in peanuts from Australia (Pitt and Hocking, 2006). More economical application rates should be tested in the future to determine the lowest effective rate. The present study demonstrated that nontoxigenic A. flavus AFCHG2 has the potential to become a management tool for the biocontrol of aflatoxin contamination of peanuts in Argentina.
Acknowledgements We are grateful to Aceitera General Deheza and Cotagro for kindly providing the commercial fields used in this study. The authors thank Ariel Bessone and Sebastian Rosso for technical assistance. This study was supported by grants from EC KBBE-2007-222690-2 MYCORED and Secretaría de Ciencia y Técnica, Universidad Nacional de Río Cuarto (SECyT-UNRC 2009–2010). Alaniz Zanon, M.S., Chiotta M.L. and Giaj-Merlera G. are fellows of CONICET and Barros, G. and Chulze, S. are members of the Research Career of CONICET.
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