I<.. ,_' w, .. ~ , , ' f ". g E.. . r t ' is j .. ' ,r s: [ CONTENTS Page INTRODUCTION .................................................. MATERIALS AND METHODS.......................................5 RESULTS........................................................8 DISCUSSION .................................................... 3 18 Preharvest Contamination ............................... 18 Postharvest Contamination............................... 18 Genetic Control........................................ 19 SUMMARY ...................................................... LITERATURE CITED............................................. 20 21 FIRST PRINTING 3.5M, NOVEMBER 1985 ACKNOWLEDGMENTS The authors gratefully acknowledge the assistance of the following: Emmett L. Carden, superintendent of the Gulf Coast Suhstation; W B. Webster, superintendent of the Tennessee Valley Substation; W E. Brown, former superintendent of the Brewton and Monroeville Experiment Fields; J. R. Akridge, superintendent of the Brewton and Monroeville Experiment Fields; J. A. Pitts, superintendent of the Chilton Area Horticulture Substation; J. T Eason, superintendent of the Sand Mountain Suhstation; J. K. Boseck (deceased), former superintendent of the Tennessee Valley Substation; W. A Griffey, superintendent of the Piedmont Substation; J. W Langford, retired, former superintendent of the Plant Breeding Unit; F T Glaze, retired, former superintendent of the Prattville Experiment Field; and J. G. Starling, retired, former superintendent of the Wiregrass Substation. Information contained herein is available to all persons without regard to race, color, sex, or national origin. Response of Corn Hybrids to Aflatoxin Formation byAspergillus flavus' NORMAN D. DAVIS, CLIFFORD G. CURRIER, and URBAN L. DIENER2 INTRODUCTION vus (yellow mold) was first reported in 1920 by Taubenhaus (36), who also recognized that the fungus was a saprophyte that required kernel damage in the milky state for invasion. From 1930 to 1959, similar observations were made by investigators in Florida (15), Illinois (21), Kansas (26), and Indiana (39) prior to the discovery of the toxic effects of aflatoxin in peanuts in 1961 (22) and its chemical characterization in 1963 (2). Moldy corn toxicosis, probably caused by aflatoxin, occurred in the fall of 1952 with 24 outbreaks being recorded in Georgia; 23 of these affected swine and one affected cattle (34). Burnside et al. (4) isolated A. flavus and Penicillium rubrum from the toxic corn and found that these two fungi, when cultured on sterilized corn and fed to swine, reproduced symptoms of moldy corn toxicosis. Aflatoxin in corn was considered primarily a stored grain problem during the 1960's. Anderson et al. (1) first demonstrated preharvest contamination of corn with aflatoxin in 1971 following a comprehensive survey and sampling program involving essentially all of the corn-producing areas of the United States. Aflatoxin was found in the first field sampling when the corn was in the late milk stage of development. However, there appeared to be no further increase in aflatoxin levels in corn left standing in the field up to 3 months after normal harvest time. The highest incidence of aflatoxin was found in 'This report is based on research supported by Regional Project S-175, "Mycotoxins of Corn and Other Feed Grains." 2 Professor of Botany, Plant Pathology, and Microbiology; former Research Associate in Agronomy and Soils (now in Agronomy Department at New Mexico State University); and Professor of Botany, Plant Pathology, and Microbiology. CONTAMINATION OF CORN (Zea mays L.) by Aspergillusfla- 4 ALABAMA AGRICULTURAL EXPERIMENT STATION corn from the warmer and more humid growing regions, i.e. the Southeastern States. In 1972, aflatoxin was detected in 30 percent of 1,283 truckloads (12,000 tons) of white corn (1971 crop) delivered to an elevator from seven counties in southeastern Missouri (33). Other investigations soon confirmed the contention that preharvest contamination of corn by A. flavus and aflatoxin was widespread and prevalent in the Southeast (17,23,24,30,32). Obviously, utilization of corn hybrids resistant to invasion by A. flavus and aflatoxin formation would be the most direct way to prevent or minimize aflatoxin contamination; consequently, considerable research has been conducted to identify such resistance in corn hybrids. In one experiment (41), 10 full-season and 10 short- to mid-season hybrids from three locations in the Georgia Coastal Plains were evaluated for aflatoxin levels resulting from natural infection by A. flavus during 1974-76. In each group, one hybrid had significantly more aflatoxin than the other 9 hybrids, but no differences were detected among 30 dent and 15 sweet corn inbreds. In a 1979-80 study (35,43), four widely grown hybrids and eight open-pollinated varieties were grown in seven Southeastern States and Hawaii to determine susceptibility to preharvest aflatoxin contamination. It was found that aflatoxin incidence and levels were correlated with location and crop year, but not with genotype, except for one highly susceptible variety. In another study (25), 26 corn hybrids from field trials in South Carolina were evaluated for interrelationships among aflatoxin level, moisture content at harvest, and bright greenish-yellow (BGY) fluorescence resulting from natural field infection by A. flavus. Results showed that kernels of short-season hybrids contained elevated levels of aflatoxin, moisture, and BGY fluorescence as compared to mid-season and full-season hybrids. No hybrid showed significant resistance to aflatoxin. The effects of kernel injury, irrigation, inoculation of uninjured kernels with A. flavus spores, and natural insect damage on aflatoxin formation in 15 commercial hybrids (5 each of short-, mid-, and fullseason) were evaluated during 1979 and 1980 in South Carolina (16). No hybrid showed consistently lower aflatoxin levels in comparison to the other 14 hybrids, regardless of whether kernels were injured prior to inoculation. Also, there was no difference in the level of contamination of the three maturity groups whether irrigated or not. In a study by Thompson et al. (38), developing kernels of eight maize single crosses (two A-inbreds X four B-inbreds) were inoculated with A. flavus after kernel injury via pinboard in four environments and assayed for aflatoxin. Two B-inbreds averaged 50 percent less aflatoxin RESPONSE OF CORN HYBRIDS TO AFLATOXIN FORMATION 5 than the other two B-inbreds (17-23 p.p.m. vs. 30-42 p.p.m.). The authors, therefore, suggested from this study that susceptibility or resistance to aflatoxin accumulation in developing kernels was under genetic control. Widstrom et al. (40) grew an eight-line diallel of sweet corn single crosses and a nine-line diallel of dent single crosses in replicated experiments for 3 years (1978-81) to identify resistant genotypes for incorporation into a breeding population for recurrent selection. Ears were knife-inoculated 20 days after full silk with an A. flavus spore suspension and grain was analyzed at maturity for aflatoxin contamination. The data led these authors to conclude that two or three inbreds within each set performed well enough as single crosses to be used as sources of resistance. They also noted that experimental inoculation methods seldom produced enough contamination to allow them to identify resistant genotypes. Thus, data obtained by injury plus A. flavus spore inoculation of kernel, ear, and silk were suspected of masking practical field resistance that might be present as well as failing to yield infection levels adequate to differentiate among genotypes (20). The research reported in this bulletin was initiated in 1976 on a small scale, but after the aflatoxin epidemic of 1977 and the development of the fluorometric-iodine rapid screen method of analysis (12), large numbers of hybrids were screened for possible resistance to natural field contamination. This report presents results of aflatoxin analyses of samples of 215 commercial hybrids and breeding lines from 20 seed companies that were grown at 12 locations throughout Alabama during the 6 years from 1976 to 1981. MATERIALS AND METHODS Corn hybrid yield trials were conducted at 12 outlying units of the Alabama Agricultural Experiment Station. Locations in southern Alabama were: Brewton Experiment Field, Brewton; Gulf Coast Substation, Fairhope; Monroeville Experiment Field, Monroeville; and Wiregrass Substation, Headland. Locations in central Alabama were: Black Belt Substation, Marion Junction; Lower Coastal Plain Substation, Camden; Piedmont Substation, Camp Hill; Prattville Experiment Field, Prattville; and E. V. Smith Research Center, Shorter. Locations in northern Alabama were: Sand Mountain Substation, Crossville; Tennessee Valley Substation, Belle Mina; and Upper Coastal Plain Substation, Winfield. Corn hybrids were grown with recommended plant populations, fertilizer rates, and cultural practices (5-10). Corn ears were shucked 6 ALABAMA AGRICULTURAL EXPERIMENT STATION TABLE 1. CORN HYBRIDS CROWN FOR 1-3 YEARS AT ONE OR MORE LOCATIONS IN ALABAMA, 1976-81 Hybrid ident. and years x location tested Asgrow RX-90 ............. 5 RX-112 ............ 2 RX-114 ............ 7 RX-140 ............ 1 RX-140A ........... 9 RX777 ............ 2 RX-909 ............ 5 Aztec SX-640 ............. 1 SX-644 ............. 1 Big D (Taylor) 2 2249 .............. 4204 .............. 2 4791 .............. 5 4862 .............. 12 6986 .............. 2 7220 .............. 2 Cargill (Security) 495 ............... 4 949 ............... 4 951 ............... 1 967 ............... 1 88-111 ............. 1 SS-112 ............. 8 Coker (Greenwood) 18................ 17 18A ............... 2 13 19................ 19A .............. 19 21 ................ 2 44 ................ 2 45 ................ 1 54................ 13 4034 .............. 1 DeKalb XL-71............. 2 XL-72AA ........... 8 XL-72BB ........... 2 XL-78 ............. 15 XL-80A ............ 3 XL-8OB............4 XL-82 ............. 6 XL-95 ............. 2 XL-390 ............ 2 XL-390A ........... 3 2 XL-390B ........... XL-395 ....... 9 XL-395A ........... 7 2 XL-808 ............ XL-1214 ........... 1 XL-1295 ............ 1 EX-8914 ........... 1 85275.............. 1 Hybrid ident. and years x location tested Funks C-4323 .......... C-4522 .......... G-4525 .......... G-4574 .......... C-4606-1 .......... C-4636 .......... G-4657 .......... C-4689 .......... G-4709 .......... G-4733 .......... G-4740A ......... G-4768-W ......... C-4787-W ......... C-4848-2 .......... 28753 ........... 29018 ........... 29092 ........... 29258 ........... Golden Harvest H-2606 .......... H-2660-W ......... H-2665-W ......... H-2666 .......... H-26830.......... H-2686A .......... H-2740A .......... H-2745 .......... H-2750 .......... H-2655 .......... Goldkist (Green) GK-695 ........... GK-748 ........... GK-875 ........... GK-915 ........... GK-925 ........... GK-955 ........... CK-1055 .......... CK-1175 .......... FFR-2325 ......... Gutwein 62 ............... 72 .............. 74 .............. 2875 ............ 2880 ............. 2910 ............. MDM-2885... Jacques (Wilstar) JX-177 ............ JX-179 ............ JX-180 ............ 1 2 5 4 34 2 2 7 8 3 20 2 4 8 4 2 1 20 5 8 2 3 6 2 5 1 1 3 1 9 2 11 1 6 1 2 1 18 8 6 5 2 22 3 5 2 10 Hybrid ident. and years x location tested Jacques (Wilstar) cont'd. 1 JX-227 ............. JX-247 ............. 4 W-200 ............. 2 W-300 ............. 7 5555 .............. 7 6662 .............. 9 McCurdy 72-24..............3 72-44A ............. 4 75-200 ............. 5 75-210 ............. 3 76-92 .............. 2 76-101 ............. 7 77-87............. 18 79-78 .............. 5 81-50 .............. 6 82-25..............2 1 X-890 .............. MSX-88 ............ 2 Northrup-King-McNair PX-72 ............. 1 4 PX-74 ............. PX-83............. 1 PX-87............. 8 2 PX-664 ............ PX-675............ 7 1 PX-707 ............. PX-7L8W...........4 13 PX-723 ............ 5 488 ............... 10 X-170 ............. X-233 .............. 3 2 EXP-3232 .......... P-A-G 751 ............... 5 SX-17A ............ SX-98 ............. SX-333 ............ SX-346............. SX-351 ............. SX-373 ............. 262-193 ............ 11 14 17 3 3 5 5 Paymaster 5 UC-8201 ........... 15 UC-9451 ........... 8 UC-9532 ........... 21 UC-9792 ........... 2 UC-9797 ........... 4 UC-9902 ........... 12 12052 ............. 12052A ............ 1 Continued RESPONSE OF CORN HYBRIDS TO AFLATOXIN FORMATION 7 TABLE 1 (CONTINUED). CORN HYBRIDS GROWN FOR 1-3 YEARS AT ONE OR MORE LOCATIONS IN ALABAMA, 1976-81 Hybrid ident. and years x location tested Pioneer 511-A ............. 519 ............... 3009 .............. 3040 .............. 3152 .............. 3160 .............. 3179 .............. 3183 .............. 3184 .............. 3311 .............. 3320 .............. 3535 .............. X-7227 ............ 27 3 20 8 2 10 5 5 14 1 12 2 1 Hybrid ident. and years x location tested RBA 111 ............... 122 ............... 2 1 Hybrid ident. and years x location tested Trojan T-1120 ............ T-1189 ............ T-1230 ............ TXS-113........... TXS-119A ......... USSAG 1515 .............. 2020 .............. 2315 .............. 2461 .............. 7501 .............. 2 5 7 1 3 14 15 4 17 2 2 Ring Around .............. 2 1401 AroundTXS-116........... 1504 .............. 1604 .............. 2501 .............. 2502 .............. 2601 .............. 3602 .............. 3605-W ........... 7 10 20 11 3 3 by hand at harvest, placed in burlap sacks, labelled with plot numbers, and dried to approximately 14-16 percent grain moisture content. Grain was removed from cobs with motorized shellers, weighed, and the moisture content (range 14 percent ± 2 percent) determined. A 1-pound sample of shelled corn was then taken from each hybrid, placed in a paper sack, which was labelled, stapled, and sent to Auburn University. All samples were ground in a hammermill to pass a 1-mm screen and stored at 15.5 percent moisture and 95-104°F until subsampled for aflatoxin analyses. Thirty-eight corn hybrids were grown for 4-6 years at one or more locations, sampled, and analyzed for aflatoxin: Coker 16, 22, 56, 77B; DeKalb XL-72B, 80, 394; Funks G-795W-1, 4507, 4507A, 4611, 4747W-1, 4776, 4810, 4864, 4949A, 5945; Golden Harvest H2500, 2775; McCurdy 67-14, 84AA; Northrup-King-McNair PX-79, 95, S338, X-300, 508; Paymaster UC-8951; Pioneer 3030, 3145, 3147, 3368A, 3369A; Ring Around 1501, 1502, 2602W; and Trojan TXS-114, 115A, 119. In addition, 177 corn hybrids that were grown for only 13 years are listed in table 1. Seed were supplied by seed companies for the agronomic trials, and the removal of old hybrids and the addition of new ones for testing were partly the decision of the seed producers. In the first 2 years of this study, a random selection of hybrids for analysis from several locations was made by E. L. Carden. However, after the severe aflatoxin outbreak of 1977, every hybrid grown at 12 locations was sampled and analyzed for several years. The number of hybrids times locations that were sampled and analyzed, table 1, varied from 1 to 34, with over 82 percent of the hybrids being tested less than 10 times; 31 hybrids were evaluated 10 to 34 times for aflatoxin. 8 ALABAMA AGRICULTURAL EXPERIMENT STATION The number of corn hybrid samples analyzed each year for aflatoxin were: 95 (1976), 86 (1977), 481 (1978), 668 (1979), 572 (1980), and 450 (1981) for a total of 2,352 analyses. Samples of corn were analyzed for aflatoxin by the Pons aqueous acetone method for cottonseed (29), as modified and described in AOAC Methods of Analysis section 26.052-26.060 in 1976 and 1977 (3). Analyses in 1978-81 were made by the fluorometric-iodine rapid screen method (FL-IRS) developed at Auburn by Davis and Diener (11,12) with one modification (13). RESULTS Data on the aflatoxin levels of 177 corn hybrids grown only 1-3 years at 12 locations are not presented, although the hybrids are identified in table 1. The pattern of data on aflatoxin occurrence of the hybrids in table 1 was essentially the same as that recorded in tables 2, 3, and 4. In years of low aflatoxin occurrence (1978, 1979), these hybrids showed little or no aflatoxin contamination. In 1977 and 1980, which were epidemic years for aflatoxin in Southeastern corn, they showed high levels (100-350 p.p.b) as did the other reported hybrids, tables 2, 3, and 4. In 1981, most hybrids had low levels of aflatoxin contamination (10-50 p.p.b.) at nearly every location, as did those listed in tables 2, 3, and 4. Data from the aflatoxin analyses of 38 corn hybrids grown at 12 locations in at least 4 of the 6 years from 1976 to 1981 are presented in tabular form, tables 2-4, by regional location within Alabama. Table 2, which shows the level of aflatoxin contamination of 26 corn hybrids grown in four southern locations near Brewton, Fairhope, Monroeville, and Headland, reveals high mean aflatoxin levels in the epidemic years of 1977 and 1980 of 1,180 and 209 p. p. b., respectively, compared with aflatoxin levels that were much lower in 1976, 1978, 1979, and 1981. In 1976, when only 19 samples of 15 hybrids, table 2, were analyzed, only 1 sample from Brewton was heavily contaminated (200 p.p.b.), while samples of 2 other hybrids from Headland contained about 50 p. p.b. of aflatoxin BI. In 1978, Headland was the only southern location which had samples appreciably contaminated, with 5 hybrids averaging 198 p. p.b., while 18 others were uncontaminated. In 1979, certain samples from Monroeville were thought to have been contaminated postharvest because of the high aflatoxin level that averaged 319 p.p.b. Only 3 hybrids from Headland were heavily contaminated and 18 were uncontaminated. Also, the 23 hybrids at Brewton and the 19 hybrids at Fairhope were uncontami- RESPONSE OF CORN HYBRIDS TO AFLATOXIN FORMATION 9 nated that year. In 1981, nearly every hybrid was contaminated, but at levels averaging only 19 p.p.b, in 66 hybrids at three locations (a trial was not carried out at Headland in 1981). In 1977, corn was under severe drought stress and high insect infestation throughout the State, resulting in practically every hybrid planted at all locations being contaminated with aflatoxin at levels exceeding 100 p.p.b. In 1977, four samples from Monroeville averaged 2,500 p.p.b. and 6 hybrids from Brewton averaged 300 p.p.b. Armyworm infestation was so severe at the other two locations that samples were deemed worthless for experimental purposes. In 1980, 91 of 99 samples tested contained aflatoxin at levels exceeding 100 p.p.b., with a mean of 210 p.p.b. Drought (water stress) exacerbated the level ofaflatoxin contamination as well as the incidence of contamination of all hybrids tested in 1980. Finally, the incidence of aflatoxin was high in 1981, but aflatoxin levels were low, averaging only 19 p.p.b. Both aflatoxin level and incidence were low in 1976, 1978, and 1979. Table 3 presents the aflatoxin contamination data for 21 corn hybrids grown in five central Alabama locations (Camden, Camp Hill, Shorter, Marion Junction, and Prattville). Mean aflatoxin levels were comparatively high in 1977 and 1980 (47 and 195 p.p.b., respectively) compared with only 5 p.p.b. in 1978, 9 p.p.b. in 1979, and 28 p.p.b. in 1981. The high mean level of aflatoxin calculated for 1976 (170 p.p.b.) is an anomaly and should be disregarded as this was skewed by a single sample's value of 2,000 p.p.b. from Shorter, probably due to postharvest contamination. Generally, in the five central Alabama locations, the incidence of aflatoxin contamination was high in 4 of the 6 years (1976, 1977, 1980, 1981), but the mean level of contamination was many times higher in 1980 than in 1981 (195 vs. 28 p. p. b.). Table 4 provides data on 22 corn hybrids grown in the three northern locations near Bella Mina, Crossville, and Winfield. Here, a high incidence and high mean level of aflatoxin contamination occurred in 1976 (413 p.p.b.), 1977 (66 p.p.b.), and 1980 (178 p. p.b.). Three highly contaminated samples (1,000-3,333 p. p. b.) from Crossville, believed to have resulted from postharvest contamination, distorted the data for 1976. Omitting these three samples from this one location, the mean aflatoxin level for 1976 was 21 p.p.b., similar to the low levels at other locations in 1978 (8 p.p.b.), 1979 (2 p.p.b.), and 1981 (24 p.p.b.). The incidence of aflatoxin-contamination was high in 1981 in the northern locations, as it was in the central and southern locations. Contamination was generally higher in hybrids grown at Crossville in 1980 than at the other northern locations. 10 ALABAMA AGRICULTURAL EXPERIMENT STATION Table 5 presents a summary of the means of the data on aflatoxin occurrence in Alabama in corn hybrids by region and year. The epi- demic years of 1977 and 1980 are obvious from these data. It is also obvious that the problem was more severe in the southern locations than elsewhere in the State. In addition, the number of samples ana- lyzed for each mean is indicated. Reasons for the notably smaller number of samples tested for aflatoxin in 1976 and 1977 are explained under materials and methods. TABLE 2. AFLATOXIN CONTAMINATION OF CORN HYBRIDS GROWN 4-6 YEARS AT FOUR SOUTHERN ALABAMA LOCATIONS, 1976-81 1976 Coker 16 Brewton .................. Fairhope .................. Monroeville ............... Headland ................. Coker 22 Brewton .................. Fairhope .................. Monroeville ............... Headland ................. Coker 77B Brewton .................. Fairhope .................. Monroeville ............... Headland ................. DeKalb XL-80 Brewton .................. Fairhope .................. Monroeville ............... Headland ................. DeKalb XL-394 Brewton .................. Fairhope .................. Monroeville ............... Headland ................. Funks G-4507A Brewton .................. Fairhope .................. Monroeville ............... Headland ................. Funks G-4611 Brewton .................. Fairhope .................. Monroeville ............... Headland ................. Funks G-4810 Brewton .................. Fairhope .................. Monroeville ............... Headland ................. 0 0 60 0 0 0 0 0 0 - 1977 2,000 6,000 100 - Aflatoxin B1 in p.p.b. 1978 1979 1980 0 0 0 0 0 0 0 475 0 0 5 0 0 0 0 0 0 0 0 0 0 46 5 0 0 0 0 0 0 7 59 0 360 0 0 460 0 0 0 480 42 0 300 0 0 70 0 0 500 0 0 0 340 0 0 0 350 0 115 114 200 96 105 108 208 120 250 360 199 375 105 102 200 75 105 108 260 106 135 120 350 465 261 288 235 145 400 138 450 20 1981 15 15 58 0 14 48 17 - 20 18 8 17 19 5 10 12 14 14 14 5 18 4 Continued RESPONSE OF CORN HYBRIDS TO AFLATOXIN FORMATION TABLE 2 (CONTINUED). AFLATOXIN CONTAMINATION OF CORN HYBRIDS CROWN 4-6 YEARS AT FOUR SOUTHERN ALABAMA LOCATIONS, 1976-81 11 1 Variety and location -- --- -- -- 1976 1977 250 Aflatoxin B1 in p. p.b. 1978 1979 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0 0 0 0 150 0 0 390 0 0 570 - 1980 180 108 300 160 103 102 172 160 90 60 73 1981 9 36 11 20 42 23 18 6 16 - Funks G-4864 Brewton................ Fairhope ................ Monroeville ............. Headland............... Funks G-4949A Brewton................ Fairhope ................ Monroeville............. Headland............... Golden Harvest H2500 Brewton................ Fairhope ................ Monroeville............. Headland............... Golden Harvest H2775 Brewton................ Fairhope ................ Monroeville................0 Headland..................40 McCurdy 67-14 Brewton................ Fairhope ................ Monroeville Headland............... McCurdy 84AA Brewton................ Fairhope ................ Monroeville............. Headland............... McNair S-338 Brewton ................ Fairhope ................ Monroeville ............. Headland ................... 0 McNair X-300 Brewton ................. Fairhope ................ Monroeville ............. 0 Headland ................... Northrup King PX-79 Brewton ................. Fairhope ................ Monroeville ............. Headland ................ Northrup King PX-95 Brewton ................. Fairhope ................ Headland ................ Pioneer 3030 Brewton ................... Fairhope ................ Monroeville ............. Headland ................ 1,240 0 0 750 0 0 0 90 0 0 370 0 0 0 110 0 0 0 560 0 0 0 150 0 0 0 120 0 117 175 101 196 310 252 350 225 405 400 167 300 170 108 106 255 140 110 120 190 255 125 33 196 265 105 112 196 240 9 10 4, 11 20 12 10 10 21 10 32 28 12 38 34 Continued 1,000 200 0 0 0 0 18 - 0 - 150 Monroeville ............. - 200 0 1,000 0 0 300 0 12 12 ALABAMA AGRICULTURAL EXPERIMENT STATION TABLE 2 (CONTINUED). AFLATOXIN CONTAMINATION OF CORN HYBRIDS CROWN 4-6 YEARS AT FOUR SOUTHERN ALABAMA LOCATIONS, 1976-81 Variety and location 1976 1977 I Aflatoxin B1 in p. p. b. 1978 1979 1980 0 0 220 0 0 0 570 0 0 109 150 165 240 256 299 250 175 120 110 - 228 - 600 400 150 400 125 208 238 260 465 0 0 230 120 110 37 65 37 71 106 200 70 266 298 210 225 209 40 76 39 37 1981 10 38 30 18 10 37 38 10 10 15 41 15 14 18 14 10 18 6 13 24 68 14 39 77 39 1 Pioneer 3145 Brewton.0 Fairhope .................. Monroeville................0 Headland.0 Pioneer 3147 Brewton.0 . .. Fairhope ......... Monroeville.9 Headland.0 Pioneer 3368A Brewton...................0 Fairhope .................. Monroeville................4 Headland.0 Pioneer 3369A Brewton.0 Fairhope .................. Monroeville................0 Headland.130 Ring Around 1501 Brewton...................... -Fairhope . . . . . .. . . . -Monroeville ........ Headland ......... -Ring Around 1502 Brewton.0 Fairhope .................. Monroeville . . . . . . . . -Headland ................... 0 Trojan TXS-114 Brewton...................... Fairhope . . . . . .. . . . -Monroeville ........ -Headland ......... Aflatoxin mean inp.p.b. -- - 0 0 0 - 0 0 1,000 0 0 0 0 10 0 0 166 0 0 0 330 1,300 0 175 0 0 0 0 0 0 0 250 of samples!/year............. Total varieties evaluated/year Brewton................... Fairhope................... Monroeville................ Headland.................. 15 11 0 10 8 1,180 10 0 10 1 12 39 43 40 73 RESPONSE OF CORN HYBRIDS TO AFLATOXIN FORMATION TABLE 3. AFLATOXIN CONTAMINATION OF CORN HYBRIDS CROWN 4-6 YEARS AT FIVE CENTRAL ALABAMA LOCATIONS, 1976-81 13 1 Variety and location ,._____ ._ 1976 1977 Aflatoxin B1 in p.p. b. 1978 1979 1980 5 0 38 0 2 0 0 0 0 0 0 0 0 0 0 0 4 0 0 15 0 0 0 0 0 0 49 0 26 10 120 110 70 240 277 185 60 118 100 186 69 266 246 166 302 320 96 295 80 360 100 325 163 283 300 170 284 160 280 168 146 350 190 460 288 1981 11 34 24 10 108 12 3 18 14 152 18 15 11 45 20 10 45 21 4 26 30 Continued (oker 16 Camden ................ Camp Hill................ Shorter................. Marion Junction ............. 4 Prattville ................ Coker 22 Camden................. Camp Hill................ Shorter................. Marion Junction.......... Prattville ................... 120 Coker 56 Camden ................ Camp Hill ............... Shorter...................2,000 Marion Junction .......... Prattville ................ Coker 77B Camden ................ Camp Hill................ Shorter-.......... Marion Junction .......... Prattville ................ Funks G-795-W-1 Camden ................ Camp Hill................ Shorter................. 4 Marion Junction ............. Prattville ................ Funks G-4507A Camden ................. Camp Hill.................. 20 Shorter ................. Marion - 20 200 0 - 1 0 27 0 0 0 0 0 0 0 2 0 22 0 0 0 0 28 0 0 0 0 1 0 0 0 - Prattville ................ Funks G-4611 Camden ................. Camp Hill ................ Shorter ................. Marion junction ............. 200 - - 0 0 0 150 0 0 41 0 20 0 0 0 0 0 0 0 0 0 3 0 Prattville ................ Funks G-4776 Camden ................. Camp Hill ................ Shorter ................. Marion junction .......... 0 Prattville ................ Funks G-4810 Camden ................. Camp Hill................... Shorter ................. Marion junction .............. 0 - - Prattville ................ junction .......... - 14 ALABAMA AGRICULTURAL EXPERIMENT STATION TABLE 3 (CONTINUED). AFLATOXIN CONTAMINATION OF CORN HYBRIDS GROWN 4-6 YEARS AT FIVE CENTRAL ALABAMA LOCATIONS, 1976-81 Variety and location 1976 1977 Aflatoxin B in p.p.b. 1978 1979 1980 0 0 25 0 0 0 27 0 0 0 0 0 10 0 0 0 - 1981 12 - Funks G-4949A Camden ................... Camp Hill ................. Shorter ................... Marion Junction ............ Prattville .................. Funks G-5945 Camden ................... Camp Hill ................. Shorter ................... Marion Junction ............ Prattville .................. McCurdy 67-14 Camden ................... Camp Hill ................. Shorter ................... Marion Junction ............ Prattville .................. McNair X-300 Camden ................... Camp Hill ................. Shorter ................... Marion Junction ............ Prattville .................. 0 0 0 200 0 - 60 0 - 0 0 0 5 0 0 30 0 0 3 0 0 0 0 0 0 3 0 155 180 196 50 223 150 95 115 195 298 180 115 300 460 288 55 19 2 48 34 30 20 - McNair 508 Camden ................... Camp Hill ................. Shorter ................... Marion Junction ............ Prattville .................. Pioneer 3145 Camden ................... Camp Hill ................. Shorter ................... Marion Junction ............ Prattville .................. Pioneer 3147 Camden ................... CampHill................. Shorter ................... Marion Junction ............ Prattville .................. Pioneer 3368A Camden ................... Camp Hill ................. Shorter ................... Marion Junction ............ Prattville ................... Pioneer 3369A Camden ................... Camp Hill ................. Shorter ................... Marion Junction ............ Prattville .................. 0 4 - 0 - 0 9 0 0 12 9 0 0 0 0 100 0 0 0 0 2 - 0 28 0 40 0 0 0 0 0 0 0 0 0 30 0 0 0 0 - 134 160 300 80 102 225 193 160 250 290 196 290 45 140 -- 22 8 45 36 14 17 26 14 12 - - 199 0 20 19 45 16 64 Continued - - 6 0 0 - 0 0 0 0 - - - 0 219 98 38 303 RESPONSE OF CORN HYBRIDS TO AFLATOXIN FORMATION 15 1 TABLE 3 (CONTINUED). AFLATOXIN CONTAMINATION OF CORN HYBRIDS CROWN 4-6 YEARS AT FIVE CENTRAL ALABAMA LOCATIONS, 1976-81 Variety and location Ring Around 1502 1976 1977 Aflatoxin B1 in p. p.hb. 1978 1979 1 0 5 0 18 0 0 5 0 0 0 0 0 0 0 0 0 35013 0 24 35 0 24 1980 125 200 199 110 290 310 250 135 110 260 50 196 40 40 1 35 42 1981 11 16 17 33 19 Camden ................... Camp Hill ................. Shorter Marion Junction ............ Prattville .................. Trojan TXS-114 Camden ................... Camp Hill ................. Shorter Marion Junction ......... Prattville ............... Trojan TXS-119 Camden ................... Camp Hill ................. Shorter.0 Marion Junction ......... Prattville ......... 33 18 5 120 28.2 40 0 50 38 38 Afltxnlatoxin mean in p. p.b. of samples!/year............. 170 Total varieties evaluated/year 0 Camden.................... 10 Camp Hill.................. 10 Shorter.................... Marion Junction.............. 9 Prattville.................... 6 46.7 9 0 0 0 0 4.6 34 39 47 35 1 9.0 39 39 74 31 41 TABLE 4. AFLATOXIN CONTAMINATION OF CORN HYBRIDS CROWN 4-6 YEARS AT THREE NORTHERN ALABAMA LOCATIONS, 1976-81 Variey an loctionAflatoxin Vait adlcain 1976 Coker 16 Belle Mina ................ 0 Crossville............... 0 Winfield................... Coker 22 Belle Mina................... 0 2,000 Crossville ................. Winfield ................ Coker 56 20 Belle Mina................. Crossville ............... Winfield ................. DeKalb XL-72-B Belle Mina.............. Crossville ................ Winfield ................ DeKalh XL-80 Belle Mina.................... 20 1977 1978 0 0 0 0 0 0 0 0 47 0 B1 in p. p~. b. 1979 1980 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 84 112 57 59 140 61 54 122 78 88 160 85 78 190 83 1981 20 20 10 20 27 9 22 9 1,000 100 Crossville ................... Winfield ................ 20 - 50 0 0 44 Continued 16 16 ALABAMA AGRICULTURAL EXPERIMENT STATION TABLE 4 (CONTINUED). AFLATOXIN CONTAMINATION OF CORN HYBRIDS GROWN 4-6 YEARS AT THREE NQBTHERN ALABAMA LOCATIONS, 1976-81 Varity nd ocaionAflatoxin Vait adlcain 1976 DeKalb XL-394 Belle Mina ................ Crossville ................. 1,000 Winfield.1 Funks G-795-W-1 Belle Mina Crossville.0 Winfield...................33 Funks G-4507 Belle Mina.0 Crossville.0 Winfield...................100 Funks G-4611 0 Belle Mina................. Crossville ................. Winfield...................0 Funks G-4747-W-1 Belle Mina.............. Crossville................... Winfield................ Funks G-4810 Belle Mina ................ Crossville.0 Winfield. .................. McCurdy 67-14 Belle Mina ................ Crossville.20 Winfield. .................. McCurdy 84-AA Belle Mina ................ Crossville ................ Winfield ................ McNair X-300 20 Belle Mina................. Crossville.................. 67 . . . . . .. . . W infield Paymaster UC8951 Belle Mina ........ Crossville...................... Winfield ................ Pioneer 3147 Belle Mina.................... Crossville . . . . . . . . . Winfield 1977 1978 0 0 0 BW p. p. b. in 1979 0 0 16 0 90 0 0 0 0 0 0 0 41 1980 71 200 51 200 193 200 268 275 300 228 244 288 75 108 214 168 210 90 1,100 64 250 264 355 86 110 90 228 276 266 236 110 276 100 55 100 244 236 288 1981 47 24 11 12 13 15 15 25 22 17 10 23 36 20 68 15 19 33 55 32 12 20 35 26 27 Continued 60 0 0 0 0 .100 0 0 20 0 40 0 0 30 - 0 0 0 3,3330 0 0 0 0 228 0 0 0 22 0 0 0 32 0 .................... 0 Pioneer 3369A Belle Mina ........ Crossville . . . . . . . . . Winfield....................... Ring Around 1501 Belle Mina - 0 - 0 0 0 0 ...... ... - Crossville . . . . . . . . . Winfield ................ - - 0 - RESPONSE OF CORN HYBRIDS TO AFLATOXIN FORMATION TABLE 4 (CONTINUED). AFLATOXIN CONTAMINATION OF CORN HYBRIDS GROWN 4-6 YEARS AT THREE NORTHERN ALABAMA LOCATIONS, 1976-81 17 Variety and location Ring Around 1502 Belle Mina ................ Crossville ................. Winfield .................. Ring Around 2602-W Belle Mina ................ 1976 1977 Aflatoxin B in p.p.b. 1978 1979 1980 0 0 - 1981 14 25 22 75 17 8 14 20 23.5 38 14 39 - 0 - 0 0 0 0 106 137 58 - Crossville ................. Winfield .................. Trojan TXS-114 Belle Mina ................ Crossville ................. Winfield .................. Trojan TXS-115A Belle Mina ................ Crossville ................. W infield .................. Aflatoxin mean in p.p.b. of samples!year ............. Total varieties evaluated/year Belle Mina ................. Crossville ................. Winfield .................. 412.5 10 12 9 12 0 0 65.7 20 18 19 46 0 0 0 8.2 43 36 24 4 0 0 0 0 0 0 2.4 74 76 51 240 268 264 288 100 127 69 178.2 43 86 43 TABLE 5. AFLATOXIN CONTAMINATION OF CORN HYBRIDS BY REGIONS AND YEARS IN ALABAMA Region of State and no. of sites Southern (4) ............. Central (5) .............. Northern (3) ............. 1976 1977 Aflatoxin mean in p.p.b.' 1978 1979 1980 12 (95) 5 (77) 8 (51) 110 (90) 9 (99) 2 (62) 210 (99) 195 (81) 178 (63) 1981 19 (66) 28 (53) 24 (41) 16 (19) 1,180 (10) 32 (14)23 47 (06) 20 (13) 66 (22) 'Numbers in parenthesis are numbers of hybrids tested. 2 One sample omitted because of apparent postharvest contamination. 3 Three samples omitted because of apparent postharvest contamination. 18 ALABAMA AGRICULTURAL EXPERIMENT STATION DISCUSSION Preharvest Contamination Preharvest contamination of corn with aflatoxin generally occurs in the range of 20-150 p.p.b., although many samples show no visible infection. However, under stress conditions induced by drought, high temperatures, and insect infestations, preharvest aflatoxin contamination can, on rare occasions, reach high levels (1,000-2,000 p.p.b.). Preharvest contamination may occur when A. flavus colonizes corn silks during the first 2 weeks of silking and when it invades the developing kernels 4-13 days after pollination (18,28). Such infection is favored by high temperatures of 86-93°F (19). The effect of water stress (drought) apparently is to increase the amount of inoculum (spore loads) rather than plant susceptibility (18,28,37). In most years, only a relatively small number of kernels are contaminated, but they may contain high levels of aflatoxin. Alternately, infection may occur following direct inoculation of kernels injured by insects and carrying spores of A. flavus, which subsequently produces moderate to high levels of aflatoxin in the damaged kernels. Postharvest Contamination Aflatoxin can be a serious postharvest problem in the South and anywhere in the world when commodities such as corn are harvested moist, or are not promptly dried to safe storage moisture. Corn, other grains, peanuts, and other crops must be dried to safe storage moisture after harvest to prevent fungal deterioration. Safe storage moisture then must be maintained in facilities adequate to prevent moisture buildup and thus prevent fungus (mold) growth and subsequent toxin formation. Safe storage moisture in the case of corn is approximately 13 percent. Aflatoxin levels generally become high in postharvest-contaminated corn compared to preharvest-contaminated corn, frequently attaining levels of 2,000 to 5,000 p.p.b. of aflatoxin B1 in Southeastern States. In 1965, Auburn research verified the first instance of aflatoxin contamination of corn in Alabama (unpublished data) when a farmer near Selma lost most of his newly-farrowed pigs after feeding corn that contained up to 8,000 p.p.b. of aflatoxin from postharvest contamination. High moisture corn was harvested before maturity and left undried in a closed bin for several days, after which it was diluted to a level of 150-200 p.p.b., which was apparently not detrimental when fed to 120-pound feeder pigs and lactating sows, but killed the more susceptible suckling pigs. RESPONSE OF CORN HYBRIDS TO AFLATOXIN FORMATION 19 Genetic Control In the investigation reported here, analysis of aflatoxin data for 6 years of over 200 corn hybrids grown throughout Alabama, tables 2, 3, and 4, indicates there was no resistance to aflatoxin formation in any hybrid tested. In 1977 and 1980, which were epidemic years with high levels of aflatoxin being common throughout the Southeast, all hybrids tested were contaminated with moderate to high levels of aflatoxin. In these 2 years, preharvest contamination resulted in levels high enough to cause serious problems for both corn and animal agribusiness (14,27). A considerable acreage of the corn crop in those years was plowed under because of the combined effect of low yield and preharvest aflatoxin contamination. Corn from most locations showed little or no contamination in 1976, 1978, and 1979,-except at southern locations of Headland in 1978 and Monroeville in 1979. Also, low levels of 10 to 50 p.p.b. were common at all locations in all hybrids in 1981, which was a year of high incidence coupled with low levels of aflatoxin contamination. The mean levels of aflatoxin contamination in corn hybrids by region were relatively low in all regions in 1976, 1978, 1979, and 1981, table 5, except for the southern region in 1979. Aflatoxin contamination was high in all regions in 1977 and 1980, but it was highest in south Alabama. The high incidence and high level of aflatoxin periodically occurring in preharvest corn in the southern region was generally the result of stress caused by a combination of high temperature, low rainfall, insect infestation, and low moisture-holding capacity of the area soils. After extensive research, Widstrom and Zuber (42), apparently believing that aflatoxin production in corn is under genetic control, attributed the inability to repeat differences in aflatoxin levels in preharvest commercial maize hybrids in comparisons over locations and years to be the major obstacle in developing a genetically resistant hybrid. Since the nature and mechanisms of resistance have not been elucidated, and since their results have not been consistently reproduced, it seems that little real progress has been made in developing hybrids with direct genetic resistance to aflatoxin contamination in corn. Nevertheless, development of genetic resistance to A. flavus invasion and aflatoxin formation in corn kernels remains the most practical way to seek control of the aflatoxin problem in corn. Resistance has been highly successful for the control of several fungal pathogens of corn. Unfortunately, A. flavus is not an aggressive plant pathogen. Instead, it is primarily a saprophyte or weak parasite, and thus, it is 20 ALABAMA AGRICULTURAL EXPERIMENT STATION possible that control by plant breeding for resistance may not be attainable by traditional experimental procedures. Although the authors conclude that genetic resistance to aflatoxin contamination has not been demonstrated to exist in commercial maize hybrids, the important search for resistance should not be abandoned. Possible innovative and unique sources of resistance may be obtained through application of the tools of of biotechnology. For example, recent research (31) has demonstrated the presence of extrachromosomal elements, including double-stranded RNA (dsRNA), in a nontoxigenic strain of A. flavus. When treated with cycloheximide (an antifungal antibiotic), this strain became a toxin producer. Thus, genetic elements that prevent aflatoxin biosynthesis may be present in this strain ofA. flavus. Attempts are now underway to transfer these genetic elements to toxigenic strains ofA. flavus to determine if they will prevent aflatoxin formation by normally toxigenic strains of these fungi. If this research is successful, then by accomplishing transfer of the inhibitory genetic elements (ds-RNA, DNA plasmids, or whatever) to corn or other higher plants via protoplast fusion or other techniques, we might create a source of resistance to aflatoxin formation for utilization by the plant breeder. Thus, the possibility of utilizing genetic resistance to aflatoxin contamination still exists. SUMMARY Aflatoxin contamination was determined for up to 215 corn hybrids grown at 12 locations in Alabama during 1976-81. In 1977,corn from the southern region of the State averaged 1,180 p.p.b. aflatoxin B1 , and that from the central and northern regions averaged 47 to 66 p.p.b., respectively. In 1980, each region averaged approximately 200 p.p.b. Levels of contamination were mostly insignificant in 1978, with regional averages ranging from 5 to 12 p.p.b. in the central and northern regions. Contamination levels in 1976 and 1981 were low, but relatively uniform throughout the State, ranging from 16 to 32 p.p.b. The principal conclusions from this investigation were that there was no resistance to aflatoxin formation in any hybrid tested, and significant aflatoxin levels generally accompanied stress caused by high temperature, low rainfall, low moisture-holding capacity of sandy soils, and insect infestation. RESPONSE OF CORN HYBRIDS TO AFLATOXIN FORMATION 21 LITERATURE CITED (1) (2) (3) ANDERSON, H.W, E.W. NEHRING, AND W.R. WICHSER. 1975. Aflatoxin Contamination of Corn in the Field. J. Agric. Food Chem. 2:775-782. ASAO, T, G. BUCHI, M.M. ABDEL-KADER, S.B. CHANG, E.L. WICK, AND 1980. Official Methods of G.W. WOGAN. 1963. Aflatoxins B and G. J. Am. Chem. Soc. 85:1706-1707. ASSOCIATION OF OFFICIAL ANALYTICAL CHEMISTS. Analysis, 13th ed., Ch. 26, Natural Poisons. Assoc. Off. Anal. Chem., Washington, D. C. (4) BURNSIDE, J.E., W.L. SIPPEL, J. FORGACS, WT. CARLL, M.B. ATWOOD, AND E.R. DOLL. 1957. A Disease of Swine and Cattle Caused by Eating Moldy Corn. II. Experimental Production with Pure Cultures of Molds. J. Am. Vet. Res. 18:817-824. (5) CARDEN, E.L. 1976. Performance of Corn Varieties in Alabama, 1976. Dept. of Agronomy and Soils, Dept. Ser. 32. Ala. Agr. Exp. Sta., Auburn Univ. 43 pp. . 1977. Performance of Corn Varieties in Alabama, 1977. Dept. (6) of Agronomy and Soils, Dept. Ser. 39. Ala. Agr. Exp. Sta., Auburn Univ. 42 pp. (7) CURRIER, C.G. 1978. Performance of Corn Hybrids in Alabama. Dept. of Agronomy and Soils, Dept. Ser. 46. Ala. Agr. Exp. Sta., Auburn Univ. 42 pp. . 1980. Performance of Corn Hybrids in Alabama, 1979. (8) Dept. of Agronomy and Soils, Dept. Ser. 53. Ala. Agr. Exp. Sta., Auburn Univ. 42 pp. . 1981. Performance of Corn Hybrids in Alabama, 1980. Dept. of Agronomy and Soils, Dept. Ser. 59. Ala. Agr. Exp. Sta., Auburn Univ. 43 pp. . 1982. Performance of Corn Hybrids in Alabama, 1981. Dept. (10) of Agronomy and Soils, Dept. Ser. 70. Ala. Agr. Exp. Sta., Auburn Univ. 49 pp. (11) DAVIS, N.D. AND UL. DIENER. 1979. A Fluorometric-lodine (FL-I) Method for Measuring Aflatoxin in Corn. J. Appl. Biochem. 1:115-122. . 1979. A Fluorometric Iodine Rapid (12) Screen Method for Aflatoxin in Corn. J. Appl. Biochem. 1:123-126. , M.L. GUY, AND U.L. DIENER. 1981. Improved Fluorometric(13) Iodine Method for Determination of Aflatoxin in Corn. J. Assoc. Off. Anal. Chem. 64:1074-1076. (9) (14) DIENER, U.L., R.L. ASQUITH, AND J.W.W DICKENS. 1983. Aflatoxin and As- pergillusflavus in Corn. So. Coop. Ser. Bull. 279. Ala. Agr. Exp. Sta., Auburn Univ. 112 pp. (15) EDDINS, A.H. 1930. Corn Diseases in Florida. Fla. Agr. Exp. Sta. Bull. 210:135. (16) (17) FORTNUM, B.A. AND A. MANWILLER. 1985. Effects of Irrigation and Kernel Injury on Aflatoxin B1 Production in Selected Maize Hybrids. Plant Dis. 69:262-265. HESSELTINE, C.W., O.L. SHOTWELL, WE KWOLEK, E.B. LILLEHOJ, WK. JACKSON, AND R.J. BOTHAST. 1976. Aflatoxin Occurrence in 1973 Corn at Harvest. II. Mycological Studies. Mycologia 68:341-353. 22 ALABAMA AGRICULTURAL EXPERIMENT STATION (18) JONES, R.K., H.E. DUNCAN, AND P.B. HAMILTON. 1981. Planting Date, Harvest Date, and Irrigation Effects on Infection and Aflatoxin Production by Aspergillusflavus in Field Corn. Phytopathology 71:810-816. (19) , G.A. PAYNE, AND K.J. LEONARD. 1980. Factors Influencing Infection by Aspergillusflavus in Silk-Inoculated Corn. Plant Dis. 64:859-863. (20) KING, S.B. AND J.R. WALLIN. 1983. Methods for Screening Corn for Resistance to Kernel Infection and Aflatoxin Production by Aspergillus flavus. In U. L. Diener, R. L. Asquith, and J. W. Dickens, (ed.) So. Coop. Ser. Bull. 279, Ala. Agr. Exp. Sta., Auburn Univ. pp. 77-80. (21) (22) KOEHLER, B. 1959. Corn Ear Rots in Illinois. Ill. Agr. Exp. Sta. Bull. 639. 87 Pp. LANCASTER, M.C., EP JENKINS, J.M. PHILP, K. SARGEANT, A. SHERIDAN, J. (23) O'KELLY, AND R. B.A. CARNAGHAN. 1961. Toxicity Associated with Certain Samples of Groundnuts. Nature 192:1095-1097. LILLEHOJ, E.B., WE KWOLEK, D.I. FENNELL, AND M.S. MILBURN. 1975. Aflatoxin Incidence and Association with Bright Greenish-Yellow Fluorescence and Insect Damage in a Limited Survey of Freshly Harvested HighMoisture Corn. Cereal Chem. 52:403-412. , G.M. SHANNON, O.L. SHOTWELL, AND C.W. HESSELTINE. 1975. Aflatoxin Occurrence in 1973 Corn at Harvest. I. A Limited Survey in the Southeastern U.S. Cereal Chem. 52:603-611. , A. MANWILLER, T.B. WHITAKER, AND M.S. ZUBER. 1983. (24) (25) Hybrid Differences in Estimation of Preharvest Occurrence of Bright Greenish-Yellow Fluorescence and Aflatoxin in Corn. J. Environ. Qual. 12:216-219. (26) MELCHERS, L.E. 1956. Fungi Associated with Kansas Hybrid Seed Corn. Plant Dis. Rep. 40:500-506. (27) NICHOLS, T.E., JR. 1983. Economic Impact of Aflatoxin in Corn. In U.L. Diener, R. L. Asquith, and J.W. Dickens, (ed.) Aflatoxin and Aspergillus flavus in Corn. So. Coop. Ser. Bull. 279. Ala. Agr. Exp. Sta., Auburn Univ. pp. 67-71. (28) PAYNE, G.A. 1983. Nature of Field Infection of Corn by Aspergillusflavus. In U. L. Diener, R. L. Asquith, and J. W. Dickens, (ed.) Aflatoxin and Aspergillus flavus in Corn. So. Coop. Ser. Bull. 279, Ala. Agr. Exp. Sta., Auburn Univ. pp. 16-19. (29) (30) PoNs, WA., JR. AND L.A. GOLDBLAT. 1965. The Determination of Aflatox- ins in Cottonseed Products. J. Am. Oil Chem. Soc. 42:471-475. G.W, J. TUITE, AND R.W. CALDWELL. 1974. Aspergillusflavus and Aflatoxin in Preharvest Corn from Indiana in 1971 and 1972. Cereal Chem. 51:595, 600-604. (31) SCHMIDT, ER., N.D. DAVIS, U.L. DIENER, AND PA. LEMKE. 1983. Cycloheximide Induction of Aflatoxin Synthesis in a Nontoxigenic Strain of Aspergillusflavus. Biotechnology 1:794-795. (32) SHOTWELL, O.L., M.L. GOULDEN, E.B. LILLEHOJ, WE KWOLEK, AND C.W. HESSELTINE. 1977. Aflatoxin Occurrence in 1973 Corn at Harvest. III. Aflatoxin Distribution in Contaminated, Insect-Damaged Corn. Cereal Chem. 54:620-626. RAMBO, RESPONSE OF CORN HYBRIDS TO AFLATOXIN FORMATION 23 (33) (34) , W.E KWOLEK, M.L. GOULDEN, L.K. JACKSON, AND C.W. HESSELTINE. 1975. Aflatoxin Occurrence in Some White Corn Under Loan, 1971. I. Incidence and Level. Cereal Chem. 52:373-380. SIPPEL, W.L., J.E. BURNSIDE, AND M.B. ATWOOD. 1953. 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Rep. 45:212-215. WIDSTROM, N.W., D.M. WILSON, AND WW. MCMILLIAN. 1984. Ear Resis- tance of Maize Inbreds to Field Aflatoxin Contamination. Crop Sci. 24:11551157. (41) , B.R. WISEMAN, W.W. MCMILLIAN, WE KWOLEK, E.B. LILLEHOJ, M.D. JELLUM, AND J. H. MASSEY. 1978. Evaluation of Commercial and Experimental Three-Way Corn Hybrids for Aflatoxin B, Production Potential. Agron. J. 70:986-988. (42) (43) AND M.S. ZUBER. 1983. Sources and Mechanisms of Genetic Control in the Plant. In U. L. Diener, R. L. Asquith, and J. W. Dickens (ed.) So. Coop. Ser. Bull. 279, Ala. Agr. Exp. Sta., Auburn Univ. pp. 72-76. ZUBER, M.S., L.L. DARRAH, E.B. LILLEHOJ, L.M. JOSEPHSON, A. MANWILLER, G.E. Scorr, R.T. GUDAUSKAS, E.S. HORNER, N.W. WIDSTROM, D.L. THOMPSON, A.J. BOCKHOLT, AND J.L. BREWBAKER. 1983. Comparison of Open-Pollinated Maize Varieties and Hybrids for Preharvest Aflatoxin Contamination in the Southern United States. Plant Dis. 67:185-187. Alabama s Agricultural Experiment Station System AUBURN UNIVERSITY \\ ith an ayric nitiral CXvenr sewes reseatrch unit in iuti( sto(il "tr, 0 Auhurn Li ni e r,,itx thte needs, of tield crop, livestock, lorestr\, and horticultural producers in (.ac h reg~ion in AIalancit Everx c itizen of thte State has a stake inl this research Ipi' )~t i, siince tm advantagc from niew and t )rce co)nomical NNJVS 01 produc ing and haninchg Lttrm~ piroduti ( onsumnlg 0 di- i8s® rectly henetits the pulic. ® Main Agricultural Experiment Station, Auburn. AE. V. Smith Research Center, Shorter. Tennessee Valley Substation, Belle Mina. Sand Mountain Substation, Crossville. North Alabama Horticulture Substation, Cullman, Upper Coastal Plain Substation, Winfield. Forestry Unit, Fayette County. Chilton Area Horticulture Substation, Clanton. Forestry Unit, Coosa County. Piedmont Substation, Camp Hill. Plant Breeding Unit. Tallassee. Forestry Unit, Autauga County Prattville Experiment Field, Prattville. Black Belt Substation, Marion Junction. The Turn ipseed- Ikenberry Place, Union Springs. Lower Coastal Plain Substation, Camden. Forestry Unit, Barbour County. Monroeville Experiment Field, Monroeville. Wiregrass Substation, Headland. Brewton Experiment Field, Brewton. Solon Dixon Forestry Education Center, Covington and Escambia counties. 20. Ornamental Horticulture Substation, Spring Hill. 21. Gulf Coast Substation, Fairhope. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.