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II. Background and Identification of Aflatoxins as Contaminants of Corn

II. Background and Identification of Aflatoxins as Contaminants of Corn

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THE AFLATOXIN PROBLEM WITH CORN GRAIN



22 1



flavus were responsible for infecting corn and initiating disease symptoms

(Dalrymple, 1893; Mayo, 189I ). Unique field conditions are required to enhance

fungal infections of the corn ear, allowing contamination to become a serious

problem during feeding and storage (Koehler, 1938, 1942). Moisture, temperature, and insects were demonstrated to be important factors influencing ear

infections, but very little importance was placed on Aspergillus spp. as contributing to the disease symptoms resulting from eating infected ears. Reports of

aflatoxicosis in animals by Sippel et af. (1953) and Burnside et a f . (1957)

initiated research that began to focus on the Aspergilli as a primary source of the

feeding problems. However, contamination was considered as being limited to

stored corn (Quasem and Christensen, 1958) and the syndrome in livestock was

referred to as “symptoms of disease” rather than toxicity (Burnside et al., 1957).

A serious effort to identify the “causal agents” of toxicity occurred as a result

of an outbreak of turkey “X” disease during 1960 in England (Blount, 1961), and

identification of the aflatoxins initiated an entirely new area of scientific investigation, that of mycotoxicology. Several animal species were soon reported as

being adversely affected by ingestion of aflatoxin, including cattle (Garrett et af.,

1968), which were also reported by Burnside et al. (1957).



A. DETECTION

AND QUANTIFICATION

OF AFUTOXINS

Methods for the detection and quantification of aflatoxins in peanut were

developed in the early 1960s and reported by Coomes et al. ( 1964). The basic

procedures for detection and quantification were modified for use on corn and

other crops; the early developments were reported by Pons and co-workers (Pons

et af., 1966). The procedures facilitated several surveys of marketed corn in the

mid-to-late 1960s in which low amounts of aflatoxin were found (Shotwell et a l . ,

1969b, 1970, 1971).

Field studies in 1971 and 1972 identified aAatoxin contamination of corn as a

preharvest problem (Anderson et d.,1975). Studies followed that were designed

to investigate the extent of preharvest contamination in com-growing regions of

the United States (Lillehoj et a l . , 1975d). Field contamination was judged to be

more serious in the southern corn-growing regions than elsewhere in the United

States. The difference between regions was partially attributed to ear-feeding

insect activity (Widstrom et a l . , 1976). Lillehoj and co-workers laid groundwork

for the investigation of genetic differences among hybrids and kernel starch types

(Lillehoj et al., 1975d, 1976~).

A realization that preharvest aflatoxin Contamination of corn also placed a

major portion of the Corn Belt at risk led to a status report with possible solutions

being presented to the 30th Annual Corn and Sorghum Research Conference in

1975 (Lillehoj and Zuber, 1975). Another status report by Zuber and Lillehoj



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N. W. WIDSTROM



( 1979) introduced control measures including genetic resistance to infection and

contamination, reduction of plant stress, and insect control. The second status

report was prompted in large part by the calamitous contamination of the 1977

corn crop, especially in the South (Wilson et al., 1979). The high aflatoxin

contamination of the 1977 crop was a rare occurrence; however, the reduction in

contamination for subsequent years (McMillian et al., 1980b, 1985b) must not

be viewed as a diminution of the problem, but rather as fluctuations of chronic

contamination that is always a threat in limited areas of the southern corn growing region of the United States (Table I).

Indirect methods were often used for determining the presence of aflatoxins in

corn fed to animals prior to the 1960s. Standardized procedures to analyze for

aflatoxin content in feeds began to emerge by the middle of the next decade

(Pons, 1976). Detection of aflatoxins in feed was often done by bioassaying

sensitive animals such as ducklings (Sargeant ef al., 1961), chicks (Brown and

Abrams, 1965), and rainbow trout (Brekke et al., 1977).



Table I

Average Yearly Levels of Aflatoxin Contamination

for Corn Grown in the Coastal Plain of Georgia,

1977-1994



Year

I977

I978

1979

1980

1981

I982

1983

I984

1985

1986

1987

I988

I989

I990

1991

1992

I993

1994



Concentration of aflatoxin

(ng g-')

622

57

68

204

91

92

I28

37

48

190

82

137

26

217

39



35

71



6



Source: McMillian et a/. (1985b). and unpublished



data.



THE AFLATOXIN PROBLEM WITH CORN GRAIN



223



An association between bright greenish-yellow fluorescence (BGYF) and the

presence of aflatoxin exists, but the relationship is not considered reliable since

the BGYF is due to kojic acid, a secondary metabolite of A . flavus (Marsh e t a / .,

1969). The simplicity and inexpensiveness of the BGYF test attracted its use,

especially before accurate measurements for aflatoxin were available. Occasional

successes when using BGYF (Shotwell et al., 1972, 1975b; Rambo et al., 1975)

resulted in a suggestion for its use as a presumptive test (Shotwell and

Hesseltine, 198 I ) for initial elimination of some samples in surveys. Attempts to

use BGYF as a routine indicator of toxin contamination have met with limited

success (Shotwell et al., 1975b), however, and perhaps have been inconclusive

(Dickens and Whitaker, 1981), because many isolates ofA.Javus do not produce

aflatoxin but do produce BGYF (Fennel1 er d.,

1973). The variability associated

with both testing for aflatoxins (Whitaker et a / . , 1979) and determinations of

BGYF particles (Calvert ef al., 1983) certainly contributes to their frequent

failure as indicators of one another. The BGYF phenomenon has also been

associated with insect damage, yet another reason why it has been ineffective as a

precise indicator of aflatoxin contamination (Rambo et al., I974b; Kwolek and

Shotwell. 1979).

Techniques developed by Pons et al. (1966, 1973) for determining aflatoxin

content in other agricultural products were gradually improved by slight modifications (Stubblefield, 1979). Variations of the minicolumn technique, introduced by Holaday (1968) were also utilized in the years following (Romer et al.,

1979). By the early 1970s, Detroy et a/. (197 I ) and others (Changes in Official

Methods of Analysis, 1972) had developed thin-layer chroniatographic (TLC)

quantification techniques. Liquid chromatographic (LC) and TLC procedures

complement one another (Trucksess and Wood, 1994) and are among the important official standard methods for analyses of aflatoxins (Official Methods of

Analysis of the AOAC, 1975). The TLC method has been modified, with success, to meet the needs of laboratories in developing countries (Guzman de Pefia

er a/., 1992). Good resolution of aflatoxins is also achieved by the LC method, a

high-pressure liquid chromatography (HPLC) procedure of Pons ( 1976), later

adapted for use on corn (Pons, 1979) and modified by Thean et a / . (1980). A

comparison among blind samples at several laboratories was made by Park ef al.

( 1990) in an effort to standardize procedures. The comparison of the LC and TLC

methods resulted in a conclusion that TLC tended to overestimate concentrations

when amounts of aflatoxin were less than 20 ng g - I (Beaver et a l . , 1990).

Other methods of aflatoxin analysis have been proposed, including the

fluorometric-iodine method of Davis and Diener for regular evaluations (Davis

and Diener 1979a) and for rapid screening (Davis and Diener, 1979b) and several

immunochemical methods (Chu, 1990). The immunochemical methods have

become popular recently because results can be obtained more quickly and at less

cost than for other methods (Trucksess and Wood, 1994). The immunoaffinity



224



N. W. WIDSTROM



column procedure developed by Trucksess et al. (1989, 1991) has been adopted

by numerous laboratories in industry as well as in other research facilities. The

method was compared with liquid chromatography for accuracy and sensitivity

by Beaver et al. (1991) and found to be reliable at aflatoxin concentrations

greater than 5 ng g-1.



B. THESCOPEOF CORNCONTAMINATION

The first surveys conducted on grain to test for the presence of aflatoxin were

made on samples obtained from large marketing centers in the United States:

Chicago, Illinois, New Orleans, Louisiana, and Omaha, Nebraska. The initial

survey by Shotwell et al. (1969a) did not include corn, but their finding that corn

was an excellent substrate for A. flavus resulted in corn being included in subsequent surveys (Shotwell et al., 1969b, 1970, 1971). Only 48 of 2117 corn

samples were found to be aflatoxin positive, and of those, only four contained

more than 20 ng g-I aflatoxins.

Reports of aflatoxicosis in North Carolina broiler chickens (Smith and Hamilton, 1970) and toxic hepatitis of swine and cattle in the southern United States

(Wilson et al., 1967) prompted Shotwell e? al. (1973) to examine southerngrown corn from commercial markets. Aflatoxins ranging from 6 to 348 ng g-I

occurred in 2 1 of the 60 samples collected. A pattern of heaviest contamination

in samples from the southeastern states began to emerge. One of six samples of

1971 white corn grown in the southeastern comer of Missouri had adatoxin

contamination of 400 ng g-l (Lillehoj et al., 1975a) and 8 of 163 samples grown

in southern Indiana in 1972 contained aflatoxin-contaminated kernels (Rambo et

al., 1974a). Documentation of preharvest contamination of corn was obtained

from the 1972 crop grown in southwest Georgia (Anderson er al., 1975) when

aflatoxin contamination was found on randomly sampled ears of both white and

yellow corn.

Sporadic reports of aflatoxin contamination in midwest corn began to appear in

the mid 1970s (Rambo et al ., 1974a; Riesselmann and Doupnik, 1975; Lillehoj

et al., 1976b). The reports were not localized or confined to any type of corn in

that contamination of 1972 dent corn was determined in 8 of 163 samples in

Indiana (Rambo et al., 1974a), as much as 30 ng g-' aflatoxins were found in

1973 Nebraska popcorn (Riesselman and Doupnik, 1975), and I1 of approximately 6000 ears of I975 Iowa corn had single-ear Contamination of 1- 1560 ng

g-' (Lillehoj et al., 1977). When corn from several locations was compared,

however, the southern locations, especially Georgia, North Carolina, and Texas,

always showed the heaviest contamination (Lillehoj et al., 197%; Zuber et al.,

1976). Samples of 1973 and 1974 corn grown in South Carolina had 24-209 and

0-281 ng g-I aflatoxins, respectively, while 1974 Florida corn was contami-



THE AFLATOXIN PROBLEM WITH CORN GRAIN



225



nated with 3-1218 ng g-1 (Shotwell et al., 1977; Lillehoj et ul., 1976~).One

instance of contamination found in white corn grown in 1971 in southeast Missouri drew much attention because white corn is often used for food products.

Among 1283 truckloads, 165 had greater than 20 ng g-' and 29 loads exceeded

100 ng g - l aflatoxin (Shotwell et al., 1975a).

National attention was focused on the aflatoxin problem in 1977 when heavy

contamination occurred throughout the southeastern states. Formal reports on the

extent of contamination of the 1977 crop were published for the states of Alabama (Gray et al., 1982), Georgia (McMillian et al., 1978), and North Carolina

(Hesseltine et al., 1981), although corn grown in the other southern states was

similarly affected. The 1977 midwestern crop, however, had a very limited

amount of contamination (Shotwell et al., 1980), and five of eight states in the

midwest were reported to have escaped completely, based on the survey. The

state of Georgia continued to monitor its corn to determine preharvest contamination, and later reported survey results of 1977 through 1982 (McMillian et al.,

1985b). This survey clearly demonstrated that the preharvest problem in Georgia

is chronic, but contamination vanes greatly from year to year (Widstrom et a l . ,

1984b); that is, contamination was heavy in 1977 and 1980, moderate in 1981

and 1982, and significantly less in 1978 and 1979 (Table I). Preharvest aflatoxin

contamination of the corn crop is chronic only in the South and Southeast, and is

not considered a threat in many areas of the United States, but it has been

reported in no fewer than 21 states (Wilson and Payne, 1994).

Contamination of the corn crop in other countries is quite possibly more

serious than that in the United States because a greater proportion of the corn

grown in many countries is used as a human food source (Jelinek, 1987; Jelinek

et al., 1989). Campbell and Stoloff (1974) discussed the implications of mycotoxins on human health while others studied their effects on domestic animals

(Wilson et af., 1967) and other test animals such as the rat (Carnaghan, 1967).

The concern, therefore, is worldwide for two reasons: first, because corn is

shipped routinely to importing countries, and second, because corn produced and

consumed within numerous countries throughout the world has been reported as

contaminated (Wood, 1989). The Philippines (Ilag et al., 1976), France (Galtier

et ul., 1977), Yugoslavia (Balzer et al., 1977; Durakovic ef al., 19841, Egypt

(Farag et al., 1980; Qutet et al., 1983), Mexico (Martinez, 1979; Zuber et a l . ,

1986), Brazil (Sabin0 et al., 1989, Zuber et a l . , 1986), Haiti (Castor et al.,

1987), India (Bilgrami et al., 1981a; Zuber et al., 1986), Australia (Blaney,

1981), Thailand (Shank et al., 1972a; Zuber et al., 1986), and Bolivia, Colombia, and Costa Rica (Zuber et al., 1986) have all reported contamination of their

corn. Africa, however, probably has a more widespread problem than any other

continent and has reported contamination in seven of its countries: Uganda

(Alpert el al., 1971), Zambia (Lovelace and Nyathi, 1977), Kenya (Ngindu et

ul., 1982), Nigeria (Okoye, 1986), Mozambique (Purchase and Goncalves,



226



N. W. WIDSTROM



1971), Tanzania (Seenappa and Nyagahunga, 1982), and Ghana (Zuber et al.,

1986). The greatest concern is for people in countries that grow much of their

own corn, use corn as a staple food in their diet, and have no organizational setup to monitor corn grown and consumed at the local level. These areas include

Mexico and several countries in Africa and Central and South America.



111. CONDITIONS IMPACTING ASPERGILLUS F L A W S

GROUP INFECTION AND AFMTOXIN ACCUMULATION

Numerous environmental and cultural conditions have an influence on infection and aflatoxin production processes which occur in the corn kernel when

exposed to spores of the A . f l a w s group. The gamut of these conditions has

changed little since being reviewed by Lillehoj (1983), but our understanding of

the importance of each and how they interact has received considerable attention

in research conducted since (Widstrom, 1992). A thorough knowledge of conditions that we cannot control, which exist prior to planting a corn crop, as well as

those over which we have some control may indeed make the difference between

whether a crop escapes or suffers contamination by aflatoxin.



A. THEINFLUENCE

OF FACTORS

BEYOND GROWER

CONTROL

Assuming that a grower is restricted to growing corn in a limited area, weather

and soil type factors are quite firmly fixed. The grower, therefore, must learn to

cope with or compensate for these uncontrollable conditions, if they are unfavorable to the production of a contaminant-free crop.



1. Weather

The prevalence of Aspergillus ear molds has long been associated with dry

weather, although wet weather provides no assurance that ear molds will not be

present (Taubenhaus, 1920). His findings are in agreement with the fact that the

Aspergillus as a group of fungi seem to appear before other kinds of fungi when

kernels have a low moisture content, 5140 g H,O kg-' dry matter (Koehler,

1938). The first confirmation of the incidence of aflatoxin being found in preharvest grain also pointed to a higher incidence of aflatoxin contamination of corn

grown in the warmer regions of the United States (Anderson et al., 1975).

The report by Anderson et al. (1975) initiated a number of studies to more

clearly define weather-related differences in aflatoxin incidence and amounts as

they relate to varied corn-growing regions in the United States (Lillehoj et al.,



THE AFLATOXIN PROBLEM WITH C O W GRAIN



227



1975d; Zuber et ul., 1976). The general conclusions of these studies were that

both incidence and amounts of aflatoxin in the corn sampled increased from north

to south and that the increase in southerly locations in the United States was

definitely related to temperature and possibly also related to regional differences

in precipitation (Lillehoj et ul., I978b). Weather-related regional differences

within states were suggested in many of the earlier reports as a reason why

differences in infection and incidence of BGYF and aflatoxin can occur between

regions (Lillehoj and Hesseltine, 1977).

Several surveys were conducted on corn from the Midwest during the 1960s

and early 1970s (Shotwell, 1977). Most of the surveys located a few samples

with low levels of aflatoxin, with the exception of the white corn harvest of 1971

in southeastern Missouri. Nearly one-third of the truckloads sampled from stored

corn from this harvest had detectable levels of aflatoxin. Samples of southerngrown corn, however, often were found to have contamination incidences of 4050% (Shotwell, 1977). Apparent regionalization of the heaviest contamination

encouraged recommendations by the extension service that growers should make

a serious effort to avoid drought stress during production of the crop (Duncan,

1979).

Any notion that aflatoxin contamination was nearly always confined to the

southern corn-growing region was dispelled by results from surveys of the 1977

crop when more than 18% of 87 samples from the drought-stressed crop in

central Iowa had amounts >20 ng g-1 (Zuber and Lillehoj, 1979). While heavy

contamination occurred locally in 1972 in the Midwest, levels of aflatoxin in

1977 southern-grown corn can be described as no less than disastrous (Wilson et

a/., 1979; Manwiller and Fortnum, 1979; Gray ef a/., 1982). From that point

forward, research on a solution or control of the problem was begun in earnest.

The problem was not so serious in 1978 (McMillian et al., 1980b), but 2 successive years of heavy contamination in I980 and 198I Georgia corn (McMillian ef

al., 1985b) convinced any remaining skeptics that chronic contamination, at

some level, existed for southern-grown corn.

One common denominator of field studies has been that high temperatures are

associated with greater amounts of aflatoxin contamination of field samples

(Jones et al., 1980; Zuber et al., 1983; Hill et a / . , 1985). High temperatures are

also nearly always an important component of drought and the plant stress

associated with drought. Drought stress has been commonly given as a major

component of contamination in those years when aflatoxin levels were high

(Davis et a / . , 1985). The persistence of conclusions that include drought and

plant stress as major components of contamination is not surprising, since detailed studies of weather-associated factors have concluded that high temperature

and low humidity, that is, evaporation or transpiration losses, are significantly

correlated with heavy contamination of corn sampled directly from the field at

harvest (Widstrom et a/. , 1990). Weather variables, in addition to temperature



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II. Background and Identification of Aflatoxins as Contaminants of Corn

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