Atoxigenic Aspergillus flavus endemic to Italy for biocontrol of aflatoxins in maize

Transcription

1 BioControl DOI /s Atoxigenic Aspergillus flavus endemic to Italy for biocontrol of aflatoxins in maize Antonio Mauro Paola Battilani Peter J. Cotty Received: 16 September 2014 / Accepted: 22 September 2014 Ó International Organization for Biological Control (outside the USA) 2014 Abstract Effective biological control of aflatoxinproducing Aspergillus flavus with atoxigenic members of that species requires suitable A. flavus well adapted to and resident in target agroecosystems. Eighteen atoxigenic isolates of A. flavus endemic in Italy were compared for ability to reduce aflatoxin contamination of maize in laboratory studies. Reduction in aflatoxin B 1 concentrations ranged from 61 to 90 % with the most effective five atoxigenics, belonging to five different vegetative compatibility groups, causing reductions similar to, or greater than, those achieved by an atoxigenic that is the active ingredient in a product registered in the USA for aflatoxin management. The atoxigenic isolates were effective against each of the six Italian aflatoxin-producers tested and both with equal proportions of toxigenic and Handling Editor: Choong-Min Ryu. A. Mauro P. Battilani Institute of Entomology and Plant Pathology, Università Cattolica del Sacro Cuore, Via Emilia Parmense 84, Piacenza, Italy antomau14@libero.it P. Battilani paola.battilani@unicatt.it P. J. Cotty (&) U.S. Department of Agriculture-Agricultural Research Service, School of Plant Sciences, The University of Arizona, Forbes Building, Room 303, 1140 East South Campus Drive, Tucson, AZ 85721, USA pjcotty@ .arizona.edu atoxigenic isolates and when atoxigenic isolates were only inoculated at 25 % the concentration of toxigenic. Equal proportions provided the highest percent reduction in contamination, but at the 4:1 ratio atoxigenics were the most efficient. The identified atoxigenic isolates will be of value as active ingredients in biocontrol products for reduction of aflatoxins in maize produced on the Italian Peninsula. Keywords Aflatoxin AFB 1 reduction VCGs Competition Biocontrol Biopesticides Introduction Aflatoxins are highly toxic metabolites produced by several Aspergillus species (Klich 2007; McKean et al. 2006). There are several naturally occurring aflatoxins, and the most toxic, aflatoxin B 1 (AFB 1 ), is a genotoxin classified by the International Agency for Research on Cancer as a group 1A human carcinogen (IARC 1982, 2002). Chronic exposure to low aflatoxin concentrations may result in immune suppression and cancer and has been linked to growth retardation. Ingestion of high concentrations, sometimes exceeding 1 ppm, has resulted in acute symptoms like hepatitis, liver necrosis, and death (Cardwell and Henry 2006). In most countries, aflatoxin concentrations in foods and feeds are strictly regulated (Payne and Yu 2010). By limiting access to markets,

2 A. Mauro et al. regulations influence the value of both domestic and imported crops contaminated by aflatoxins, as well as animal products contaminated by carryover of aflatoxins from feed (Wu and Khlangwiset 2010). In Italy in 2003 high levels of aflatoxin M 1, the hydroxylated animal metabolite of AFB 1, were detected in milk. This dairy contamination resulted from the feeding of domestically-produced aflatoxin contaminated maize (Giorni et al. 2007; Piva et al. 2006). Aflatoxin contamination of maize is primarily caused by A. flavus in Italy (Giorni et al. 2007). This species is divided into many genetic groups called vegetative compatibility groups (VCGs) by a heterokaryon incompatibility system (Bayman and Cotty 1993). Isolates of A. flavus from Italy vary in aflatoxinproducing ability, VCG, morphology of sclerotia, and production of conidia (Giorni et al. 2007; Mauro et al. 2013). There is less variability among isolates belonging to the same VCG than to isolates belonging to different VCGs (Bayman and Cotty 1993). Biological control strategies, utilizing active ingredients including bacteria, yeast, and filamentous fungi have been developed with the goal of limiting preharvest aflatoxin contamination (Dorner et al. 1999; Yin et al. 2008; Hell and Mutegi 2011). Among these active ingredients, several atoxigenic A. flavus have proven to be the most effective (Amaike and Keller 2011). Atoxigenic isolates of A. flavus are used to displace aflatoxin producers during crop development and, in so doing, reduce aflatoxin contamination (Cotty and Bayman 1993; Cotty 2006). Alteration of the population structure of the A. flavus infecting crop tissues so that aflatoxin-producers are less common, is the primary mechanism through which atoxigenic strains mitigate contamination. Changes to fungal population structures can have both multi-year and area-wide benefits (Cotty 2006; Cotty et al. 2008). However, secondary mechanisms may also be responsible for a small proportion of the efficacy (Mehl and Cotty 2010; Huang et al. 2011). Currently, there are two biopesticides with atoxigenic A. flavus active ingredients that are registered with the US Environmental Protection Agency for use on commercial crops. These biopesticides are Aspergillus flavus AF36 (registered for use on pistachio, maize, and cottonseed) and Afla-Guard Ò (registered for use on peanut and maize). In addition, Aflasafe TM NG, with four atoxigenic strains of A. flavus as the active ingredients, is provisionally registered for commercial use on maize in Nigeria with the NAFDAC by the International Institute of Tropical Agriculture (IITA, In each case, the atoxigenic A. flavus that are active ingredients are endemic to the target regions where the biopesticides are used. The current study applied laboratory assays on maize to the selection of atoxigenic A. flavus of potential value as active ingredients of biological control products directed at preventing aflatoxin contamination of maize in Italy. Previously identified atoxigenic isolates endemic to maize producing regions of Italy were compared for ability to reduce aflatoxin contamination of maize during competition with aflatoxin-producers from Italy and the US. Several promising atoxigenic isolates endemic to Italian maize fields were selected for further development. Materials and methods Aspergillus flavus isolate, culture conditions and storage Seven aflatoxin-producing and 18 atoxigenic isolates of A. flavus originating from Italy were used in this study (Table 1). Isolates collected in four districts of northern Italy from 2003 to 2009 (Giorni et al. 2007) were previously examined for aflatoxin production and categorized into VCGs (Mauro et al. 2013) and included in the culture collection of the Institute of Entomology and Plant Pathology of Università Cattolica del Sacro Cuore of Piacenza, Italy. Aspergillus flavus NRRL 21882, the active ingredient of the Afla-Guard Ò biopesticide (Dorner and Lamb 2006) anda. flavus AF13 (wildtype of ATCC 96044), a consistent aflatoxin producer (Cotty 1989), both originating from the US, were also included in these studies for comparison. Isolates were transferred by single spore, cultured on 5/2 agar (5 % V8 juice, 2 % agar, ph 5.2) and incubated at 31 C for 5 6 days prior to transferring culture plugs (eight to ten plugs, 3 mm diameter) to vials containing sterile water (2.5 ml) for storage at 8 C(Probst et al.2011). Co-infection of viable corn kernels with atoxigenic and aflatoxin-producing isolates of A. flavus Conidia from six day old cultures (31 C, dark) were collected from agar surfaces with cotton swabs,

3 A. flavus for biocontrol of aflatoxins in maize Table 1 Aspergillus flavus isolates used in the current study Isolate a VCG b c AFB 1 Location District d Year e A2049 IT12 Padova V 2003 A2066 IT8 Mantova L 2003 A2085 IT6 Reggio ER 2004 Emilia A2087 na Reggio ER 2004 Emilia A2088 IT9 Reggio ER 2004 Emilia A2090 IT9 Reggio ER 2004 Emilia A2096 IT9 Reggio ER 2004 Emilia A2098 IT6 Bologna ER 2004 A2100 IT18 Piacenza ER 2004 A2102 na Piacenza ER 2004 A2103 IT15 Piacenza ER 2004 A2105 IT6 Modena ER 2004 A2313 IT23 Ravenna ER 2009 A2319 IT22 Cremona ER 2009 A2320 IT22 Cremona ER 2009 A2321 IT19 Parma ER 2009 A2322 IT34 Modena ER 2009 A2323 IT23 Ravenna ER 2009 NRRL na Georgia (USA) 1991 A2039 IT11? Lodi L 2003 A2062 IT13? Venezia V 2003 A2068 IT26? Udine FVG 2003 A2097 IT10? Modena ER 2004 A2295 IT3? Cremona ER 2008 A2300 IT5? Cremona ER 2008 AF13 YV13? Arizona (USA) a All isolates originated from Italy except the NRRL and AF13 which originated from the USA b Vegetative Compatibility Group; na = not available c AFB 1 :(±): aflatoxin and no aflatoxin production d Italian regions: ER = Emilia Romagna, FVG = Friuli Venetia Giulia, L = Lombardy, V = Veneto e Year of isolation suspended in sterile distilled water, and spore concentrations were adjusted as described by Probst et al. (2011). Briefly, a turbidity meter (Model ; Orbeco-Hillige, Farmingdale, NY, USA) was used to measure the turbidity of conidia suspensions and conidial concentrations that were extrapolated from a nephelometric turbidity unit (NTU) versus CFU curve (Y = X, where X = NTU and Y = conidia ml -1 ). Conidial concentration for each isolate was adjusted to 10 5 conidia ml -1. Undamaged kernels of Pioneer hybrid 33B50 were surface sterilized in warm water for 45 s at 80 C (Mehl and Cotty 2010) and dispensed in sterilized glass flasks (10 g of maize per 250-ml flask). Flasks were sealed with plugs containing gas-permeable membranes (BugStopper Plugs, Whatman, Piscataway, NJ, USA) to prevent humidity loss and allow gas exchange. After sterilization, maize moisture level was quantified with a HB43 Halogen Moisture Analyzer (Mettler Toledo, Columbus, OH, USA) and the volume of water in which the atoxigenic and toxigenic spore suspensions were applied to the kernels was adjusted to bring the maize water content to 25 %. Conidia of atoxigenic and toxigenic isolates were added to the flasks simultaneously and then the flasks were gently agitated to allow kernel coating. Control flasks received the same quantity of water but containing only toxigenic spores. Inoculated maize was incubated at 31 C for seven days in the dark. The trials had a completely randomized design with four replicates. All experiments were performed twice. Influence of atoxigenic isolates on aflatoxin contamination of viable maize kernels was examined in three sets of co-infection studies. In the first set of experiments, all 18 Italian atoxigenic isolates were compared with NRRL for ability to reduce contamination of kernels inoculated with AF13. In the second set of experiments, two atoxigenic isolates (A2321 and A2085) belonging to different VCGs and originating from Italy were tested for ability to prevent aflatoxin contamination by six aflatoxin-producing isolates of A. flavus from Italy. The aflatoxin producers were the isolates that both produced the highest aflatoxin concentrations in a previous study (Mauro et al. 2013) and belonged to a distinct VCG. Atoxigenic isolate A2085 belongs to the VCG (IT6) most frequently infecting maize produced in Italy (Mauro et al. 2013) and A2321, belonging to VCG IT19, was the atoxigenic isolate most effective at reducing aflatoxin contamination in the first set of experiments, even if it was not significantly different from A2085. Viable maize kernels were surface sterilized, as described above, and inoculated (10 5 conidia per

4 A. Mauro et al. flask) with either one of the six aflatoxin-producing isolates alone or simultaneously with both one of the aflatoxin-producers and one of the two atoxigenic isolates (10 5 conidia per flask each). In the third set of experiments, the ratio of atoxigenic isolate to aflatoxin-producer was varied in order to assess if the relative proportion of aflatoxinproducer inoculum influenced the efficiency with which aflatoxin contamination was prevented by the atoxigenic isolates. The five atoxigenic isolates most effective at reducing aflatoxin contamination in the first set of experiments and NRRL from North America were compared. As in the prior two sets of experiments, viable maize kernels were inoculated simultaneously with both atoxigenic and aflatoxinproducing isolates. Aflatoxin-producers were inoculated at 10 5 conidia per flask and atoxigenic isolates were inoculated at 1x, 0.5x, and 0.1x the quantity of conidia of the aflatoxin-producers. Inoculated kernels were incubated as described for the first set of experiments. Aflatoxins quantification At the end of the incubation period, kernels were blended in 50 ml 80 % methanol and the homogenate was passed through Whatman No. 4 filter paper and spotted directly on thin-layer chromatography (TLC) plates (Silica gel 60; EMD, Darmstadt, Germany) beside aflatoxin standards (AFB 1, AFB 2, AFG 1 and AFG 2 ; Aflatoxin Mix kit-m; Supelco Bellefonte, PA, USA). Plates were developed in ethyl ether:methanol:water (96:3:1), air-dried, and aflatoxins were visualized under 365-nm UV light. Aflatoxins were quantified directly on TLC plates with a scanning densitometer (TLC Scanner 3; Camag Scientific Inc, Wilmington, NC, USA). The limit of detection was 20 lg kg -1 (Probst et al. 2011). Production and quantification of cyclopiazonic acid Cyclopiazonic acid (CPA) production was evaluated only for the five atoxigenic isolates most effective at reducing AFB 1 concentration. Two CPA producers (Giorni et al. 2007), A2062 (IT13) and A2068 (IT26), were included as positive controls and uninoculated substrate as a negative control. Isolates were tested on undamaged kernels following the method previously described for AFB 1 production (Mauro et al. 2013) and on Czapek Agar (CZ) (Giorni et al. 2007). CPA from kernels was extracted according to Urano et al. (1992) and quantified according to Zambonin et al. (2001). The method reported by Giorni et al. (2007) was used to extract and quantify CPA from CZ. Statistical analyses Randomized complete block designs with four replicates were used in all experiments. Aflatoxin concentrations were log-transformed and percentages were arcsine-transformed prior to performing analysis of variance (ANOVA) with the general linear model procedure of SAS (version 9.2; SAS Institute, Cary, NC, USA). Mean separations were performed with Tukey s Honestly Significant Difference (HSD) test (Pagano and Gauvreau 2000; P = 0.05). Mean percentage reduction in aflatoxin content between maize inoculated with aflatoxin producers alone and maize co-inoculated with atoxigenic isolates and aflatoxin-producers were calculated as [1 - (total aflatoxin in maize co-inoculated with both aflatoxinproducer and atoxigenic isolate/total aflatoxin in maize inoculated with the aflatoxin producer alone)] The aflatoxin producer was AF13 in the first and third experiments and one of five Italian aflatoxin-producers in the second experiment. The concept of Treatment Efficiency was developed in order to quantitatively compare the relative ability of atoxigenic A. flavus to inhibit aflatoxin contamination during coinfection of maize with an aflatoxin producer when the two fungi are introduced to the crop at different inoculum concentrations. Treatment Efficiency (E) was calculated following the formula E = R/[A/(A 1 T)], where R is the percent aflatoxin reduction, A is the quantity of conidia of the atoxigenic strain applied and T is the quantity of conidia of the aflatoxin-producer applied. An efficiency of 1.0 indicates that equal proportions of the atoxigenic isolate and the aflatoxin-producer results in an aflatoxin concentration fifty percent of that in maize inoculated with the aflatoxin producer alone. Values of E [ 1.0 indicate that aflatoxin reduction is more than proportional to the inoculum added. Increased E values indicate an improvement in the amount of aflatoxin reduction each conidium achieves. All the experiments were repeated twice.

5 A. flavus for biocontrol of aflatoxins in maize Results Efficacy of Italian atoxigenic isolates Co-inoculation of aflatoxin-producer AF13 with any of the 18 atoxigenic A. flavus isolates from Italy resulted in significantly lower maize kernel AFB 1 concentrations than inoculation of maize kernels with AF13 alone (Table 2; n = 160, F = 18.10, df = 19, 140, P \ ). Kernels inoculated with AF13 alone averaged 146 mg kg -1 AFB 1 while between 61 and 90 % (n = 160, F = 34.92, df = 19, 140, P \ ) lower aflatoxin concentrations were detected in kernels co-inoculated with one of the Italian atoxigenic isolates. Half (nine) of the Italian isolates reduced aflatoxin concentrations more than 80 %. The five most effective isolates belonged to five different VCGs, originated from two districts, Lombardy (one VCG) and Emilia Romagna (four VCGs), and were collected in 2003 (one isolate), 2004 (three isolates) and 2009 (one isolate) (Table 1). Variation in susceptibility to atoxigenic isolates among aflatoxin producers from Italy The two atoxigenic isolates, A2085 and A2321, effectively reduced AFB 1 contamination of viable maize kernels inoculated with any of six representative aflatoxin-producing isolates from Italy. Kernels inoculated with one of the aflatoxin producers alone became Table 2 Ability of 18 atoxigenic isolates of Aspergillus flavus endemic to Italy to reduce aflatoxin B 1 production in maize Isolate a VCG b mg kg -1 AFB 1 (SE) c,e R (%) (SE) d,e A2321 IT19 15 (2.3) H 90 (1.5) A A2103 IT15 21 (3.5) GH 85 (2.2) AB A2090 IT9 22 (3.3) FGH 85 (2.1) ABC A2066 IT8 23 (2.6) EFGH 85 (1.6) ABC NRRL na 24 (3.5) EFGH 84 (2.2) ABC A2085 IT6 25 (4.2) EFGH 83 (2.7) ABC A2088 IT9 27 (2.9) EFG 82 (2.0) ABC A2105 IT6 27 (3.5) DEFG 81 (2.3) ABC A2313 IT23 28 (3.5) DEFG 81 (2.2) ABCD A2098 IT22 29 (3.0) CDEFG 80 (1.9) ABCD A2096 IT6 30 (2.5) CDEFG 79 (1.5) BCD A2102 na 32 (1.8) BCDEFG 78 (1.5) BCDE A2319 IT22 33 (4.4) BCDEFG 78 (2.8) BCDE A2049 IT12 39 (2.5) BCDE 73 (1.4) CDEF A2320 IT9 41 (6.7) BCDE 72 (4.2) CDEF A2087 na 47 (1.8) BCD 68 (1.2) DEF A2322 IT34 51 (2.4) BC 65 (1.5) EF A2323 IT23 54 (10.2) BC 62 (7.5) EF A2100 IT18 57 (2.6) B 61 (2.1) F AF13 YV (4.5) A 0 G a Atoxigenic isolates were inoculated with AF13 at conidia ml -1. NRRL is the active ingredient of the Afla-Guard Ò biopesticide. AF13 produces high amounts of aflatoxins b Vegetative Compatibility Groups of isolates; na = not available c Aflatoxin B 1 concentration (mg kg -1 ) after seven days incubation of infected kernels co-inoculated with atoxigenic and aflatoxinproducing isolates d Percentage aflatoxin reduction (R) = [1 - (total aflatoxin in co-inoculation/total aflatoxin in AF13)] e Values, mean (SE), in a column not followed by a common letter are significantly different by Tukey s HSD test (P \ 0.05). ANOVA was significant (AFB 1 concentration: n = 160, F = 18.10, df = 19, 140, P \ ; Percentage aflatoxin reduction: n = 160, F = 34.92, df = 19, 140, P \ ). Combined data from two independent trials are presented

6 A. Mauro et al. contaminated with mg k -1 gafb 1.Differences among aflatoxin producers were significant (n = 48, F = , df = 5, 42, P \ ). Otherwise AFB 1 concentration in the co-inoculum with the atoxigenic isolate A2085 ranged from 7 to 22 mg kg -1 (n = 48, F = 16.35, df = 5, 42, P \ ) and from 5 to 17 mg kg -1 with the isolate A2321 (n = 48, F = 28.77, df = 5, 42, P \ ). Contamination was reduced % by co-inoculation with atoxigenic isolate A2085 (n = 48, F = 8.34, df = 5, 42, P \ ) and % by co-inoculation with A2321 (n = 48, F = 7.31, df = 5, 42, P \ ). A2085 caused similar reductions (60 68 %) in aflatoxin content in maize kernels inoculated with five of the aflatoxin producers but was more effective against A2039, the Italian A. flavus isolate that produced the highest concentrations of aflatoxins. Contamination produced by A2039 was reduced by 78 % (Table 3). Isolate A2321 was also most effective against A2039 causing a 83 % reduction in aflatoxins. A2321 was significantly (P \ 0.05) more effective than A2085 at reducing contamination of two aflatoxin producers (A2062 and A2300). There was also a positive correlation between the extent to which maize inoculated with the aflatoxin-producer became contaminated and the efficacy of the atoxigenic isolate for both A2085 (n = 6, r = 0.947, P \ ), and A2321 (n = 48, r = P \ ). Influences of the ratio of atoxigenic isolate to aflatoxin-producer on aflatoxin production Maize kernels inoculated with equal quantities (1:1 ratio) of conidia from aflatoxin-producing and atoxigenic isolates became contaminated with significantly less AFB 1 than kernels inoculated at a 4:1 ratio with four times more conidia from an aflatoxin-producer than from an atoxigenic isolate (34 vs. 73mgkg -1, on average) (Table 4). With the 1:1 ratio, AFB 1 ranged from 20 to 44 mg kg -1 (n = 64, F = 29.71, df = 6, 57, P \ ) while with the 4:1 ratio, AFB 1 ranged from 56 to 86 mg kg -1 (n = 64, F = 35.37, df = 6, 57, P \ ). Percent reduction ranged between 24 and 50 % (n = 64, F = 44.12, df = 6, 57, P \ ) for the 4:1 ratio and from 61 to 83 % (n = 64, F = 59.41, df = 6, 57, P \ ) for the 1:1 ratio. However, on average, atoxigenic isolates had significantly greater efficiency (percent reduction adjusted for the proportion of the inoculum composed by the atoxigenic) when composing only 20 % of the infecting population (as in the 4:1 treatment) than when composing 50 % of the infecting population (as in the 1:1 treatment) (Table 4). Table 3 Reduction of two atoxigenic isolates of Aspergillus flavus from Italy in maize contaminated with aflatoxins produced by six Italian isolates of A. flavus (atoxigenic and toxigenic were inoculated at the same ratio) Toxigenic isolates mg kg -1 AFB 1 b mg kg -1 AFB 1 (SE) c,e R (%) (SE) d,e A2085 a A2321 a A2085 a A2321 a A (2.7) A 22 (3.5) A 17 (1.8) A 78 (3.1) A 83 (1.8) A A (1.7) BC 11 (0.5) BC 8 (0.6) B 68 (2.1) B 78 (2.2) AB* A (1.8) B 16 (1.2) AB 16 (0.9) A 60 (3.0) B 61 (2.3) C A (1.8) C 11 (0.9) BC 8 (0.6) B 61 (1.8) B 71 (2.8) BC A (1.2) D 8 (0.4) CD 7 (0.8) BC 65 (1.6) B 71 (4.0) BC A (0.7) E 7 (0.3) D 5 (0.4) C 60 (2.3) B 72 (3.0) BC* a A2085 and A2321 atoxigenic isolates were co-inoculated simultaneously with each toxigenic isolate at the same conidial concentration ( conidia ml -1 ) b Aflatoxin B 1 concentration (mg kg -1 ) after seven days in infected kernels inoculated with toxigenic isolates c Aflatoxin B 1 concentration (mg kg -1 ) after seven days in infected kernels co-inoculated with atoxigenic and toxigenic isolates d Percentage aflatoxin reduction (R) = [1 - (total aflatoxin in co-inoculation/total aflatoxin in respective toxigenic isolate)] e Values, mean (SE), in a column not followed by a common letter are significantly different by Tukey s HSD test (P \ 0.05). ANOVA was significant (AFB 1 concentration toxigenic isolates alone: n = 48, F = , df = 5, 42, P \ ; AFB 1 concentration co-inoculum A2085: n = 48, F = 16.35, df = 5, 42, P \ ; AFB 1 concentration co-inoculum A2321: n = 48, F = 28.77, df = 5, 42, P \ ; Percentage aflatoxin reduction A2085: n = 48, F = 8.34, df = 5, 42, P \ ; Percentage aflatoxin reduction A2321: n = 48, F = 7.31, df = 5, 42, P \ ). * indicate significant (P \ 0.05) differences among atoxigenic isolates by Student s t test. Combined data from two independent trials are presented

7 A. flavus for biocontrol of aflatoxins in maize Table 4 Influence of atoxigenic and toxigenic ratios of Aspergillus flavus on reduction of aflatoxin B 1 produced by toxigenic isolates of A. flavus Isolate a mg kg -1 AFB 1 (SE) c,f R (%) (SE) d,f E (SE) e,f (4:1) b (1:1) b (4:1) b (1:1) b (4:1) b (1:1) b A (2.1) B 44 (4.1) B* 24 (2.0) D* 61 (3.9) B 1.20 (0.100) C 1.21 (0.077) B A (4.3) CD 31 (5.6) B* 38 (3.9) BC* 72 (5.0) AB 1.90 (0.194) B 1.45 (0.100) AB A (1.6) BCD 37 (6.4) B* 33 (1.3) BCD* 67 (5.8) B 1.66 (0.063) BC 1.35 (0.116) AB* A (4.0) D 34 (6.4) B* 39 (3.8) AB* 70 (5.0) AB 1.97 (0.190) AB 1.39 (0.099) AB* A (3.5) E 20 (3.3) C* 50 (3.2) A* 83 (3.0) A 2.52 (0.160) A 1.65 (0.059) A* NRRL (4.9) BC 40 (4.0) B* 26 (4.7) CD* 65 (3.7) B 1.30 (0.236) C 1.29 (0.074) B Mean 73 (2.0) 34 (2.2) * 35 (1.8) * 70 (2.0) 1.76 (0.092) 1.39 (0.040) * AF (1.9) A 113 (1.9) A a NRRL is the active ingredient of the Afla-Guard Ò biopesticide. AF13 produces aflatoxins and it represents the positive control b Atoxigenic and toxigenic isolates were co-inoculated at total conidia ml -1 in two ratios: 80 % toxigenic and 20 % atoxigenic (4:1) and equal proportions (1:1) c Aflatoxin B 1 concentration (mg kg -1 ) seven days after inoculation in kernels co-inoculated with each of atoxigenic and toxigenic isolates d Percentage aflatoxin reduction (R) = [1 - (total aflatoxin in co-inoculation/total aflatoxin in AF13)] e Efficiency (E) was calculated following the formula: E = R/[A/(A? T)], where R is the aflatoxin reduction and the denominator term represents the percentage of atoxigenic in each treatment f Values, mean (SE), in a column not followed by a common letterare significantly different by Tukey s HSD test (P \ 0.05). ANOVA was significant (AFB 1 concentration ratio 1:1: n = 64, F = 29.71, df = 6, 57, P \ ; AFB 1 concentration ratio 4:1: n = 64, F = 35.37, df = 6, 57, P \ ; Percentage aflatoxin reduction ratio 1:1: n = 64, F = 59.41, df = 6, 57, P \ ; Percentage aflatoxin reduction ratio 4:1: n = 64, F = 44.12, df = 6, 57, P \ ; Efficiencies ratio 1:1: n = 64, F = 84.89, df = 6, 57, P \ ; Efficiencies ratio 4:1: n = 64, F = 46.71, df = 6, 57, P \ ). * indicate significant (P \ 0.05) differences between the 1:1 and 4:1 ratios by Student s t test. Combined data from two independent trials are presented Efficiencies of isolates tested at 4:1 and 1:1 ranged from 1.20 to 2.52 (n = 48, F = 46.71, df = 6, 57, P \ ) and from 1.21 to 1.65 (n = 48, F = 84.89, df = 6, 57, P \ ), respectively. The lowest and the highest efficiencies for both treatments were expressed by A2066 and A2321, respectively. However, significant differences in efficiency between the two ratios for individual fungi occurred only for the atoxigenic isolates A2090, A2103 and A2321. Cyclopiazonic acid The CPA production by the two positive controls, A2062 and A2068, on both substrates tested was always higher than 1 lg g -1. No CPA was detected in the kernels and in CZ medium inoculated with individual isolates A2066, A2085, A2090, A2103 and A2321. Discussion Aflatoxin contamination is a health and economic problem worldwide with annual losses exceeding 500 million US dollars (CAST 2003; Yu et al. 2005). As a result, considerable effort has been devoted in developing strategies to prevent aflatoxin contamination in the field. Use of cultivars with reduced susceptibility to aflatoxin contamination (Henry et al. 2009) and optimisation of agronomic practices to reduce the effects of drought and heat stress (Bruns 2003) can reduce the severity of aflatoxin contamination in maize. However, these techniques are frequently insufficient to reduce crop aflatoxin concentrations to levels mandated by regulations. Biological control based on the use of atoxigenic isolates of A. flavus is effective at reducing aflatoxin contamination on cotton, peanuts, pistachio nuts and maize (Cotty and Antilla 2003; Doster et al. 2014; Atehnkeng et al. 2008; Yin et al. 2009). This technique has rapidly

8 A. Mauro et al. become the preferred method for aflatoxin mitigation in some areas of the USA. However, the technique has be unavailable in Europe due to lack of atoxigenic A. flavus based biopesticides utilizing active ingredients endemic to and effective in European agroecosystems. In northern Italy, where aflatoxin contamination of maize has been economically important since 2003, A. flavus communities associated with maize production have been characterized (Giorni et al. 2007; Mauro et al. 2013) and atoxigenic A. flavus endemic to Italy identified. However, the current report is the first to quantitatively compare atoxigenic A. flavus isolates from Italy for the ability to limit aflatoxin contamination during infection of viable maize kernels by aflatoxin-producers and to provide bases for selection of isolates for field development. The efficacy of the Italian atoxigenic isolates in reducing aflatoxin contamination of maize is similar to that reported for atoxigenic strains from North America (Mehl and Cotty 2010) and Africa (Probst et al. 2011). The extents to which 14 of the 18 Italian isolates reduced contamination were statistically equivalent to NRRL 21882, the active ingredient of a product registered for use in aflatoxin mitigation on maize and groundnuts in USA. Only four of the isolates were significantly less efficacious in reducing AFB 1 contamination. These results suggest there are many atoxigenic VCGs of A. flavus distributed across Italy of potential value as active ingredients in biocontrol products directed at preventing aflatoxin contamination. Ability to displace aflatoxin-producers is probably a multigenic trait (Mehl and Cotty 2010) with one aspect possibly requiring mycelial contact between the atoxigenic isolate and the aflatoxin producer (Huang et al. 2011). Variance in competitive ability may reflect adaptive differences among the isolates. Such adaptive differences can be related to variation in fungal life strategy. Isolates with a predominantly sporulating life strategy may provide superior displacement of aflatoxin-producers during epidemic increases in A. flavus populations, compared with isolates that express the Ramify and Hold life strategy (Mehl and Cotty 2010). Ramify and Hold isolates have increased plant tissue invasion associated with reduced sporulation. Ramify and Hold isolates will out-compete isolates adapted to rapidly sporulate in the viable kernel assays. This is just one reason, laboratory assays only provide an initial tool for selecting biocontrol agents. To be the most effective in preventing contamination, atoxigenic isolates must be adapted to both the crop and the target environment (Cotty 2006; Cotty et al. 2008). Distribution of atoxigenics in the agroecosystem is one measure of adaptation that was previously established for isolates evaluated in the current study. However, field studies on efficacy and persistence under standard agronomic practices in the target region are necessary in order to establish which isolates are best for incorporation into aflatoxin management products. The current study identified the best isolates of Italian origin for advancement to that next necessary step in the development of atoxigenic technology for Italy. Studies of atoxigenic strain efficacy typically evaluate activity against a single or a few aflatoxin producing isolates. However, aflatoxin contamination events are caused by complex communities of aflatoxin producers (Cotty et al. 2008). The current study demonstrates that individual atoxigenic isolates from Italy can reduce aflatoxin contamination by the aflatoxin-producers that cause the majority of aflatoxin contamination of maize in Italy (Mauro et al. 2013). Isolates A2085 and A2321 displayed the greatest efficacy in reducing aflatoxin contamination. Each of these atoxigenic isolates was effective at reducing aflatoxin contamination by diverse aflatoxin producers in viable maize kernels. Efficacy of both atoxigenics against several aflatoxin producers (Table 3) suggests these fungi may have utility against widely differing communities of aflatoxin producing fungi and, as a result, be useful in reducing aflatoxin contamination throughout Italy. Across Italian maize production areas there is a strong desire for aflatoxin management tools as a result of recent contamination episodes (Piva et al. 2006; Battilani et al. 2008a, b; Mauro et al. 2013). The concept of Efficiency (E) is introduced here in order to quantify the relative amount of aflatoxin reduction achieved per atoxigenic conidium. An efficiency of 1.0 indicates a fifty percent reduction in aflatoxins from equal proportion of aflatoxin-producing and atoxigenic conidia. Values of E [ 1.0 indicate that aflatoxin reductions are more than proportional to the percent of the overall inoculated A. flavus conidia composed of the atoxigenic isolate. Thus, an increased E value indicates a greater efficiency with which the atoxigenic isolate decreases aflatoxin content during coinfection with an aflatoxin-producer. In the current

9 A. flavus for biocontrol of aflatoxins in maize study, increased E was associated with maize inoculation with lower proportions (toxigenic:atoxigenic 4:1) of atoxigenic isolates. This suggests atoxigenics act more efficiently when present at low proportions and achieve greater reductions than would be expected based on their frequencies in the environment alone. Greater values of E for lower proportions of an atoxigenic isolate were also observed by Degola et al. (2011). It is unclear from the current work if the greater efficiencies associated with reduced proportions of atoxigenic isolates result from more efficient competition and displacement of aflatoxin producers or greater activity by one of the previously hypothesized second mechanisms of aflatoxin inhibition (Cotty and Bayman 1993; Mehl and Cotty 2011; Huang et al. 2011). The current study identified several atoxigenic isolates that have potential as biocontrol agents for mitigating aflatoxin contamination in Italy. The most effective five Italian atoxigenic isolates caused reductions in the contamination of viable maize kernels similar to an isolate that is the active ingredient of a commercial biopesticide registered for aflatoxin prevention in the USA. These five most effective isolates also did not produce CPA. Cyclopiazonic acid is a mycotoxin with low toxicity compared to aflatoxins. However, there has been disagreement among researchers developing biological control as to the importance of CPA production and/or lack of production to selection of active ingredients in biocontrol products (Dorner and Lamb 2006; Probst et al. 2011). All the identified atoxigenics are endemic in the target regions in Italy. As a result, these atoxigenic isolates are thought to offer several advantages over introduced isolates including improved environmental safety and better adaptation to the target region (Probst et al. 2011). Greater adaptation to the Italian agroecosytems should mean both increased efficacy in the target area and greater carryover between crops. The characteristics associated with endemic isolates well adapted to Italian agro-ecosystems will facilitate development of area-wide management directed at eliminating farmer risk from aflatoxins. Acknowledgments Antonio Mauro carried out this work within the Doctoral School on the Agro-Food System (Agrisystem) of Università Cattolica del Sacro Cuore (Italy). The authors are grateful to the government of Emilia Romagna for supporting this work, to the Research Centre for Crop Production (CRPV) for the collaboration and to Dr. Ramon Jaime-Garcia for assistance with statistical analyses. References Amaike S, Keller NP (2011) Aspergillus flavus. Annu Rev Phytopathol 49: Atehnkeng J, Ojiambo PS, Ikotun T, Sikora RA, Cotty PJ, Bandyopadhyay R (2008) Evaluation of atoxigenic isolates of Aspergillus flavus as potential biocontrol agents for aflatoxin in maize. Food Addit Contam 25: Battilani P, Barbano C, Piva G (2008a) Aflatoxin B 1 contamination in maize related to the aridity index in North Italy. World Mycotoxin J 1: Battilani P, Pietri A, Barbano C, Scandolara A, Bertuzzi T, Marocco A (2008b) Logistic regression modelling of cropping systems to predict fumonisin contamination in maize. J Agric Food Chem 56: Bayman P, Cotty PJ (1993) Genetic diversity in Aspergillus flavus: association with aflatoxin production and morphology. Can J Bot 71:23 34 Bruns HA (2003) Controlling aflatoxin and fumonisin in maize by crop management. J Toxicol-Toxin Rev 22: Cardwell FK, Henry SH (2006) Risk of exposure to and mitigation of effects of aflatoxin on human health: a West African example. Toxin Rev 23: CAST (2003) Mycotoxins: risks in plant, animal, and human systems. Task Force Report 139. Council for Agricultural Science and Technology, Ames, Iowa, USA Cotty PJ (1989) Virulence and cultural characteristics of two Aspergillus flavus strains pathogenic on cotton. Phytopathology 79: Cotty PJ (2006) Biocompetitive exclusion of toxigenic fungi. In: Barug D, Bhatnagar D, van Egmond HP, van der Kamp JW, van Osenbruggen WA, Visconti A (eds) The mycotoxin factbook. Wageningen Academic Publishers, Wageningen, The Netherlands, pp Cotty PJ, Antilla L (2003) Managing aflatoxins in arizona. United States Department of Agriculture, Agricultural Research Service, New Orleans, LA, USA Cotty PJ, Bayman P (1993) Competitive exclusion of a toxigenic strain of Aspergillus flavus by an atoxigenic strain. Phytopathology 83: Cotty PJ, Probst C, Jaime-Garcia R (2008) Etiology and management of aflatoxin contamination. In: Leslie JF, Bandyopadhyay R, Visconti A (eds) Mycotoxins: detection methods, management, public health, and agricultural trade. CABI Publishing, Oxfordshire, UK, pp Degola F, Berni E, Restivo FM (2011) Laboratory tests for assessing efficacy of atoxigenic Aspergillus flavus strains as biocontrol agents. Int J Food Microbiol 146: Dorner JW, Lamb MC (2006) Development and commercial use of afla Guard (Ò), an aflatoxin biocontrol agent. Mycotoxin Res 21:33 38 Dorner JW, Cole RJ, Wicklow DT (1999) Aflatoxin reduction in corn through field application of competitive fungi. J Food Prot 62:

10 A. Mauro et al. Doster MA, Cotty PJ, Michailides TJ (2014) Evaluation of the atoxigenic Aspergillus flavus strain AF36 in pistachio orchards. Plant Dis 98: Giorni P, Magan N, Pietri A, Bertuzzi T, Battilani P (2007) Studies on Aspergillus section Flavi isolated from maize in northern Italy. Int J Food Microbiol 113: Hell K, Mutegi C (2011) Aflatoxin control and prevention strategies in key crops of Sub-Saharan Africa. Afri J Microbiol Res 5: Henry WB, Williams WP, Windham GL, Hawkins LK (2009) Evaluation of maize inbred lines for resistance to Aspergillus and Fusarium ear rot and mycotoxin accumulation. Agron J 101: Huang C, Jha A, Sweany R, DeRobertis C, Damann KE Jr (2011) Intraspecific aflatoxin inhibition in Aspergillus flavus is thigmoregulated, independent of vegetative compatibility group and is strain dependent. PLoS ONE 6:e23470 International Agency for Research on Cancer (1982) The evaluation of the carcinogenic risk of chemicals to humans. International Agency for Research on Cancer, Lyon, France International Agency for Research on Cancer (2002) IARC monographs on the evaluation of the carcinogenic risks to humans: some traditional herbal medicines, some mycotoxins, naphthalene and styrene, vol 82. International Agency for Research on Cancer, Lyon, France, pp Klich MA (2007) Aspergillus flavus: the major producer of aflatoxin. Mol Plant Pathol 8: Mauro A, Battilani P, Callicott KA, Giorni P, Pietri A, Cotty PJ (2013) Structure of an Aspergillus flavus population from maize kernels in northern Italy. Int J Food Microbiol 162:1 7 McKean C, Tang L, Tang M, Billam M, Wang Z, Theodorakis CW, Kendall RJ, Wang JS (2006) Comparative acute and combinative toxicity of aflatoxin B 1 and fumonisin B 1 in animals and human cells. Food Chem Toxicol 44: Mehl HL, Cotty PJ (2010) Variation in competitive ability among isolates of Aspergillus flavus from different vegetative compatibility groups during maize infection. Phytopathology 100: Mehl HL, Cotty PJ (2011) Influence of the host contact sequence on the outcome of competition among Aspergillus flavus isolates during host tissue invasion. App Environ Microbiol 77: Pagano M, Gauvreau K (2000) Principles of biostatics. Duxbury, Pacific Grove, CA, USA Payne GA, Yu J (2010) Ecology, development and gene regulation in Aspergillus flavus. In: Machida M, Gomi K (eds) Aspergillus: molecular biology and genomics. Caister Academic Press, Norfolk, UK, pp Piva G, Battilani P, Pietri A (2006) Emerging issues in southern Europe: aflatoxins in Italy. In: Barug D, Bhatnagar D, van Egmond HP, van der Kamp JW, van Osenbruggen WA, Visconti A (eds) The mycotoxin factbook: food and feed topics. Wageningen Academic Publisher, Wageningen, The Netherlands, pp Probst C, Bandyopadhyay R, Price LE, Cotty PJ (2011) Identification of atoxigenic Aspergillus flavus isolates to reduce aflatoxin contamination of maize in Kenya. Plant Dis 95: Urano T, Trucksess MW, Matusik J, Dorner JW (1992) Liquid chromatographic determination of cyclopiazonic acid in corn and peanuts. J AOAC Int 75: Wu F, Khlangwiset P (2010) Health economic impacts and costeffectiveness of aflatoxin reduction strategies in Africa: case studies in biocontrol and postharvest interventions. Food Addit Contam 27: Yin YN, Lou T, Michailides TJ, Ma ZH (2009) Molecular characterization of toxigenic and atoxigenic Aspergillus flavus isolates, collected from peanut fields in China. J Appl Microbiol 107: Yin YN, Yan LY, Jiang JH, Ma ZH (2008) Biological control of aflatoxin contamination of crops. J Zhejiang Univ Sci B 9: Yu J, Cleveland TE, Nierman WC, Bennett JW (2005) Aspergillus flavus genomics: gateway to human and animal health, food safety, and crop resistance to diseases. Rev Iberoam Mycol 22: Zambonin CG, Monaci L, Aresta A (2001) Determination of cyclopiazonic acid in cheese samples using solid phase microextraction and high performance liquid chromatography. Food Chem 75: Antonio Mauro performed the experiments reported in the current article while spending one year of his doctoral studies at the University of Arizona. Dr. Mauro currently manages, for a company, both field activities directed at quantifying efficacy of candidate biocontrol agents in Italy and development of technical contributions required for product registration. Paola Battilani is a professor in plant pathology at Università Cattolica del Sacro Cuore. Her research group focuses on the ecology and plant-pathogen interactions of mycotoxin producing fungi. Knowledge acquired is used to predict and prevent fungal growth and mycotoxin contamination in sustainable agriculture. She is a section editor for the World Mycotoxin Journal. Peter J Cotty is a research plant pathologist with the Agricultural Research Service, United States Department of Agriculture, embedded in the School of Plant Sciences of the University of Arizona in Tucson. Dr. Cotty frequently includes farmers, affected industries, and international collaborators in his laboratory s work on aflatoxins, aflatoxin-producing fungi, and the contamination process. Several of his laboratory s practical advances in aflatoxin management have become widely used.