1554 Journal of Food Protection, Vol. 77, No. 9, 2014, Pages 1554–1562 doi:10.4315/0362-028X.JFP-13-474
Susceptibility to Aflatoxin Contamination among Maize Landraces from Mexico ALEJANDRO ORTEGA-BELTRAN,1{ MANUEL D. J. GUERRERO-HERRERA,2 ALEJANDRO ORTEGA-CORONA,2 VICTOR A. VIDAL-MARTINEZ,3 AND PETER J. COTTY4* 1School of Plant Sciences, University of Arizona, Tucson, Arizona 85721, USA; 2Secretarı´a de Agricultura, Ganaderı´a, Desarrollo Rural, Pesca y Alimentacio´n (SAGARPA), Instituto Nacional de Investigaciones Forestales, Agrı´colas y Pecuarias (INIFAP), Campo Experimental Norman E. Borlaug (CENEB), Ciudad Obrego´n, Sonora 85000, Me´xico; 3SAGARPA, INIFAP, El Centro de Investigacio´n Regional del Noroeste (CIRNO), Santiago Ixcuintla, Nayarit 63300, Me´xico; and 4U.S. Department of Agriculture, Agricultural Research Service, School of Plant Sciences, University of Arizona, Tucson, Arizona 85721, USA
MS 13-474: Received 4 November 2013/Accepted 11 April 2014
ABSTRACT Maize, the critical staple food for billions of people, was domesticated in Mexico about 9,000 YBP. Today, a great array of maize landraces (MLRs) across rural Mexico is harbored in a living library that has been passed among generations since before the establishment of the modern state. MLRs have been selected over hundreds of generations by ethnic groups for adaptation to diverse environmental settings. The genetic diversity of MLRs in Mexico is an outstanding resource for development of maize cultivars with beneficial traits. Maize is frequently contaminated with aflatoxins by Aspergillus flavus, and resistance to accumulation of these potent carcinogens has been sought for over three decades. However, MLRs from Mexico have not been evaluated as potential sources of resistance. Variation in susceptibility to both A. flavus reproduction and aflatoxin contamination was evaluated on viable maize kernels in laboratory experiments that included 74 MLR accessions collected from 2006 to 2008 in the central west and northwest regions of Mexico. Resistant and susceptible MLR accessions were detected in both regions. The most resistant accessions accumulated over 99% less aflatoxin B1 than did the commercial hybrid control Pioneer P33B50. Accessions supporting lower aflatoxin accumulation also supported reduced A. flavus sporulation. Sporulation on the MLRs was positively correlated with aflatoxin accumulation (R ~ 0.5336, P , 0.0001), suggesting that resistance to fungal reproduction is associated with MLR aflatoxin resistance. Results of the current study indicate that MLRs from Mexico are potentially important sources of aflatoxin resistance that may contribute to the breeding of commercially acceptable and safe maize hybrids and/or open pollinated cultivars for human and animal consumption.
The center of origin of maize, the critical staple food for billions of people across the globe, is located in Mexico. Molecular analyses indicate that maize was domesticated from teosinte in a single event around 9,000 YBP (24). Remnants associated with ancient human shelters in the Central Balsas Valley of Mexico indicate the use of primitive maize by pre-Hispanic communities around 8,700 YBP (30). Today, across rural Mexico, a great array of 59 maize landraces (MLRs) is harbored in living genetic libraries that were passed through generations by indigenous people in traditional agroecosystems (44). Genetic diversity and diverse ecological adaptations exist within and among MLR populations; each MLR has unique chemical and physical characteristics shaped over thousands of years by human preference for foods, feeds, and other uses (37, 39). * Author for correspondence. Tel: 520-626-5049; Fax: 520-626-5944; E-mail:
[email protected]. { Present address: Department of Plant Pathology, University of California, Davis, Kearney Agricultural Research and Extension Center, Parlier, CA 93648, USA.
Maize is frequently contaminated with aflatoxins, which are highly toxic, carcinogenic, and teratogenic mycotoxins, when maize ears become infected with Aspergillus flavus and closely related fungi (3, 22, 29, 47). Other important crops susceptible to aflatoxin contamination include cottonseed, chilies, peanuts, and tree nuts (9, 14, 21, 38). Aflatoxins are detrimental to the health of both humans and domesticated animals (5, 35, 43), causing hepatocellular carcinoma, acute hepatitis, cirrhosis, immune system suppression, and other negative effects (14, 23, 29, 51). High temperature (.27uC) and relative humidity (.85%) are ideal for proliferation of aflatoxin-producing fungi and subsequent crop contamination (2, 3, 47). Thus, maize production in tropical and subtropical regions increases the likelihood of contamination. Fungi produce four major aflatoxins: B1, B2, G1, and G2 (35); aflatoxin B1 is the most prevalent and also the most toxic (19, 34). Several Aspergillus section Flavi species produce aflatoxins. Within a species, aflatoxin-producing ability may be consistent and high (i.e., Aspergillus parasiticus) or may be highly variable, with many individuals producing little or no aflatoxin (i.e., A. flavus). Some species produce only B
J. Food Prot., Vol. 77, No. 9
AFLATOXIN SUSCEPTIBILITY AMONG MAIZE LANDRACES FROM MEXICO
aflatoxins, whereas others produce both B and G aflatoxins (8, 9, 17, 19). Traditional plant disease management methods are inconsistent in prevention of aflatoxin contamination. Optimized cultural practices combined with pesticide applications have not been effective and may negatively impact environmental safety (2). Isolates of A. flavus that do not produce aflatoxins (atoxigenic) are currently used in both North America and Africa as biopesticides that competitively displace aflatoxin-producing fungi and, in so doing, reduce aflatoxin accumulation, typically by more than 80% (11, 12, 15, 16). Development of maize hybrids with increased preharvest resistance to both fungal reproduction and aflatoxin accumulation (3) may both supplement the biocontrols and provide a low-cost, easily dispersed independent management alternative. Although maize resistance to aflatoxin contamination has been sought for well over three decades and resistant germplasm has repeatedly been identified (1, 7, 26, 27, 40, 42, 45, 48–50, 52), commercially acceptable resistant cultivars have not been developed. Traditional agroecosystems are important sources of germplasm for development of cultivated plants (28), and MLRs are potential sources for aflatoxin resistance (31). However, it is unclear if sources of resistance exist within the great genetic diversity of heirloom MLRs traditionally maintained by people across Mexico. The extent to which exposure to aflatoxins is influenced by consumption of traditional MLRs compared to advanced high-yielding hybrids is unknown. MLRs are consumed within producing villages and, as such, are not checked for aflatoxin contamination. Influences of MLRs on aflatoxin exposures and resulting health impacts in extant rural Mexican communities have not been studied. The current study sought to assess variability in susceptibility to aflatoxin contamination within and among MLRs endemic to Mexico by evaluating the ability of A. flavus to contaminate mature viable maize grain from a diverse sampling of 74 accessions of eight MLRs from the central west and northwest regions of Mexico. The results suggest that diversity in susceptibility to aflatoxin contamination occurs both within and among MLRs. Several accessions express high levels of resistance, with accumulation of less than one-tenth the aflatoxin concentration accumulated in the commercial hybrid control. MATERIALS AND METHODS Germplasm. MLR accessions were collected from the central west and northwest regions of Mexico, at elevations ranging from 100 to 2,100 m (Fig. 1). The first region included locations in the states of Sinaloa and Nayarit in central west Mexico. Twenty-two accessions of MLR Tabloncillo were collected from this region in 2006. The second region was sampled in 2007 and 2008 in various locations across the state of Sonora in northwest Mexico. Collections from the second region included 52 accessions from eight MLRs: Tabloncillo (18), Blando de Sonora (10), Onaven˜o (7), Vanden˜o (6), Chapalote (3), Reventador (4), Tuxpen˜o (2), and Dulcillo (2). Samples were imported into the United States under a U.S. Department of Agriculture (USDA) Animal and Plant Health Inspection Service permit and were maintained at the USDA Agricultural Research Service Aflatoxin Research Laboratory in
1555
FIGURE 1. Map of Mexico with collection sites for maize landrace (MLR) accessions. Symbols may represent more than one MLR accession. Black stars, black diamonds, and black triangles correspond to 2006, 2007, and 2008 sampling sites, respectively. the School of Plant Sciences, University of Arizona, Tucson. MLR accessions were dried in a forced-air oven at 60uC for 2 days and then were stored in sealed bags and kept under refrigeration (4uC) until use. The commercial hybrid standard used in each experiment was Pioneer maize hybrid P33B50 (from here on referred to as P33B50; Pioneer Hi-Bred International Inc., Johnston, IA). Inoculum. A. flavus isolate AF13 from agricultural soil in Arizona (9) is frequently used in maize resistance studies (4, 5); it was used in the current study because of its well-characterized virulence (9, 25) and consistent production of high concentrations of aflatoxins in maize (4, 25). Conidia from 5- to 7-day-old cultures (31uC, dark) grown on 5-2 agar (5% V-8 juice, 2% bactoagar [pH 5.2]) (9) were collected with a cotton swab and suspended in sterile deionized water. Conidial suspensions were quantified by turbidity using an Orbeco-Helling digital direct reading turbidimeter (Orbeco Analytical Systems Inc., Farmingdale, NY) and a nephelometric turbidity unit (NTU) versus CFU standard curve (y ~ 49,937x; x ~ NTU, y ~ spores per ml) (25) and were diluted to a final inoculum concentration of 2 | 106 conidia per ml. Inoculation of maize material. Mature kernels of each MLR were subjected to screening for postharvest resistance to aflatoxin contamination and for variation in the extent to which sporulation by A. flavus is supported during infection of viable kernels. Three experiments were conducted. In the first experiment, MLRs collected in 2006 (22 entries total) were compared with grain of P33B50. The second experiment compared the first set of MLRs collected in 2007, P33B50, and the most resistant and most susceptible MLRs from 2006 (14 entries total). The third experiment compared the second set of MLRs collected in 2007, MLRs collected in 2008, the most susceptible MLR from 2006, and P33B50 (44 entries total). Grain was sorted to remove damaged and undersized kernels. Sorted kernels (10 g) were surface disinfected by immersion in hot water (80uC, 40 s), aseptically blotted free of surface moisture, and allowed to sit at room temperature in a biological safety cabinet for
1556
ORTEGA-BELTRAN ET AL.
10 to 15 min before transfer to sterile 250-ml Erlenmeyer flasks. Kernel water content was determined with a HB43 halogen moisture analyzer (Mettler Toledo, Columbus, OH). Spore suspensions were adjusted and combined with the appropriate volume of water to bring kernel water content to 25%. Kernels were inoculated with approximately 350,000 spores per g. After inoculation, flasks were covered with gas-permeable stoppers (BugStopper, Whatman, Piscataway, NJ) and positioned in a randomized complete block design (31uC, 5 days, dark). Five kernels of each maize entry were incubated (31uC, 5 days) after surface disinfection on modified rose Bengal agar (10) to verify kernel viability and absence of A. flavus. Two independent trials were performed for each experiment. The number of replicates was partially dependent on the quantity of grain available. Each replicate consisted of a single flask. Aflatoxin quantification. Fungal growth was terminated by addition of 50 ml of methanol. Flasks were then swirled by hand (60 s) to dislodge the spores from the kernels. One milliliter was removed for spore quantification. The remainder was blended with a laboratory blender (Waring Commercial, Torrington, CT) for 15 s in a 110-ml stainless steel blending jar (MC-2) for aflatoxin quantification. The analytical mill was washed thoroughly with 80% ethanol between replicates to avoid crosscontamination of aflatoxin B1 among replicates. Homogenates were directly spotted (4 ml) in duplicate alongside aflatoxin standards on thin-layer chromatography plates (silica gel 60, EMD Millipore, Darmstadt, Germany) and developed with diethyl ether–methanol–water (96:3:1). Plates were visualized under UV light (365 nm), and presence or absence of aflatoxins was scored visually. Aflatoxins were quantified directly on thinlayer chromatography plates with a scanning densitometer (CAMAG TLC Scanner 3) and quantification software (winCats 1.4.2) (both from Camag AG, Muttenz, Switzerland) as previously described (13, 32). Samples from which aflatoxin B1 was not initially detected were combined with 100 ml of water and extracted twice with 25 ml of methylene chloride. Extracts were passed through a bed (25 g) of anhydrous sodium sulfate contained in fluted Whatman no. 4 filter paper, combined, and evaporated to dryness (13). Residues were solubilized in an appropriate volume of methylene chloride for accurate densitometry and quantified as above. The limit of quantification for all experiments was 20 mg/kg. Variability among MLRs in support of A. flavus reproduction. Production of conidia was quantified to determine A. flavus reproduction on MLRs. Spore suspensions (1 ml of the 50 ml total) washed from infected kernels were diluted 20-fold with 50% methanol. Turbidity was quantified in NTUs as described above. Relationship between resistance to fungal reproduction and aflatoxin accumulation. The relationship between aflatoxin B1 production and reproduction by AF13 was examined using data from all three experiments. Data from three different experiments were incorporated in a single analysis; for each experiment, MLR values were converted to percentages of the value for P33B50, the susceptible commercial hybrid used in all experiments. This allowed inclusion of data in a single analysis, using data from tests in which aflatoxin and/or NTU values differed markedly (i.e., NTU values for P33B50 were 79, 44, and 49 in the first, second, and third experiments, respectively). As a result of the transformation, values for P33B50 were 100% for all three experiments and were excluded from the analysis.
J. Food Prot., Vol. 77, No. 9
Statistical analyses. Aflatoxin B1 concentrations in mg/kg and spores per g in NTUs were log transformed to normalize variances. Experiments were performed with completely randomized designs, and resulting data was subjected to analysis of variance and means were separated with Fisher’s least significant difference test. All statistical tests were performed using SAS 9.2 (SAS Institute, Cary, NC). A relative resistance factor to toxin (RRFT) for each accession was calculated to allow comparisons among experiments (RRFT ~ P33B50 aflatoxin B1 accumulation/ MLR aflatoxin B1 accumulation). RRFT values less than 1 indicate greater aflatoxin accumulation in the MLR than in P33B50. A relative resistance factor to reproduction (RRFR) for each accession was also calculated to allow comparisons among experiments (RRFR ~ AF13 reproduction in P33B50/AF13 reproduction in MLR). Pearson’s correlation analysis using the CORR procedure was used to test relationships between aflatoxin accumulation and fungal reproduction.
RESULTS AND DISCUSSION Aflatoxin accumulation. Significant differences were detected among MLR accessions in aflatoxin B1 accumulation in each of the three experiments; and, in each case, results from the two independent trials were similar, allowing results to be combined. P33B50 grain consistently accumulated significantly (P , 0.05) more aflatoxin than the most resistant accession. Across the three experiments, aflatoxin B1 accumulation ranged from 0 to 164 mg/kg (Table 1). The first experiment was composed only of accessions of MLR Tabloncillo. Large differences in aflatoxin accumulation were detected among accessions of this MLR. MLR 2006-23 was the most resistant, supporting accumulation of only 5 mg/kg aflatoxin B1, significantly less than the other 14 maize entries, including P33B50. In experiment 1, P33B50 allowed the highest quantity of aflatoxin B1 (116 mg/kg), more than twice the aflatoxin B1 accumulated in 17 of the included MLRs accessions. RRFT of accessions in this experiment ranged from 1.6 to 22.2. The second and third experiments were composed of a more diverse collection of MLRs than the first (six and eight MLRs in the second and third experiments, respectively). Vanden˜o 2007-06, the most resistant accession in the second experiment, accumulated less than 20 mg/kg aflatoxin B1 and significantly (P , 0.05) differed from other accessions in the experiment. Accession 2006-23, the most resistant from experiment 1, accumulated 19 mg/kg aflatoxin B1 in experiment 2. Blando de Sonora 2007-37, the most resistant accession in the third experiment, accumulated less than 6.5 mg/kg aflatoxin B1, significantly (P , 0.05) less than any of the other 43 accessions in experiment 3 (Table 1). In the second experiment, RRFT for accessions ranged from 0.93 to 152,049 (although 2007-06 did not support detectable aflatoxin accumulation, a value of 20 mg/kg aflatoxin B1 was assigned to this accession for the purpose of calculating the RRFT). In the third experiment, nine accessions accumulated more aflatoxins than P33B50 and had RRFT values below 1. RRFT ranged from 0.64 to 15.81 in experiment 3 (Table 1). Zuber et al. (1983) found that open pollinated varieties were generally more susceptible to preharvest aflatoxin contamination than hybrids. This is contrary to results of the
Tabloncillo Tabloncillo
Tabloncillo
Tabloncillo
Tabloncillo Tabloncillo Tabloncillo Tabloncillo Tabloncillo Tabloncillo Tabloncillo Tabloncillo Tabloncillo
Tabloncillo Tabloncillo Tabloncillo Tabloncillo Tabloncillo Tabloncillo Tabloncillo Tabloncillo Tabloncillo Tabloncillo Tabloncillo Blando de Sonora Chapalote Onaven˜o Tabloncillo
Tabloncillo
Vanden˜o
2006-03
2006-04
2006-05 2006-07 2006-08 2006-09 2006-10 2006-11 2006-11 2006-11 2006-13
2006-14 2006-15 2006-16 2006-17 2006-18 2006-19 2006-20 2006-21 2006-22 2006-23 2006-23 2007-01 2007-02 2007-03 2007-04
2007-05
2007-06
Raceb
2006-01 2006-02
Maizea
San Juan
Pinto amarillo
Pepitilla Jazmı´n Platanen˜o Maı´z blanco Zorrita de ocho Carrasco Zorrita Zorrita Carrasco Blanco bola Blanco bola Maı´z blando Chapalote Pirineo Pinto morado
Morado hı´brido Morado blanco rojo Blanco negro morado Chaquira chinabime Criollo amarillo Maı´z morado Blandito amarillo Serrano rojo Serrano morado Maı´z negro Maı´z negro Maı´z negro Maı´z ventanilla
Common namec
La Palmita, NAY, N 21u519300, W 104u42935.10 La Palmita, NAY, N 21u519300, W 104u42935.10 La Palmita, NAY, N 21u519300, W 104u42935.10 La Palmita, NAY, N 21u519300, W 104u42935.10 La Palmita, NAY, N 21u519300, W 104u42935.10 La Palmita, NAY, N 21u519300, W 104u42935.10 La Palmita, NAY, N 21u519300, W 104u42935.10 La Palmita, NAY, N 21u519300, W 104u42935.10 Cie´nega del Mango, NAY, N 22u00938.30, W 104u48904.60 Santa Marı´a, SIN, N 23u06931.30, W 105u41953.40 Maloya, SIN, N 22u55959.30, W 105u35925.40 Buenavista, SIN, N 22u57900.10, W 105u36910.40 Maloya, SIN, N 22u55959.30, W 105u35925.40 Cabaza´n, SIN, N 23u54945.70, W 106u30920.80 Cabaza´n, SIN, N 23u54945.70, W 106u30920.80 El Rinco´n, SIN, N 24u15930.60, W 106u47912.60 Paredo´n Colorado, SIN, N 24u05945.60, W 106u40946.90 Ipucha, SIN, N 24u20904.60, W 106u45953.20 Boca de Arroyo, SIN, N 25u19934.30, W 107u18949.70 Boca de Arroyo, SIN, N 25u19934.30, W 107u18949.70 Cochibampo, SON, N 27u10919.80, W 108u49931.30 Cochibampo, SON, N 27u10919.80, W 108u49931.30 Cochibampo, SON, N 27u10919.80, W 108u49931.30 Labor de Santa Lucı´a, SON, N 26u54934.30, W 108u50928.40 Labor de Santa Lucı´a, SON, N 26u54934.30, W 108u50928.40 Labor de Santa Lucı´a, SON, N 26u54934.30, W 108u50928.40
El Roble, NAY, N 21u30934.80, W 104u28902.40
La Palmita, NAY, N 21u519300, W 104u42935.10
La Palmita, NAY, N 21u519300, W 104u42935.10 La Palmita, NAY, N 21u519300, W 104u42935.10
Locationd
280
280
234 177 122 177 128 128 245 126 348 198 198 462 462 462 280
253 253 253 253 253 253 253 253 705
1,221
253
253 253
Above sea level (m)
2
2
1 1 1 1 1 1 1 1 1 1 2 2 2 2 2
1 1 1 1 1 1 2 3 1
1
1
1 1
Expte
—*
1.5*
7.1* 3.8 4.0 6.6 2.4 4.6* 2.5* 2.9 1.8 22.2* 7.9* 1.6* 0.9 1.4* 1.3
9.2* 2.4 5.5* 1.8 15.0* 1.6 1.1 0.8 2.2
2.2
2.8
1.8 3.7
RRFTf
G
D
BCD
CD
A
DE
FG
D
AB
ABC
BCD
CD
AB
ABCD
ABC
ABC
D
AB
ABCDE
ABC
AB
BCD
AB
BCD
AB
CD
ABC
ABC
ABC
AB
Toxin LSD testg
14.6*
3.6*
79.0* 26.3* 56.4* 35.9* 8.1* 9.9* 15.8* 65.8* 7.7* 8.2* 38.0* 3.9* 11.4* 5.2* 9.4*
8.4* 49.4* 65.8* 43.9* 65.8* 3.9* 2.4* 3.1* 19.8*
2.9*
12.0*
10.1* 39.5*
Spores/g RRFRh
F
CD
EF
DE
F
CD
F
DE
D
FG
DEFG
DEF
DE
FG
FG
EFG
G
DEFG
GH
B
C
FG
FG
FG
FG
DE
B
DEFG
FG
DEFG
Spores/g LSD testi
TABLE 1. Quantity of aflatoxin B1 produced by Aspergillus flavus AF13 on viable surface-disinfected kernels of 74 maize landrace accessions and a commercial hybrid incubated at 31uC for 5 days
J. Food Prot., Vol. 77, No. 9 AFLATOXIN SUSCEPTIBILITY AMONG MAIZE LANDRACES FROM MEXICO
1557
Reventador Blando de Sonora Reventador Tuxpen˜o Blando de Sonora Tabloncillo Reventador Tabloncillo Tabloncillo Tabloncillo Tabloncillo Tabloncillo
2007-09 2007-10 2007-11 2007-13 2007-14 2007-16 2007-17 2007-19 2007-20 2007-21 2007-22 2007-23
Mayobachi
Blando de Sonora Dulcillo Onaven˜o Tabloncillo
Tabloncillo Blando de Sonora Tabloncillo Blando de Sonora Tabloncillo Dulcillo Onaven˜o Onaven˜o
2007-31 2007-33 2007-34 2007-35
2007-36 2007-37 2007-38 2007-39 2008-02 2008-10 2008-14 2008-15
1,440 1,440
Rı´o Huahuasari, SON, N 28u30928.20, W 108u48928.10 Rı´o Huahuasari, SON, N 28u30928.20, W 108u48928.10 El Trigo de Corodepe, SON, N 28u18933.60, W 108u47933.60 El Trigo de Corodepe, SON, N 28u18933.60, W 108u47933.60 El Trigo de Corodepe, SON, N 28u18933.60, W 108u47933.60 El Trigo de Corodepe, SON, N 28u18933.60, W 108u47933.60 Agua Blanca, SON, N 28u329140, W 108u55928.40 Agua Blanca, SON, N 28u329140, W 108u55928.40 La Mesa, SON, N 28u239390, W 108u35918.50 Mesa del Campanero, SON, N 28u19950.50, W 109u00932.10 Ye´cora, SON, N 28u22913.10, W 108u55947.60 Ye´cora, SON, N 28u22913.10, W 108u55947.60 Ye´cora, SON, N 28u22913.10, W 108u55947.60 La Mesa, SON, N 28u239390, W 108u35918.50 Sahuaripa, SON, N 29u03909.10, W 109u13955.90 Guisamopa, SON, N 28u38943.40, W 109u06928.10 Tepache, SON, N 29u31956.10, W 109u31955.10 Tepache, SON, N 29u31956.10, W 109u31955.10
210 210 462 480 480 480 462 463 522 522 1,434 1,434
1,548 1,548 1,548 1,620 431 680 600 600
1,440 1,440 1,620 2,087
1,380
1,380
1,380
1,380
280
280
Above sea level (m)
Labor de Santa Lucı´a, SON, N 26u54934.30, W 108u50928.40 Labor de Santa Lucı´a, SON, N 26u54934.30, W 108u50928.40 La Isleta, SON, N 26u509370, W 108u549430 La Isleta, SON, N 26u51931.80, W 108u54916.70 Cochibampo, SON, N 27u10919.80, W 108u49931.30 Munihuasa, SON, N 27u08946.60, W 108u48906.30 Munihuasa, SON, N 27u08946.60, W 108u48906.30 Munihuasa, SON, N 27u08946.60, W 108u48906.30 Cochibampo, SON, N 27u10919.80, W 108u49931.30 Cochibampo, SON, N 27u10919.80, W 108u49931.30 La Estrella, SON, N 27u49940.80, W 109u14934.80 La Estrella, SON, N 27u49940.80, W 109u14934.80 Los Vallecitos, SON, N 28u25944.70, W 108u48940.20 Los Vallecitos, SON, N 28u25944.70, W 108u48940.20
Locationd
3 3 3 3 3 3 3 3
3 3 3 3
3
3
3
3
3 3
2 2 2 3 3 3 3 3 3 3 3 3
2
2
Expte
1.9 15.8* 1.2 0.7 1.4 3.2* 1.1 1.6
2.2* 9.5* 1.2 1.1
1.4
2.5*
0.9
0.9
7.4* 1.6
1.3 2.9* 0.9 1.4 1.7 4.3* 1.4 1.9 1.1 0.7 2.7* 1.1
4.5*
5.9*
RRFTf
HIJKLM
ABCDEFGHIJK
MN
GHIJKLM
AB
ABCDEFGHIJK
Q
IJKLM
ABCDEFGHIJK
BCDEFGHIJK
P
KLM
EFGHIJKL
LMN
ABCDEFG
ABCDEFGHI
DEFGHIJKL
OP
ABCDEFGHIJ
MN
ABCD
ABCDEFGHIJK
HIJKLM
BCDEFGHIJKL
NO
GHIJKLM
CDEFGHIJKL
A
EF
BCD
FG
FG
Toxin LSD testg
6.9* 26.5* 2.6* 2.9* 4.5* 12.9* 6.2* 4.1*
10.5* 23.9* 2.0* 2.9*
5.9*
2.6*
11.8*
5.4*
22.3* 6.0*
5.0* 11.7* 3.1* 2.2* 5.8* 14.0* 3.8* 5.7* 7.4* 7.6* 8.9* 12.0*
14.8*
27.3*
Spores/g RRFRh
IJ
LM
QR
IJK
FG
EF
R
LMNO
FG
C
R
NOPQR
KLM
EF
OPQR
JKL
KLM
R
PQR
MNOPQ
LMNOPQ
LMNOP
KL
HI
QR
KL
CD
C
F
D
F
F
Spores/g LSD testi ORTEGA-BELTRAN ET AL.
Pinto amarillo Maı´z blando Blanco del Pilar Maı´z blando Pinto amarillo Maı´z dulce Pinto amarillo Maı´z chermen˜o
Maı´z blando Maı´z dulce Olote delgado Pinto amarillo
Blando de Sonora Maı´z blando
2007-30
Maı´z blanco
Tabloncillo
2007-29
2007-27
2007-26
2007-24 2007-25
Vanden˜o
2007-08
Pinto amarillo
Common namec
Reventador Maı´z blando Reventador San Juan Maı´z blando Pinto amarillo Reventador Pinto morado Pinto amarillo Pinto amarillo Pinto amarillo Pinto amarillo elotero Tabloncillo Pinto amarillo Tuxpen˜o Hı´brido de Chihuahua Onaven˜o Maı´z blanco cristalino Blando de Sonora Maı´z blando
Tabloncillo
Raceb
2007-07
Maizea
TABLE 1. Continued
1558 J. Food Prot., Vol. 77, No. 9
Common namec
Onaven˜o Chapalote Onaven˜o Chapalote
Maı´z blanco Reventador Maizo´n Reventador oscuro Vanden˜o Maı´z blanco Vanden˜o Maizo´n Reventador Reventador Vanden˜o Maizo´n Tabloncillo Pinto amarillo Vanden˜o Maizo´n Tabloncillo Pinto amarillo Tabloncillo Maı´z blanco Blando de Sonora Maı´z blando Blando de Sonora Maı´z blando
Raceb
La Estancia, SON, N 29u47945.70, W 110u12943.80 La Estancia, SON, N 29u47941.40, W 110u12944.70 Aconchi, SON, N 29u499280, W 110u13930.40 Arizpe, SON, N 30u20913.10, W 110u10903.80 Arizpe, SON, N 30u20913.10, W 110u10903.80 Bacanuchi, SON, N 30u36917.60, W 110u14909.20 Nuri, SON, N 28u06955.70, W 109u19933.50 Nuri, SON, N 28u06955.70, W 109u19933.50 Mesa Tres Rı´os, SON, N 29u50930.40, W 108u42943.70 Mesa Tres Rı´os, SON, N 29u50930.40, W 108u42943.70
Moctezuma, SON, N 29u48913.80, W 109u40949.40 Hua´sabas, SON, N 29u54928.60, W 109u189040 Hua´sabas, SON, N 29u54928.60, W 109u189040 Moctezuma, SON, N 29u48913.80, W 109u40949.40
Locationd
610 606 617 850 850 1,050 368 369 1,880 1,880
630 546 546 630
Above sea level (m)
3 3 3 3 3 3 3 3 3 3 1 2 3
3 3 3 3
Expte
0.9 1.2 1.1 1.1 1.9* 1.1 0.9 1.3 1.2 1.7 1.0 1.0 1.0
0.9 1.1 1.3 0.8
RRFTf
ABCDEFGHI
AB
A
EFGHIJKLM
ABCDEFGHIJK
BCDEFGHIJK
ABCDEF
ABCDEFGHIJ
JKLM
ABCDEFGHIJ
ABCDEFGHI
ABCDEFGHIJK
ABCDEFG
ABC
CDEFGHIJKL
ABCDEFGHIJ
ABCDEFGH
Toxin LSD testg
2.4* 2.5* 6.0* 4.4* 2.8* 1.8* 5.6* 2.9* 3.7* 6.6* 1.0 1.0 1.0
5.3* 9.1* 3.2* 4.2*
Spores/g RRFRh
A
A
A
LMN
HI
FG
KL
B
EFG
IJK
KLM
EF
DE
IJ
GH
MNOPQ
JKL
Spores/g LSD testi
b
Code used to identify maize landrace (MLR) accessions used in the current study. Year collected followed by a serial number. Maize accessions were classified as belonging to MLRs based on morphological characteristics (39). c Common names of accessions may vary and/or be identical among and within MLRs dependent upon grower custom. d Location, longitude, and latitude where accession was collected. More than one accession may have been collected from a single field. NAY, Nayarit; SIN, Sinaloa; SON, Sonora. e Three different experiments were conducted with either four (experiments 2 and 3) or five (experiment 1) replicates. Each experiment was performed twice independently and, in each case, results from the two trials resulted in results sufficiently similar to allow combination of the trials. f A relative resistance factor to toxin (RRFT) was calculated by dividing aflatoxin accumulation in P33B50 with aflatoxin accumulation in MLRs. RRFT values lower than 1 indicate accessions that support greater aflatoxin accumulation than P33B50. Asterisks indicate accession values significantly different from P33B50 value from the corresponding experiment by Fisher’s protected least significant difference (LSD) test (a ~ 0.05). RRFTs were calculated with aflatoxin B1 values of either 8 or 10 replicates from two independent trials. The average of aflatoxin B1 accumulation in P33B50 was 116.1, 152.0, and 101.2 mg/kg for the first, second, and third experiment, respectively. g Means separation of log-transformed aflatoxin B1 concentrations was conducted using Fisher’s protected LSD test (a ~ 0.05). Values followed by different letters differ significantly. h A relative resistance factor to reproduction (RRFR) was calculated by dividing A. flavus spore yield on P33B50 with A. flavus spore yield on MLRs. RRFR values lower than 1 indicate accessions that support greater A. flavus reproduction than P33B50. RRFRs were calculated with NTU per gram values of either 8 or 10 replicates from two independent similar trials. Asterisks indicate accession values significantly different from P33B50 value from the corresponding experiment by Fisher’s LSD (a ~ 0.05). 1 NTU ~ 49,937 spores. The average NTU per gram in P33B50 was 79, 44, and 49 for the first, second, and third experiment, respectively. i Means separation of log-transformed NTU values was conducted using Fisher’s LSD (a ~ 0.05). Values followed by different letters differ significantly.
a
2008-31 2008-32 2008-34 2008-37 2008-40 2008-41 2008-43 2008-44 2008-45 2008-46 P33B50 P33B50 P33B50
2008-19 2008-20 2008-22 2008-28
Maizea
TABLE 1. Continued
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FIGURE 2. Relationship between spore production by Aspergillus flavus AF13 and aflatoxin production in viable MLR kernels, with values expressed as percentages of the P33B50 value for the corresponding experiment. Spore production and aflatoxin accumulation increase linearly (R2 ~ 0.358, P , 0.0001).
current study, in which open pollinated MLRs frequently accumulated lower aflatoxin concentrations than the commercial hybrid (Table 1). Relatively large standard error (SE) values were detected in most MLR accessions, regardless of the experiment, whereas relatively low SE values were detected in P33B50 across the three experiments (Table 1). Open pollination in rural fields may be responsible for a portion of the observed variability within MLRs. Thus, open pollination is not associated with increased susceptibility to aflatoxin contamination in MLRs. Traditional agro-ecosystems contribute to maintenance of traits associated with resistance to both pests and pathogens (6, 28). The current study indicates that these traits include resistance to aflatoxin contamination in MLRs. Reduced susceptibility of certain MLR accessions may have resulted from farmer selection of ears with less kernel rot. Mechanisms by which MLRs resist accumulation of aflatoxins are unknown. A. flavus reproduction. Reproduction by A. flavus on viable kernels ranged from 8,500 to 395,000 spores per g. P33B50 consistently allowed significantly (P . 0.05) more fungal reproduction than any MLR accession (Table 1). Increased fungal reproduction on kernels influences the speed of development of epidemics and, thus, the rate at which crops become infected and contaminated (25). A. flavus reproduction on MLRs was correlated with aflatoxin accumulation (R ~ 0.5336, P , 0.0001), when results from the three experiments were combined (Fig. 2). Different results were obtained when experiments were analyzed independently. The correlation coefficient (R ~ 20.08, P ~ 0.7293) between aflatoxin accumulation and fungal reproduction (spores per g) was not significant for the first experiment. In contrast, significant correlations were detected in both the second (R ~ 0.701, P ~ 0.0076) and the third experiments (R ~ 0.36, P ~ 0.0164). However, several accessions deviated from the association of resistance to reproduction with resistance to aflatoxin accumulation. In the first experiment, accession 2006-19 allowed the greatest A. flavus reproduction (138,000 spores per g), but it was among the least susceptible to aflatoxin accumulation (Table 1).
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Reproduction on each MLR was significantly (P , 0.05) less than on P33B50, regardless of experiment. RRFR ranged from 1.9 to 39.5 (Table 1). This is in contrast to aflatoxin accumulation, as several MLR accessions accumulated more aflatoxins than did P33B50. This is another demonstration that apparent long-term selection for reduced rot and fungal growth in MLRs by traditional farmers is not always associated with reduced aflatoxin accumulation. Selection for less fungal growth and reproduction is, on average, beneficial (Fig. 2), but increased resistance to aflatoxin accumulation is most reliably detected by direct quantification of aflatoxin. Similar observations have been made previously (3), and there are chromosomal regions that are associated with resistance to A. flavus reproduction but not associated with resistance to aflatoxin accumulation. Other chromosomal regions are associated with both traits (46). Multiple accessions of MLR Tabloncillo were collected in 2006; these varied widely in susceptibility to both aflatoxin contamination and fungal reproduction (Table 1). Great variability was also detected in accessions of other MLRs. Resistance to either aflatoxin contamination and/or fungal reproduction in an MLR accession might not be shared among other accessions of the same MLR. Neutral markers indicate that MLRs are genetically distinct morphological variants, and MLRs differ from both other MLRs and commercial hybrids (6, 24). However, there is diversity within MLRs, stemming either from isolation between villages or among microclimates or introgression from other Zea species. The current study detected considerable variation in susceptibility among accessions of single MLRs. This was particularly noticeable for MLR Tabloncillo, in which both highly resistant (supported undetectable quantities of aflatoxins) and highly susceptible (supported over 50,000 ppb aflatoxin B1) accessions were detected. Variability in aflatoxin susceptibility paired with overall reduced genetic variation in the MLR, as compared to Z. mays as a whole, may indicate that MLR Tabloncillo is an excellent background in which to identify genes for resistance to aflatoxin contamination. MLRs are preferred maize for human consumption in diverse communities because of adaptations to local conditions and preferred sensory characteristics (20, 33, 37, 39). This preference has the potential to influence human exposure to aflatoxins. Although a few MLRs accumulated similar quantities of aflatoxins as the commercial hybrid in this study, overall MLRs accumulated 62.2% lower concentrations of aflatoxins than the commercial hybrid, and several MLRs accumulated less than 10% the aflatoxin concentration accumulated by the commercial hybrid. Thus, use of MLRs as significant sources of calories may reduce exposure of human communities to aflatoxins as compared to consumption of modern cultivars. Adoption of high-yield modern hybrids will result in greater yields and potentially increased income. However, care must be taken to assure that hybrid adoptions are not associated with health effects from increased exposure to aflatoxins.
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AFLATOXIN SUSCEPTIBILITY AMONG MAIZE LANDRACES FROM MEXICO
Billions of people across the globe rely on maize as a primary source of food. In some developing countries, including Mexico, maize consumption contributes more than half of the total caloric intake (18). Aflatoxinproducing fungi are ubiquitous in warm production areas. However, aflatoxin concentrations of vulnerable crops, including maize, are seldom quantified, and commodities are used without knowing their suitability for human and/or animal consumption. In developed countries, aflatoxins are monitored on a regular basis, and crops exceeding permissible aflatoxin levels are removed from consumption, resulting in association of contamination with economic loss (12, 36, 41, 51). Aflatoxin contamination might be limited by integrating resistant germplasm into traditional maize breeding. Results of the current study indicate that MLR accessions (2006-23, 2007-06, 2007-07, 2007-37, among others) from Mexico are potentially important sources of aflatoxin resistance that may contribute to the breeding of commercially acceptable and safe maize hybrids and/or open pollinated cultivars for human and animal consumption. Mechanisms of resistance to aflatoxin contamination in MLRs should be elucidated in order to facilitate identification of additional lines with increased resistance and to aid development of resistant hybrids. ACKNOWLEDGMENTS This study was part of the first author’s doctoral dissertation and was funded by the USDA Foreign Agricultural Service, Scientific Cooperation Research Program, USDA Agricultural Research Project 5347-42000-0200D and by CONACyT-Mexico (scholarship 309103).
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