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III. Conditions Impacting Aspergillus Flavus Group Infection and Aflatoxin Accumulation

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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,


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



and moisture, have been suggested as having an influence on aflatoxin production

(Fortnum, 1987) and a study by McMillian et al. (1985) illustrated the complex

interrelationships that exist among moisture, insect damage, temperature, and

plant resistance to aflatoxin production. It is, therefore, not surprising that some

investigations do not always indict drought stress and temperature as being the

dominant factors producing aflatoxin contamination (Stoloff and Lillehoj, 1981).

High temperatures are consistently found to be an important factor influencing

aflatoxin contamination and fungal growth when tests are conducted under controlled conditions (Thompson et al., 1980; Holmquist et al., 1983; Hill et al.,

1985; Kingsland, 1986; Wieman et af.,1986; Payne et af., 1988b). Modifications of temperature and plant stress through irrigation or other means are always

important components of recommended control measures, even when genetics

and host plant resistance are principal emphases (Zuber and Lillehoj, 1987;

Widstrom, 1987). The first attempts at correlating specific weather within a

weekly or monthly time frame with the incidence of aflatoxins were made by

Sisson (1987). The importance of temperature and humidity within time windows was corroborated by Widstrom et al. (1990), who suggested that, contrary

to the standard recommendations for early planting, those plantings in the deep

South are believed to be at higher risk for aflatoxin contamination than those

which are delayed to change the time period for grain-filling. The most recent

comprehensive review (Payne, 1992) states that no control strategy is completely

effective for presently grown commercial hybrids when environmental conditions are extremely favorable for growth of the fungus.

2. Edaphic Factors

Recommendations made by the extension service to help minimize aflatoxin

contamination of the corn crop have always included adequate fertilization and

irrigation to provide a root zone in the soil that will impose a minimum of stress

on the plant (Georgia Extension Aflatoxin Committee, 1978; Glover and Krenzer, 1980). The suggestion of altering edaphic factors to reduce aflatoxin contamination of the crop, whether through fertilization, irrigation, or cultivation, may

relate to the fact that the soil serves as a repository for the spore load imposed on

the crop to be planted. Cultivated soils seem to carry higher spore concentrations

than others (Angle, 1987), probably increasing the likelihood of exposure to

infection and necessitating production of healthy plants that will resist infection.

Angle (1987) also demonstrated that degradation of aflatoxin occurred more

slowly in silty clay loams than in fertile silt loam soils; however, no information

was given on inactivation of A . flavus spores in those soils.

In one of the first studies involving geographical differences, contamination

was primarily attributed to the differences in weather and plant factors, but soil

factors were probably also involved (Lillehoj et ul., 1975d). The interrelation-



ships among factors have not been fully sorted out in that the choice of fields

may, for example, be based on whether soils are sandy (droughty) since they are

more likely to produce stress on corn than on sorghum (Jones, 1987). The

importance of the soil as a source of inoculum has been well documented (Lillehoj et a / . , 198Od) and has been included in reviews when control measures

were discussed (Widstrom et al., 1984b; Zuber and Lillehoj, 1987; Wilson et al.,

1989b). Martyniuk and Wagner (1978) demonstrated that management systems

such as continuous cropping have an impact on the quantity and quality of

microflora. Some tillage studies have provided mixed results; subsoiling in North

Carolina reduced aflatoxin contamination (Payne ef al., 1986), but differences

due to one, two, or three cultivations for weed control were nonsignificant in

India (Bilgrami et a / . , 1992).

Experiments involving fertilization and irrigation effects have been more definitive than those on tillage, and recommendations for cultural control of aflatoxin contamination of corn always include the need for maintaining adequate

fertility and moisture in the soil profile (Duncan, 1979; Smith, 1981; Jones,

1987; McMillian et a l . , 1991). Good nutrition of corn reduced contamination

when stresses by other factors were not present (Wilson et al., 1989a). Similar

conclusions were drawn from two independent studies in North Carolina (Jones

and Duncan, 1981; Payne et al., 1989). The interrelationship between good

fertility, available soil moisture, and other factors has been the subject of several

studies, and interaction among the influencing factors should be expected (Jones

et ai., 1981; Smith and Riley, 1992). Tremendous differences from year to year

that were encountered in some of these studies (Fortnum and Manwiller, 1985)

were undoubtedly responsible for conflicting views as to their importance to

contamination of corn and to recommendations given for management. For example, Jones et al. (1981) found the least contamination in early plantings while

samples from early plantings grown by Smith and Riley (1992) had significantly

larger amounts of aflatoxin than late plantings. Recommendations for adequate

irrigation are, of course, standard for grain production and are merely reinforced

as far as prevention of aflatoxin contamination is concerned.



An adequate knowledge of the crop and management history of the area on

which corn is to be grown is a necessary prerequisite to improving the probability

for producing an aflatoxin-free grain crop. The adage that “an ounce of prevention is worth a pound of cure” is definitely applicable to corn grown for grain.

Decisions regarding where, when, and what to plant can make the difference

between success and failure in producing profitable crops free of contamination.

Preplanting decisions are impossible to change after the crop has emerged; there-



fore, careful preseason planning is critical to reducing contamination of a corn


1. Soil Testing

Soil testing is critically necessary for evaluation of fertilizer needs of areas

where corn is to be grown. Corn requires a higher soil pH and more nitrogen than

most other crops. Maintaining an adequate soil pH and nitrogen supply is difficult in the South because the area in general has greater rainfall than the Corn

Belt, and sandier soils, lowering the pH and leaching soluble nutrients from the

root zone (Aldrich et a l . , 1975). Adjustments of soil pH with lime application

should be made well in advance of planting, before the pH reaches critically low

levels, because adjustment of pH due to liming is usually not effective during the

same growing season it is applied, and the crop will have matured before breakdown of the lime can have any noticeable effect on the pH. Soil testing and

subsequent preplant application of lime and fertilizer to alleviate deficiencies in

pH, nitrogen, and other plant nutrients enable the grower to get his crop started

with a minimum of the stresses that have been reported to predispose the crop to

aflatoxin contamination (Jones and Duncan, 1981). Deficiencies of other major

(phosphorus and potassium) and minor elements, as determined by soil test, can

normally be remedied at planting by application of a complete fertilizer blended

by commercial dealers especially for corn production.

2. Crop Rotation

Quantitative differences in soil microflora, including A , flavus, have been

found in soils that have been placed under continuous cropping of corn (Martyniuk and Wagner, 1979). Conventional tillage practices in a red clover-wheat

rotation yielded soil samples with 256 propagules of A. flaws and A . parasiticus

per gram of soil. Subsoiling, which is a more frequent practice in some rotations

than in others, has been demonstrated to be beneficial in reducing aflatoxin

contamination of corn (Payne et al., 1986).

Specific crop rotations have not been compared sufficiently to warrant recommendations other than to encourage basic rotation principles, such as the avoidance of continuous cropping. The use of cultural practices and rotations that

optimize production are those that minimize contamination and infection (Widstrom, 1992), and are routinely recommended, having proven to be effective in

practice (Wilson e t a ! . , 1989b).

Another aspect of the soil microflora associated with rotation, continuous

cropping in particular, is the formation of sclerotia on crop debris after harvest

(Wicklow et al., 1982, 1984). The resistance of these fungal structures to decomposition and quick germination could prove them to be an abundant source of

inoculum in the following year. Sclerotia from certain A. flavus isolates have


23 1

been shown to germinate in the field, providing inoculum prior to silking of the

corn crop (Wicklow and Wilson, 1986).

3. Hybrid Selection

The selection of an appropriate hybrid for planting is a vital part of the

grower’s management program to minimize aflatoxin contamination of his corn

grain crop. An earnest search was begun for hybrids that would restrict or prevent

the infection by A . jlavus and accumulation of aflatoxin in their grain (LaPrade

and Manwiller, 1976, 1977; Lillehoj et a / . , 1976~;Widstrom et al., 1978), as

soon as a preharvest contamination problem was documented (Anderson et a l . ,

1975). Although most investigators agreed that differences existed among hybrids for resistance to aflatoxin formation, questions regarding the source or

causes of that resistance were still being extensively discussed. Several of the

early studies that included numerous hybrids were designed to answer questions

other than whether hybrid differences were worthy of pursuit as a solution to the

problem (Lillehoj et al., 1982b, 1983a); therefore there were difficulties in

acquiring definitive information on hybrid differences (Lillehoj and Zuber,

1981). Some reports involving large numbers of hybrids suggested that no differences for resistance existed among hybrids (Davis et al., 1985). Prior to 1980,

most recommendations regarding hybrid selection were guarded in that growers

were encouraged to use adapted hybrids (Zuber, 1977; Zuber and Lillehoj, 1979)

alluding to the avoidance of stress during development.

Early reports associated field infection of corn ears by Aspergillus spp. with

insect infestations (Taubenhaus, 1920). The report documenting preharvest contamination of corn by aflatoxin also associated the problem with insect damage

(Anderson et a l . , 1975). Several lepidopteran insects were identified as contributors to contamination, but the European corn borer (Ostrinia nubilalis, Hiibner)

was associated with the highest concentration of aflatoxin (Widstrom et a l . ,

1975). Confirmation of the finding by Fennel1 et al. (1978) pointed toward insect

resistance as a critical factor in the choice of hybrids by the grower. Maize

weevils (Sitophilis zeamais Motschulsky) have also been shown to effectively

transport the fungus into corn ears. Therefore, resistance to weevils as well as

other insects must be a consideration in hybrid selection (McMillian et a l . ,


Husk tightness has always been a component of field resistance to insects and

as such has been shown to be an important contributor to reduced aflatoxin

contamination (Barry et a / . , 1986; McMillian et a l . , 1987; Widstrom et a l . ,

1994). While Corn Belt hybrids have loose husks for quick dry-down, most

southern-bred (adapted) hybrids in the southeastern United States have sufficient

husk coverage and tightness to discourage insect invasion. Husk traits are consequently very important in the hybrid selection process.

Genetic differences for both hybrids and inbreds in A. j a w s infection or



aflatoxin accumulation that are independent of resistance to insects and husk

traits have been clearly demonstrated (Zuber et al., 1978; Widstrom et al., 1987;

Scott and Zummo, 1988). Recent recommendations specifically suggest that

emphasis must also be placed on genetic differences that have as their basis

something in addition to adaptation, husk traits, and resistance to ear feeding

insects (Widstrom and Zuber, 1983; Zuber and Lillehoj, 1987; McMillian et al.,

1991). The grower, at this point, is still faced with basing his selection of a

hybrid on adaptation, husk traits, and resistance to insects, since hybrids with

chemically based or other resistance mechanisms have not yet been developed.

a. Plant Population

Recommendations to minimize risk of aflatoxin contamination of corn routinely caution against planting populations in excess of those optimum for yield

of a given hybrid under expected growing conditions (Georgia Extension Aflatoxin Committee, 1978; Duncan, 1979). The recommendations are made based

on avoidance of stress that is known to influence the infection and contamination

processes (Wilson et al., 1989b). There is a dearth of information on the influence of plant populations and opposing hypotheses have been proposed for

possible effects of population density (Jones, 1987). High plant populations

encourage stress due to competition for nutrients and water, while on the other

hand they also produce heavy canopies that may reduce exposure of maize ears

and silks to airborne spore load.

An additional factor associated with high plant populations, introduced by

McMillian et al. (1985a), related to the amount of free water and period of time

that free water is present on ears and silks during kernel maturation. Ears that

were repeatedly exposed to free water over a 4-week period to simulate heavy

dews common in the Southeast sustained elevated levels of aflatoxin contamination. The finding is consistent with other recent studies that have correlated net

evaporation rates during kernel maturation with the amount of aflatoxin found in

those kernels (McMillian et a l . , 1985b; Widstrom et al., 1990).

The lowest plant population sustained the highest level of aflatoxin contamination in a crop grown during monsoon when rainfall amounts varied from 5 to 10

cm week-' (Bilgrami era/., 1992). Temperature rather than moisture or humidity

was probably the determining factor for toxin contamination during this study in

which varieties rather than hybrids were compared. Optimum populations for

hybrids under varied conditions are often listed on the bags of commercially

produced seed corn. Exceeding those recommendations, especially for noninigated corn, can be an invitation to A . Javus infection and aflatoxin contamination. Lower plant populations should be seriously considered also when the crop

is planted in soils with high sand content since these soils have lower waterholding capacity and are more likely to impose drought stress than soils with high

clay content.



b. Planting Date

The earliest studies conducted in 1977 in Georgia (Georgia Extension Aflatoxin Committee, 1978) and in 1978 and 1979 in North Carolina (Jones and Duncan, 1981; Jones et a f . , 1981) indicated that the latest planting dates produced the

highest amounts of aflatoxin contamination in grain samples. Heavier contamination of South Georgia plantings in 1974-1976 than those further north was

partially attributable to later planting dates, but the differences were confounded

with location effects (Widstrom et a / ., 1978). Recommendations were made for

planting early to minimize contamination, based on these early experiments,

even though planting date was not the primary factor for investigation in the

studies (Duncan, 1979; Glover and Krenzer, 1980).

Mixed signals regarding the relationship between planting date and aflatoxin

contamination (Widstrom et a / . , 1978; Lillehoj et a/., 1980b) suggested that

planting date means were being influenced by other factors and possibly may be

location specific. Consequently, specific planting recommendations were avoided

(McMillian et al., 1985b) until a more complete pattern emerged from the then

unpublished work in Georgia (Widstrom et a / ., 1990). Recommendations to

delay planting as long as possible because later plantings on the Coastal Plain

were subject to lower contamination levels began to appear as early as 1984

(Widstrom er a / . , 1984b) and have been continued since (Wilson et al., 1989b;

McMillian et al., 1991; Widstrom, 1992)). Since delayed plantings produce

lower grain yields, a grower must weigh contamination risk against lower yields

when making a planting date decision. Additionally, the effect of reduced contamination for later plantings may not hold for other locations where temperature, soil type, etc., are different. Studies in Louisiana, however, showed a lower

level of aflatoxin contamination for the latest of three planting dates than in either

of two plantings 10 and 20 days earlier (Smith and Riley, 1992). The basic tenet

for late planting is to time planting so that the critical grain filling period,

beginning at 20 days after silking, occurs after the highest seasonal temperatures

and period of net evaporation (Widstrom et al., 1990). The data showing the

trends in sample contamination of wound-inoculated ears for planting dates

obtained over several years are given in Table 11. A general reduction in aflatoxin

Contamination of 150-200 ng g-I occurs from early to late for each approximate

15-day planting interval.

c. Resistance to Insects and Diseases

Insect damage recorded in aflatoxin studies revealed that the highest levels of

aflatoxin contamination were usually associated with heavy insect damage (Lillehoj et a / . , 1 9 8 0~;Wilson et al., 1981a). Lillehoj and Hesseltine (1977) suggested that insects were important as carriers of aflatoxin-producing fungi. Specific insects had been identified as being associated with the presence of A.

,flovus, i.e., corn earworm (Lillehoj et a / . , 1976d) and European corn borer


2 34

Table I1

Planting Dates, Grain-Fdling Periods, and Geometric Means

for Aflatoxin Concentrations of Wound-InoculatedCorn

Samples at TiPton, Georgia, 1982-1987"

Planting date

Grain filling period

27 February

18 March

31 March

15 April

1 May

15 May

29 May

13 June

30 June

14 July

29 July

I3 June- I9 July

21 June-28 July

29 June-8 August

8 July- 15 August

19 July-24 August

2 August-7 September

13 August- I 8 September

28 August-3 October

I3 September- 18 October

29 September-5 November

12 October- 17 November


Average anatoxins

(ng g - I P













Adapted from Widstrom (1992).

Geometric means are the antilogarithms of the logarithmic

means for aflatoxin concentrations.

(Lillehoj et a l . , 1976b). A comparison of several insect species and their impact

on aflatoxin contamination made by Widstrom et al. (1975) revealed that the

European corn borer was among the most effective in exacerbation of the infection and contamination processes. This insect may be an important factor in

preharvest contamination during sporadic outbreaks of contamination that occur

in the Corn Belt (Guthrie et a l . , 1982). The maize weevil, a late-season, preharvest pest of corn in the Southeast, has been identified as an effective vector of A.

j h v u s , while the wheat curl mite (Eriophyes tulipae, Keifer) was determined to

be ineffective as a vector (Barry et al., 1985). Nitidulid beetles (Nitidulidae:

Coleoptera) were associated with A . f l a w s infection of wounded kernels

(Lussenhop and Wicklow, 1990), and A. flavus contamination of corn earworm

moths was significantly correlated with aflatoxin contamination of grain sampled

from the Coastal Plain of Georgia over a 6-year period (McMillian et a l . , 1990).

No hybrid is resistant to attack by all of the insects that have been implicated as

contributors to aflatoxin contamination, but most states have performance bulletins that identify the hybrids most resistant to insects in their test areas to assist

growers in hybrid selection.

Husk tightness is an important component of plant resistance to most earfeeding insects, and several studies were initiated to determine if that trait was

also critical in reducing aflatoxin Contamination. A near-linear relationship be-



tween husk tightness and grain contamination was found among five hybrids

varying in husk tightness and evaluated under inoculated conditions when infected by corn earworm and European corn borer (Barry et ul., 1986). Similar

results were obtained by McMillian et al. (1987) and Widstrom et al. (1994),

reaffirming the importance of husk cover in regions where insects and/or contamination are chronic problems. The experiments with hybrids varying in husk

tightness provided an alternative explanation for experiments that had utilized

hybrids from varied locations. Differences in contamination that initially had

been ascribed to differences in adaptation could be more logically ascribed to

variation in husk cover. In fact, most of the differences in contamination were

probably due to husk cover since southern hybrids have much better husk protection than those developed for the Corn Belt. Lillehoj ei al. (1976~)determined

that hybrids adapted to the South and grown in the South had lesser amounts of

aflatoxin contamination than Corn Belt hybrids grown at the same location.

Resistance to insects that restricts insect activity in the ear, whether due to husk

traits or other inherited resistance traits, will reduce the resulting amount of

aflatoxin contamination in the grain. This is especially true for European corn

borer, which is also a notorious leaf-feeder (Lillehoj et a/., 1982a). Ear damage

by insects, when present, is nearly certain to be associated with the level of

aflatoxin contamination found in the grain, and has probably been an influencing

factor determining hybrid differences for contamination as a function of husk

coverage and plant resistance to those insects (Zuber er a / ., 1983; Barry et al.,


Selection of a hybrid with resistance to diseases is obvious as a means to

maximize yields, but it is a choice that will also reduce the risk of aflatoxin

contamination. The predisposition of seed to aflatoxin contamination by ear rots,

such a5 Helrninthosporium maydis, was first noted in grain samples from Georgia

in 1970 and 1971 (Doupnik, 1972). Aspergi/lus$uvus is often considered to be

an ear-rot organism in its own right, and as such is inextricably confounded with

damage by the ear-rot complex infecting corn ears in the field. Some investigators have chosen to evaluate A . flavus infection in that context (Campbell et al.,

1993). Inoculation and evaluation techniques similar to those used for other ear

rots have been employed for A . flavus (Campbell and White, 1994), but it must

be remembered that aflatoxin concentration is the ultimate trait of interest. Earrot evaluation procedures have been successful in identifying some hybrids with

resistance, even though many evaluations have been made only on a visual basis.

Attempts to separate the influence of organisms other than A. Javus that are

present on the ear have been generally confined to viewing them as competitors

for substrate and as such have been considered as a possible means of control.

This concept will be discussed in a subsequent section of this chapter. With

respect to grower selection of a hybrid, most separations of resistant types have

been based on aflatoxin contamination at harvest (Widstrom et al., 1978; Damah



et al., 1987; Kang et al., 1990; Wallin et al., 1991) or the percentage of infected

kernels at harvest (Tucker et al., 1986; Scott and Zummo, 1990a). Both resis-

tance evaluation methods have been validated as identifying many of the same

resistant germplasm sources, and therefore results from studies designed to answer other questions seem compatible, independent of the evaluation method

used (Widstrom et al., 1978, 1984c; Scott and Zummo, 1990a, 1994; Scott et

al., 1991). The greatest difficulty at this time is availability of hybrids that have

moderate levels of resistance to insects, diseases, and aflatoxin contamination,

not in identifying those hybrids.



The ultimate management tool for agronomic problems, including injury or

product quality, in plants is that of manipulation through plant breeding or other

genetic techniques designed to give control during plant growth and development. Initial efforts to locate and utilize genetic resistance to aflatoxin contamination in the corn plant were not very successful in that experimental results

were nonconclusive (Zuber, 1977; Widstrom et al., 1978; Lillehoj et al., 1980a).

Several management practices have been proposed to supplement the genetic

sources of resistance in the plant, although most investigators concede that plant

resistance will be a major component of any control package to reduce aflatoxin

contamination (Widstrom, 1992).




Components of any management package, whether or not perceived as temporary, include insect and disease control, and alleviation of conditions that contribute to stress on the plant (Lillehoj and Hesseltine, 1977; Zuber and Lillehoj,

1979, 1987). Stress relief has been viewed as a major component of resistance to

the contamination process (Lillehoj, 1983; Fortnum, 1987). The stress component must continue to receive major emphasis (Georgia Extension Aflatoxin

Committee, 1978; Duncan, 1979; Smith, 1981) throughout the life of the plant

(Fig. I).

1. Irrigation

One of the easiest methods of preventing stress on the plant is to avoid

drought. This can be accomplished by an adequate irrigation system and is a


Environmental stTesses on plants, diseases, and insects


Wind, water, and insect borne spores from colonized debris, sclerotia, and soil sources



Whorl to silking

L -Infection



and Colonization period-




t H a r v e s t period 4 4 - S t o r a g e and -b




b F t susceptible

65 time of infection 85


Approximate no. of

days post-planting




damage to grain by insects


Increasing post-infection aflatoxin accumulation


figure 1 The chronology of corn kernel infection by Aspergi/lusJ?avrts and subsequent atlatoxin contamination. Source: Widstrom ( 1992).

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