Resistance to aflatoxin accumulation in kernels of maize inbreds selected for ear rot resistance in West and Central Africa.

Abstract

Thirty-six inbred lines selected in West and Central Africa for moderate to high resistance to maize ear rot under conditions of severe natural infection were screened for resistance to aflatoxin contamination using the previously established kernel screening assay. Results showed that more than half the inbreds accumulated aflatoxins at levels as low as or lower than the resistant U.S. lines GT-MAS:gk or MI82. In 10 selected aflatoxin-resistant or aflatoxin-susceptible inbreds, Aspergillus flavus growth, which was quantified using an A. flavus transformant containing a GUS-beta-tubulin reporter gene construct, was, in general, positively related to aflatoxin accumulation. However, one aflatoxin-resistant inbred supported a relatively high level of fungal infection, whereas two susceptibles supported relatively low fungal infection. When kernels of the 10 tested lines were profiled for proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, significant variations from protein profiles of U.S. lines were observed. Confirmation of resistance in promising African lines in field trials may significantly broaden the resistant germplasm base available for managing aflatoxin contamination through breeding approaches. Biochemical resistance markers different from those being identified and characterized in U.S. genotypes, such as ones inhibitory to aflatoxin biosynthesis rather than to fungal infection, may also be identified in African lines. These discoveries could significantly enhance the host resistance strategy of pyramiding different traits into agronomically useful maize germplasm to control aflatoxin contamination.

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Journal of Food Protection, Vol. 64, No. 3, 2001, Pages 396–400
Copyright q, International Association for Food Protection
Research Note
Resistance to A?atoxin Accumulation in Kernels of Maize
Inbreds Selected for Ear Rot Resistance in West and
Central Africa
ROBERT L. BROWN,1* ZHI-YUAN CHEN,2ABEBE MENKIR,3THOMAS E. CLEVELAND,1KITTY CARDWELL,3
JENNIFER KLING,3AND DONALD G. WHITE4
1Southern Regional Research Center, U.S. Department of Agriculture, Agricultural Research Service, New Orleans, Louisiana 70179, USA;
2Department of Plant Pathology and Crop Physiology, Louisiana State University, Baton Rouge, Louisiana 70803, USA;3International Institute of
Tropical Agriculture, Ibadan, Nigeria; and4Department of Crop Sciences, University of Illinois, Urbana, Illinois 61801, USA
MS 00-162: Received 26 May 2000/Accepted 14 September 2000
ABSTRACT
Thirty-sixinbred lines selectedin West and Central Africa for moderateto high resistanceto maize ear rot underconditions
of severe natural infection were screened for resistance to a?atoxin contamination using the previously established kernel
screening assay. Results showed that more than half the inbreds accumulated a?atoxins at levels as low as or lower than the
resistant U.S. lines GT-MAS:gk or MI82. In 10 selected a?atoxin-resistantor a?atoxin-susceptibleinbreds, Aspergillus ?avus
growth, which was quanti?ed using an A. ?avus transformant containing a GUS-b-tubulin reporter gene construct, was, in
general, positively related to a?atoxin accumulation. However, one a?atoxin-resistantinbred supported a relatively high level
of fungal infection, whereas two susceptibles supported relatively low fungal infection. When kernels of the 10 tested lines
were pro?led for proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis,signi?cant variationsfrom protein
pro?les of U.S. lines were observed. Con?rmation of resistance in promising African lines in ?eld trials may signi?cantly
broaden the resistant germplasm base available for managing a?atoxin contaminationthrough breeding approaches.Biochem-
ical resistance markers different from those being identi?ed and characterized in U.S. genotypes, such as ones inhibitory to
a?atoxin biosynthesis rather than to fungal infection, may also be identi?ed in African lines. These discoveries could signif-
icantly enhance the host resistancestrategy of pyramidingdifferent traits into agronomicallyuseful maize germplasm to control
a?atoxin contamination.
A?atoxins, toxic secondary metabolites of the fungi
Aspergillus ?avus Link:Fr. and A. parasiticus Speare, are
potent carcinogens. They pose serious health hazards to hu-
mans and domestic animals because of their frequent con-
tamination of agricultural commodities, such as cottonseed,
peanuts, tree nuts, or maize (8, 15). A?atoxin contamina-
tion of maize (Zea mays L.) is a preharvest and a posthar-
vest problem; A. ?avus may infect the crop before harvest
and remain throughout harvest, storage, and use (23).
Progress made in identifying maize genotypes that re-
sist a?atoxin contamination has enhanced host resistance
strategies for eliminating or controlling a?atoxin contami-
nation (2, 3, 6, 7, 25, 28). These strategies also have ben-
e?ted from the discovery of both maize kernel pericarp and
subpericarp resistance to a?atoxin contamination (3, 6, 19).
Regarding the former, kernel pericarp wax of a resistant
genotype, GT-MAS:gk, has been implicated as potentially
a physical barrier to fungal ingress and as chemically in-
hibitory to A. ?avus growth and subsequent a?atoxin pro-
duction (19, 24). Subpericarp resistance has been investi-
gated through side-by-side comparisons of maize kernel
* Author for correspondence. Tel: 504-286-4359; Fax: 504-286-4419;
E-mail: rbrown@commserver.srrc.usda.gov.
protein pro?les of susceptible and resistant germplasm (11,
16, 17). Both constitutive and inducible proteins associated
with resistance have been identi?ed and characterized (10,
11, 16, 17, 20). It is hoped that these markers will play a
role in the development of a strategy of pyramiding differ-
ent resistance mechanisms against A. ?avus infection and
a?atoxin accumulation into agronomically useful maize
germplasm through either breeding or genetic engineering.
However, for this strategy to succeed, germplasm with
higher levels of resistance and that use different mecha-
nisms than those already identi?ed will have to be discov-
ered.
The purpose of the present study was to investigatethe
potential of 36 maize inbreds, adapted to the savanna and
mid-altitude ecological zones of West and Central Africa,
to resist a?atoxin accumulation. These lines were selected
for resistance to ear rot under conditions of severe natural
infection in their respective areas of adaptation and have
moderate to high levels of resistance to ear rot. The major
ear rot–causing fungi in these environments include Asper-
gillus, Botrydiplodia, Diplodia, Fusarium, and Macropom-
ina. To determine the potential of these inbreds to resist
a?atoxin production by A. ?avus, the kernel screening lab-
oratory assay (KSA) was used (3, 6). It also was used in

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397
conjunctionwith an A. ?avus transformant expressingEsch-
erichia coli b-glucuronidase (GUS) to assess fungal growth
in selected resistant and susceptible maize genotypes(3, 6).
The KSA has been used successfully in previous studies to
identify maize genotypes resistant to a?atoxin production
and, along with the above A. ?avus GUS tester strain, to
study resistance mechanisms and to assess fungal growth
(3, 4, 6). Sodium dodecyl sulfate-polyacrylamide gel elec-
trophoresis (SDS-PAGE) was used in the present study to
preliminarily assess these selected lines for protein pro?le
diversity and, thus, potential to contain resistance markers
varying from those observed in U.S. resistant lines. A pre-
liminary report of a?atoxin accumulation assessment has
been made (1).
MATERIALS AND METHODS
Maize entries. Thirty-six inbred lines were obtained from
the International Institute of Tropical Agriculture in Ibadan, Ni-
geria. Genotype MI82, a resistant control, was obtained from the
Department of Crop Sciences of the University of Illinois. GT-
MAS:gk, another resistant control was obtained from the U.S.
Department of Agriculture, Agricultural Research Service, Insect
Biology Population Management Research Laboratory, in Tifton,
Ga. Delta Pine G-4666 (DP), the susceptiblecontrol,was obtained
from the Department of Plant Pathology and Crop Physiology at
Louisiana State University. MI82 was identi?ed as resistant to
a?atoxin production as an inbred and as an F1 cross with suscep-
tible inbreds B73 and/or Mo17 (3, 7). GT-MAS:gk was identi?ed
as resistant in numerous ?eld trials and has been studied exten-
sively in laboratory investigations (2, 28). DP is susceptible to
the elaboration of a?atoxins (18). All kernels were kept in sealed
plastic containers at 48C until used.
Fungal strains and growth conditions. The A. ?avusisolate
(AF13) used in the evaluation of the 36 inbreds was isolated from
agricultural soils in Arizona (12, 14). It produces large quantities
of a?atoxins in developing cottonseed and maize and in culture
(5, 12). Cultures were grown at 308C in the dark on a 5% V-8
juice and 2% agar medium. Plugs (3 mm in diameter) of sporu-
lating cultures were stored at 88C on a long-term basis in 4-dram
(14.788-ml) vials containing 5 ml of deionized water (12, 13).
Conidia from 4- 7-day-old cultures suspended in deionized water
served as inocula.
An A. ?avus isolate (GAP2-4) transformed with the E. coli
GUS gene linked to an A. ?avus b-tubulin gene promoter (3) was
used to quantify fungal growth in maize kernels of selected in-
breds. Cultures were grown at 378C in the dark on potato dextrose
agar. Conidia from 4- to 7-day-oldculturessuspendedin deionized
water served as inocula.
Inbred evaluation.Kernels of 36 inbreds(dividedand tested
in four separate series, each containing nine inbreds plus two con-
trols) and of genotypesMI82 or GT-MAS:gk and DP were surface
sterilized (27) and then dipped into a suspension of AF13 conidia
(4.0 3 106conidia/ml) and evaluated using the KSA (3, 6). Each
experimental unit contained 3 kernels that were replicated 10
times. Kernels were incubatedwith AF13 using the KSA protocol
(318C, 7 days) and afterward were removed and dried in a forced-
air oven at 608C for 2 days to stop fungal activity and prepare
samples for a?atoxin analyses (3). The experimentwas performed
twice.
Inoculations with GAP2-4 and quanti?cation of GUS ac-
tivity. The 10 inbreds, four susceptible and six resistant, selected
for further evaluation using the GAP2-4 strain were, respectively,
1188, 603, 15, and KU and 1368, 502, 305, 102, 28, and 1823.
Kernels of these 10 genotypes and of MI82 and DP were surface
sterilized as above. They were inoculated by dipping them into a
spore suspension of A. ?avus GAP2-4 (4.0 3 106conidia/ml) and
were incubated at 318C for 6 days using the KSA. Each treatment
was replicated eight times, with each replicate containing three
kernels. Tests were performed twice. After incubation, kernels
were subjected to a protocol for ?uorogenic GUS quanti?cation
(4, 21). For each enzyme reaction, 50 ml of crude extract was
added to the assay buffer containing the substrate, and reactions
were stopped after 20 min. Preliminary kinetic studies were per-
formed to identify the amount of crude extract to use in each test
and a time point that fell within the linear portion of the enzyme
reaction curve. GUS activity in samples was determined with a
Gilford Fluoro IV spectro?uorometer (Corning Laboratory Sci-
ences Co., Oberlin, Ohio); excitation was at 360 nm and emission
at 455 nm. GUS activity was normalized through protein deter-
minations in crude extracts using the method of Sedmak and
Grossberg (26).
A?atoxin analyses. The a?atoxin B1content of replicates
from all tests was determined by a procedure used in an earlier
study (6). This protocol involved methylene chloride extractionof
a?atoxins from infected seed, thin layer chromatographicsepara-
tion of compounds, and quantitationof a?atoxin B1using a scan-
ning densitometer.
Statistical analyses. Analyses of a?atoxin data were per-
formed with the Statistical Analysis Software System (SAS Insti-
tute, Inc., Cary, N.C.). Treatment replicates from each test were
?rst subjected to analysis of variance followed by mean compar-
isons of log transformationsof toxin values.Transformationswere
performed to equalize treatment variances. Differences among
treatment means were determined by the least signi?cant differ-
ence test.
Protein extractionand gel electrophoresis.Dry kernels(20
g) of each of the 10 selected genotypeswere extracted for protein
using the procedureof an earlier study (11). SDS-PAGE of protein
extracts was performed using a 15% resolving gel with a 4%
stacking gel according to Laemmli (22). Low-range proteinmark-
ers (Sigma Chemical Co., St. Louis, Mo.) were used as molecular
mass standards. The gels were electrophoresed (120 V, 1.5 h),
stained with 0.125% Coomassie blue R-250 in 50% methanoland
10% acetic acid for 1 h at room temperature, and destained in
50% methanol and 10% acetic acid.
Quanti?cation of proteins. The quanti?cation of selected
proteins (14 and 34 kDa) was performed using Bio-Rad’s GS-700
Gel Densitometer (Bio-Rad Laboratories, Richmond, Calif.) and
the associated Molecular Analyst software. The data presented in
this report are the mean values of each genotype obtained in two
experiments.
RESULTS AND DISCUSSION
A?atoxin B1levels in 19 of the 36 African lines tested,
inbreds 1393, 5057, 1368, 5012, 103, 104, 502, 305, 102,
25, 20, 30, 34, 28, 2151, MmB90, 7271, 5052, and 1823,
were approximately equivalent to levels measured in resis-
tant controls, MI82 or GT-MAS:gk, in respective tests (Ta-
ble 1). Toxin levels supported by inbreds 28 and 34 were
lower than those supported by GT-MAS:gk. There was
great variation in the overall amount of a?atoxin B1pro-
duced in test series A and B compared with series C and

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J. Food Prot., Vol. 64, No. 3
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BROWN ET AL.
TABLE 1. A?atoxin accumulation in West and Central African maize genotypes
Line/toxin (ng/g)a
Series ASeries BSeries CSeries D
1188/1839 A
1394/1395 A
DP (S)/1277 AB
2097/1267 AB
1201/756 B
2096/557 BC
1393/431 CD
5057/132 D
1368/78 D
MI82 (R)/41 D
5012/23 D
603/1528 A
DP (S)/1231 A
604/1043 AB
301/1032 AB
501/963 AB
103/571 BC
MI82(R)/457 CD
104/268 CD
502/70 D
305/34 D
102/21 D
DP (S)/16.979 A
15/15.633 A
38/8830 B
39/7425 B
35/6858 B
25/5441 BC
20/4363 BC
MAS(R)/2212 CD
30/1781 DE
34/720 E
28/403 E
KU/9710 A
4205/4885 B
9484/3883 B
8071/2377 BC
DP (S)/1604 CD
2151/739 DE
MmB90/571 DE
7271/151 E
5052/129 E
MI82(R)/103 EF
1823/39 F
aValues are averages of 20 replicates from two separate tests. Data were log transformed before analyses to equalize variances.Inbreds
to be tested were divided into groups or ‘‘series’’ and tested at the same time. Each series contained a resistant and a susceptible
control.
bValues in a column followed by the same letter are not signi?cantly different (P 5 0.05) by the least signi?cant difference test.
FIGURE 1. Fungal growth measured through speci?c GUS activ-
ity in nM of methylumbelliferone(MU) produced per min per mg
of protein in 10 selectedresistantand susceptibleAfrican inbreds.
Bars represent GUS activity means 6 standard error of 16 rep-
etitions measured during two experiments. MI82 is the resistant
control; DP, the susceptible control.
D; this variability phenomenon is routinely observed in af-
latoxin studies. Con?rmation of resistance in these lines
through ?eld evaluations could greatly increase the number
of resistant inbred lines being investigated in a?atoxin-re-
sistance breeding programs and could enhance the level of
resistance accessible to breeders.
Fungal growth of the A. ?avus GUS tester strain in
kernels of 10 selected inbreds followed the typical pattern
observed in U.S. lines, being generally high in susceptible
and low in resistant inbreds. This may indicate the possible
functioning of kernel antifungal resistance mechanisms.
Typical quantities (measured in nM of methylumbelliferone
produced per min per mg of protein) of fungal growth (Fig.
1) were observed in susceptible inbreds 603 and KU and
in resistant inbreds 305, 502, 102, 1823, and 28. However,
growth of A. ?avus in kernels of susceptible inbreds 1188
and 15 was atypical. It was lower than and equal to, re-
spectively, growth in resistant control MI82. Fungal growth
in resistant inbred 1368 varied from levels normally en-
countered in resistant inbreds. The levels of A. ?avus mea-
sured in inbred 1368 were as high as those observed in
susceptible inbreds 603 and KU. The discovery of high
fungal growth in a maize line that accumulates low levels
of a?atoxins could lead to the identi?cation and character-
ization of a kernel resistance mechanism(s) directly inhib-
itory to a?atoxin biosynthesis rather than to fungal infec-
tion. This might enhance the development of maize germ-
plasm through the pyramiding of diverse resistance traits
and yield maize lines capable of sustaining resistance to
a?atoxin formation over time and in different environ-
ments. The discovery that a?atoxin-susceptible inbreds
1188 and 15 support low levels of fungal growth may pro-
vide investigatorswith the opportunityto study in vivo sub-
strate effects on a?atoxin production, an opportunity up to
now not afforded by U.S. lines under investigation.
Genetic diversity may have been demonstrated in the
electrophoretic pro?les of the African inbreds compared
with U.S. lines. Not only is there variation from the typical
pro?les as represented by MI82 and DP, but there is sig-
ni?cant diversity among the African resistant and suscep-
tible lines as well. On SDS-PAGE gels, a 14-kDa protein
band correspondingto the size of a trypsininhibitorprotein,
whose constitutive levels were previously correlated with
resistance in U.S. lines (including MI82) (11), is easily ob-
served as is a 34-kDa band corresponding to ribosome-
inactivating protein (Fig. 2). Average percentage of protein
content of the 14-kDa band for susceptible inbreds was rel-
atively high compared with that found in a previous study
involving U.S. genotypes (11) and as a group were not

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J. Food Prot., Vol. 64, No. 3 RESISTANCE TO AFLATOXIN FORMATION RELATED TO EAR ROT RESISTANCE
399
FIGURE 2. SDS-PAGE analysis of extracted kernel proteins of 10 selected African inbreds. Resistant inbred pro?les are in lanes 1
through 6 and susceptible inbred pro?les are in lanes 9 through 12. Lane 1, 1368; lane 2, 102; lane 3, 305; lane 4, 28; lane 5, 502;
lane 6, 1823; lane 7, susceptible control, DP; lane 8, resistant control, MI82; lane 9, KU; lane 10, 603; lane 11, 15; and lane 12,
1188. White arrows point to 14- and 34-kDa protein bands.
TABLE 2. Quanti?cation of the relativecontent of a 14-kDa pro-
tein band and a 34-kDa band in the total kernel protein extracts
of the 10 selected African genotypes based on analysis of 15%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis pro-
?les after staining with Coomassie brilliant blue R-250
Entries
Resistance to
A. ?avusa
Protein band
14 kDa 34 kDa
1368
102
305
28
502
1823
MI82 (C)
DP (C)
1188
15
603
KU
R
R
R
R
R
R
R
S
S
S
S
S
8.98
14.55
15.96
20.53
16.64
4.43
11.81
13.53
3.80
14.54
17.59
13.37
2.22
1.97
1.40
1.55
3.96
1.71
1.46
1.35
5.98
0.59
1.69
2.17
aR, potentiallyresistant;S, susceptible;both determined by KSA.
different from the resistant African inbreds (Table 2). The
possibility that susceptible African inbreds (unlike tested
U.S. susceptible lines) could possess high levels of this
trypsin inhibitor may indicate that traits or interactions be-
tween traits, different from those in U.S. lines, are respon-
sible for the resistance demonstrated in the African inbreds.
DP, uncharacteristically for U.S. susceptible genotypes, ex-
pressed a high level of the 14-kDa protein in these tests
and did so in previous tests as well (9).
The highest levels of the 34-kDa protein band were
seen in resistant inbred 502 and in susceptible inbred 1188
(Table 2), and as in U.S. lines (unpublisheddata), this band
was not associated with resistance. Another band, approx-
imately 23 kDa in size, was observed in pro?les of all sus-
ceptible inbreds and in resistant inbreds 1368, 102, and 28
that was not present in either the resistant or the susceptible
control (MI82 and DP, respectively). There also is variation
among resistant and susceptible African inbreds in the pro-
tein bands above the 34-kDa region. Overall, there appears
to be greater diversity in protein pro?les among the African
inbreds than between MI82 and DP, representativesof U.S.
resistant and U.S. susceptible classes, respectively. There
also appears to be quantitative differences among maize
lines in many individual protein bands. Further investiga-
tion, however, is needed to quantify and compare the ex-
pression of speci?c proteins and determine associations
with resistance.
In the last several years, it has been shown that maize
germplasm as a whole possesses genes that increase resis-
tance of the possessing genotype to a?atoxin accumulation
by A. ?avus. However, all identi?ed U.S. resistant lines dis-
play less than desirable agronomic qualities (2). This fact
highlights the need to identify and characterize speci?c re-
sistance traits that then can be transferred to commercially
desirable germplasm through marker-assisted breeding or
through maize transformation. A few resistance traits have
been characterized from resistant lines that appear to pre-
vent a?atoxin buildup indirectly through fungal growth in-
hibition (3, 11, 19); the characterization of other traits is
currently being pursued. The African inbreds evaluated in
the present study provide the potential to broaden the pres-
ent narrow base of a?atoxin-resistant maize germplasm and
enhance the levels of resistance attainable. They also may
provide resistance genes currently not expressed in U.S.
tested lines and even ‘‘weapons’’ useful against other se-
rious fungal diseases of crops.
ACKNOWLEDGMENTS
We thank Janice Pauline and Denise Hadrick for technical assistance.
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