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Control of Aflatoxin Using Atoxigenic Strains and Irrigation Management
Control of Aflatoxin Using Atoxigenic Strains and Irrigation Management
DOI: 10.1002/csc2.20710
Crop Science
ORIGINAL RESEARCH ARTICLE
Crop Breeding & Genetics
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© 2022 The Authors. Crop Science published by Wiley Periodicals LLC on behalf of Crop Science Society of America.
2009). A. flavus load itself is difficult to impossible to measure rous and 64,000 plants per hectare. Corpus Christi trials were
in plants even with tools like reverse transcription-polymerase planted at 74,000 plants per hectare in both irrigated and dry-
chain reaction (Mideros et al., 2009). Without genotype × land. College Station and Corpus Christi had two inoculation
atoxigenic applications data available, it has been necessary types (atoxigenics applied (ATOX) and no atoxigenics applied
to assume that atoxigenic strains of A. flavus would act in a [noATOX]), each with four replications, ensuring appropri-
similar manner to the toxigenic strains. However, it is reason- ate statistical power for investigating these interactions. In the
able to hypothesize that certain plant phenotypes or unseen split-split-split-plot treatments, irrigated and dryland was the
genetic interactions could induce preferential susceptibilities main split followed by the atoxigenic application split fol-
to infection of either the atoxigenic or the native toxigenic lowed by genotypes.
strains. This interaction could potentially be exploited for Fields were selected for uniformity to minimize residual
gains in the synergistic reduction of aflatoxin accumulation error from unexplained field variation. The size of the trial
through genotype × atoxigenic applications. Exploiting this was 0.70 hectares, and design was an important factor for try-
interaction could allow producers to apply atoxigenic strains ing to reduce residual (i.e., unexplained) error. Replications
in hybrids that primarily host toxigenic strains. Similarly, within trials were randomly allotted so that field variation was
tolerance to water and nutrient stress affects aflatoxin produc- minimized. Field variation has typically been associated with
tion (genotype × management; Jones, et al., 1981). While we range and row effects (also called row and column, respec-
hypothesize that correctly managing genotype × atoxigenic tively) that are generally attributed to fertilizer and flood irri-
applications × management could decrease aflatoxin by gation. In College Station, trials and replications within trials
more than 90%, we also hypothesize that ignoring these were placed so that the furrow irrigation effects were as uni-
interactions could result in aflatoxin levels being higher than form as possible. This was less of a concern in Corpus Christi
expected. due to the installation of sub-surface drip.
The specific objective of this study were to a) evaluate
the aflatoxin accumulation in diverse commercial and exper- 2.1 A. flavus inoculation
imental hybrids known to have varying levels of suscepti-
bility to aflatoxin; b) evaluate the sources of variance from Half of the plots (ATOX) were treated with the commercial
the main effects of environment, management (irrigation vs. formulation of an atoxigenic strain, Afla-Guard (Syngenta
dryland), and atoxigenic applications (atoxigenic and toxi- Crop Protection, 2017), at a rate of 22.3 kg/ha just prior to
genic inoculated vs. toxigenic only); and finally c) evalu- tasseling. All plots were then treated with A. flavus isolate
ate the proportion of variance from the interactions between NRRL 3357 grown on autoclaved corn kernels. In brief, toxi-
main effects to determine synergistic or antagonistic relation- genic inoculum was applied shortly after the majority of plots
ships between aflatoxin control measures. This study will sug- had started silking at an amount of 6 oz. per 9.14 linear row
gest how closely researchers, agricultural companies, and pro- meters, usually around 12 d after the start of silking. Inocu-
ducers should manage these control measures for real-world lum was prepared from stock A. flavus isolate NRRL 3357
reductions in aflatoxin accumulations. as previously described (Isakeit et al., 2010). Similar to Far-
fan et al. (2013), following maturity, sub-samples were taken
2 MATERIALS AND METHODS from the research combine. These samples were then ground
in a Romer mill (Romer Labs), and aflatoxin levels were mea-
Beginning in 2014, irrigated (Irr) and dryland (Dry) trials sured via the Vicam AflaTest (VICAM).
were initiated in Corpus Christi, TX, and College Station, TX.
Experimental design consisted of a modified split-plot ran- 2.2 Traits measured
domized complete block design of 32 entries in 2014, includ-
ing local commercial checks, and 28 entries the following Secondary phenotypic traits were measured at the College
years (2015, 2016, and 2017) in two locations (Corpus Christi, Station location in all years for days to anthesis (DTA), days
College Station), including local commercial checks, each to silking (DTS), plant height (PHT) from the ground to the
having two management approaches of irrigated and dryland tip of the tassel, and ear height (EHT) from the ground to the
(Irr, Dry). The number of checks varied due to seed availabil- primary ear’s node attachment point. Grain yield was mea-
ity from 11 checks in 2014, 16 checks in 2015 and 2016, and sured on a plot basis with a JD 3300 modified research com-
8 checks in 2017. Planting populations and fertilizer applica- bine retrofitted with a Harvest Master classic grain gage for
tions in College Station were 81,000 plants per hectare in the total plot weight, moisture, and test weight (Juniper Systems,
irrigated trial with a fertilization rate of 134 kg/ha on nitrogen 1993–2017), and subsamples were taken from each plot dur-
and 61 kg/ha of phosphorous while the dryland had a fertil- ing harvest. Many of the same traits were taken most years in
ization rate of 90 kg/ha nitrogen and 61 kg/ha of phospho- Corpus Christi, but the ears were hand-harvested.
870 Crop Science PEKAR ET AL.
2.3 Statistical analysis well above the 300 ng g−1 upper regulatory limit (Table 1).
Best linear unbiased predictions means in this same environ-
Statistical analysis was conducted using R (R Development ment were 218 ng g−1 . When comparing the check hybrids
Core Team, 2010). Assuming a nonnormal distribution, as to the experimental hybrids during the College Station 2016,
is almost always the case for aflatoxin measurements, all which is the season with the highest accumulation, it was
aflatoxin data were first transformed by logarithmic transfor- found that the check group had significantly higher accrual of
mation to normalize the data (Betran, et al., 2002; Williams & aflatoxin than the experimental group (227 and 134 ng g−1 ,
Windham, 2015). Models were then analyzed with both raw respectively). In Corpus Christi, levels during the 2016 sea-
and transformed data using the lme4 R package (Bates, et al., son reached levels of 1,800 ng g−1 for an individual sample
2015) and validated with JMP (JMP14, 1989–2019). Aflatox- with a mean of 73 ng g−1 . In the overall analysis, combin-
ins in ppb (Afppb) and transformed aflatoxin data (AfTrans) ing both locations for AfTrans, the highest variation explained
were both reported throughout the analysis in order to offer was from the effect of location × year (38.3%). In AFppb, this
increased perspective on actual aflatoxin concentration. variation was only 6.6%. Nonnormal data, which is almost
For these analyses, it was assumed that test hybrids were always the case with Afppb aflatoxin data, caused an anomaly
unrelated and not randomly selected; thus, repeatability was fixed by log-transformation. The treatment for Inoc was suc-
estimated instead of heritability (Equation 1). Data were first cessful on average, as the mean of samples across all environ-
analyzed jointly across all years, locations, and management ments with the atoxigenic treatment applied was 39% lower
practices to evaluate overall trends and then were analyzed than untreated control (p < .001). This was substantially less
individually by environments (location by year). In the all- than the 75 to 90% that has been reported and suggests con-
years-combined analysis, hybrids’ genetic effects were treated trol factors might not have synergistic effects (Abbas, et al.,
as fixed while all other factors were random. Factors included 2011; Brown, et al., 1991; Isakeit, et al., 2010; Isakeit, et al.,
in the model were location, year, irrigation management, and 2011).
inoculation as well as all possible interactions between these. Hybrid grain yield means ranged from 3.4 to 10.3 MT ha−1
In order to detect the contribution of variation from all factors, in each environment. Yields were highest in College Station
an all-random model was used for both full model and sepa- 2014 overall. Yield is a primary grower criterion, and the
rate year analysis. Here range and row factor effects reflect the best experimental hybrid yield was 112% of the hybrid check
terminology used in furrow irrigation where water and trac- average in the 2015 environment. Competitive advantage of
tors drive down the row. The full model was fit as a five-level the experimental hybrids could be due to the nonadaptation
full factorial design. All means were compared using Fisher’s of commercially marketed hybrid cultivars used as checks.
protected least significant difference (P = .05). Repeatability The commercial checks sold in Texas have not been bred
(h2 ) was calculated as: (as inbreds or hybrids) in Texas (Murray, et al., 2019) nor have
they been specifically selected for the unique Texas environ-
( )
GE ε
ℎ2 = 𝐺∕ 𝐺 + + (1) ments tested (Farfan, et al., 2013). Based on 2017 data, the
𝑟 𝑟𝑒 Corpus Christi 2017 location flowered significantly faster at
53 DTA and 53 DTS, while the average across all recorded
where G, GE, and Ɛ were the variance components of geno-
years was 61 DTA and 60 DTS, respectively. Later than opti-
type, genotype by environment interaction, and residual error,
mal planting was conducted for all environments to maximize
respectively, with r as number of replications and e as number
stress for increased aflatoxin pressure. Later planting also
of environments.
compressed flowering time among genotypes due to faster
Correlations were compared using Pearson correlation
heat unit accumulation, which was beneficial for applying
coefficients. Phenotypic correlations were formed on raw
more uniform inoculations. A drawback to later planting and
data, while genotypic correlations were formed from the best
compressed flowering times was that there was less variation
linear unbiased predictor (BLUP) genotypic estimates of each
present to determine how flowering time might have affected
trait extracted from the reduced model.
aflatoxin levels and grain yields.
3 RESULTS AND DISCUSSION Plant height and EHT in all environments had a BLUP
mean range of 179.3 to 252.2 cm and an EHT BLUP mean
To best discriminate genotypes and other treatment effects, range of 46.3 to 96.2 cm, suggesting variation in stress
higher environmental aflatoxin levels are useful. Here, afla- between environments that limited or favored plant growth.
toxin accumulation was sufficient to discriminate genotypic In environments with lower PHT and EHT, yield also tended
sources of variation in all years. In College Station, aflatoxin to be lower, which is commonly observed in the stressful con-
accumulation was highest during the 2016 growing season, ditions of Texas (Farfan, et al., 2013; Liu & Wiatrak, 2011;
reaching levels up to 940 ng g−1 for an individual sample, Yin, et al., 2011).
PEKAR ET AL. Crop Science 871
T A B L E 1 Summary statistics on best linear unbiased predictors (BLUPs) for transformed aflatoxin data, actual ng g-1 of aflatoxin, test weight,
yield, days to 50% anthesis, days to 50% silking, plant height, and ear height separated by location and years. Environment is a combination of
location and year for College Station (CS) and Corpus Christi (CC)
All
years CC14 CC15 CC16 CC17 CS14 CS15 CS16 CS17
Log-transformed Max 2.5 2.5 1.8 1.8 2.5 1 1.9 2.6 1.1
[Aflatoxin (ng
g−1 )+1]
Mean 1.1 2.1 0.8 1.4 1.7 0.5 0.6 1.2 0.4
Min 0.5 1.6 0.3 0.7 0.8 0.1 0.2 1.7 0.4
SD 0.3 0.2 0.4 0.2 0.4 0.2 0.4 0.3 0.2
Aflatoxin (ng Max 391.1 477.4 187.7 142.8 412.3 120.6 146.9 481.9 179.5
g−1 )
Mean 85.9 204.3 35.7 72.6 122.9 30.5 12.8 217.7 55.4
Min −30.5a 67 2.4 14.3 15.4 1.9 0.9 77.3 5.9
SD 68.1 95.4 45.1 37 96.9 30.1 27 112.3 41.2
Test weight Max 61.8 58.5 62.1 54.8 61.6 61.1 59 60.6
Mean 57.7 54.7 59.2 56.1 58.3 58.8 55.2 58.2
Min 53.3 51.1 54.4 53.4 54.8 56 51.9 55.2
SD 1.8 2 1.9 0.9 1.7 1.5 1.9 1.3
Yield (MT ha−1 ) Max 9.5 8 9.6 7.4 4.8 12.1 9.7 10.5 10.3
Mean 7.7 5.3 7.2 5.9 3.4 10.3 8.3 9 8.6
Min 3.7 3.4 5.5 5.3 2 6.6 6.8 6.8 6.7
SD 1 1.2 1 0.5 0.6 1.3 0.7 0.8 0.8
Days to anthesis Max 68.3 – – – 57.5 83.1 65.3 66.8 66
Mean 63.3 – – – 54.7 78.5 61.6 61 60.8
Min 59.2 – – – 51.9 75.4 57.1 58.1 57.2
SD 1.9 – – – 1.2 1.8 2.2 2.1 2.4
Days to silking Max 69.5 – – – 59.1 82.9 67.4 67.4 67.5
Mean 63.3 – – – 55.6 79.1 63.1 61.7 61.4
Min 59.2 – – – 53.3 75.7 57.9 58.1 57.3
SD 1.9 – – – 1.5 1.8 2.8 2.3 2.9
Plant height (cm) Max 254.9 245.5 205.2 227.3 216.3 275.1 238.6 259.7 274.2
Mean 227.8 226.8 179.3 214.5 190.8 252.2 220.2 237.3 240.3
Min 197.6 205.3 146.1 204.9 168.4 222.1 199.4 218.6 211.3
SD 11.5 11.2 15.4 5.3 10.5 12.4 9.7 10.9 13.9
Ear height (cm) Max 115.1 103 65.3 97.6 93.6 113.5 94.9 123.2 117.6
Mean 82.6 85.7 46.3 87.3 74.5 96.2 75.5 86.1 4.2
Min 57.3 71.6 31.8 74.9 61.8 78.6 63.3 68.4 59.7
SD 10.1 6.8 8.6 5.9 8.1 9.5 8.6 12.4 14.1
Note. Underlined numbers indicate the mean, minimum, and SD of the associated row.
a
A negative BLUP value was an artifact of the model fitting all years’ and locations’ combined analysis.
3.1 Variance components and repeatability: However, when aflatoxin data were transformed the major
Aflatoxin sources of variation fell into Loc × year and Loc × year × Irr,
38.3 and 30.1%, respectively. These major sources of varia-
Variance components are useful for evaluating sources of vari- tion for aflatoxin data that, while interesting, are not sources
ation in an experiment and allow repeatability to be calcu- that a farmer would have much control over. Total genotypic
lated. Location × year and the Loc × year × Irr interac- variation was 3.8% for AfTrans and 3.7% for Afppb in the
tion variables for Afppb explained 6.6 and 0%, respectively. full model (all collected data), suggesting genotype was a
872 Crop Science PEKAR ET AL.
minor to moderate driver of aflatoxin variation in this exper- 3.2 Variance components: grain yield and
iment; however, it is one a producer can easily manipulate. secondary agronomic traits
This lower genotypic variation also reflects a generally less-
susceptible set of cultivars tested here. Location × year × Irr × Within the full model, the variation explained for grain yield
Gen for transformed aflatoxin explained 3.3% variation, and was from Loc (55.4%) with only 3.3% explained by the Year.
this four-way interaction would be useful in selecting reduced Only 2.3% of variation was explained by the irrigation regime.
aflatoxin-susceptible genotypes for different environments The reduced variation from irrigation could be explained by
such as dryland in Corpus Christi versus irrigated in Col- rainfall amounts during most seasons that limited moisture
lege Station. While mean comparisons showed that atoxigenic stress from occurring. It was observed that Loc × Year × Irr
applications reduced aflatoxin, this main effect explained only had a large effect on AfTrans (30.12%) while Year and Irr
2.7% of variation for AfTrans and 4.7% for Afppb. Variation did not have a large effect on Yield. This could be explained
explained by inoculation was 2.7% and demonstrated that for by timing of rainfall or irrigations during the season which
this experiment, selecting resistant genotypes would have a may have had little beneficial effect on plant growth, but a
larger effect than applying atoxigenic strains. On transformed large increase in Aspergillus flavus growth. Genotype, what
aflatoxin data, Loc × Inoc × Gen and year × Irr × Inoc × farmers have most control over explained 27.4%, but this was
Gen both explained .2% of the variation. Demonstrating the likely inflated due to some experimental checks with low
effectiveness of atoxigenic control applications depended on yield. Location explained 66.4% variation for PHT, similar to
the different locations and genotypes examined. Due to logis- grain yield, however only 12% of the variation for EHT was
tics and knowledge of flowering, it was difficult to ensure that explained by Loc. In EHT the variation was more attributable
the atoxigenics were applied at the same developmental stage to hybrid genotype (17.7%), whereas genotype only explained
in each environment, which could have caused some of this 7.7% of the variation for PHT.
variation. Total variation explained from all variables con- Flowering variation was mostly explained by Year
taining Inoc was 5.42%. Genotype × Inoc and Loc × Inoc (DTS = 77.2%, DTA = 75.7%) indicating that days to flow-
× Gen explained .14% and .16% variation, respectively. This ering was mostly dependent on seasonal variations such as
could suggest that in this experiment, atoxigenics had dif- heat accumulations shared across College Station and Corpus
ferent effectiveness for genotypes. Comparing the atoxigenic Christi. Residual error for both DTS and DTA was 1.3 and
applications against the check hybrids versus the experimen- 1%, respectively. Comparing across all agronomic traits, the
tal hybrids it was observed that the treatment of an atoxigenic year had the greatest effect on creating experimental variabil-
application when applied to the experimental hybrids did sig- ity than nearly any other source. This was surprising given
nificantly lower the amount of aflatoxin accrued by up to 38% these two environments and the four years were relatively
(p < .001). similar to each other. This is important because environment
A substantial interaction of 30.1% was seen between Loc (year × location) is what producers have the least ability
× year × Irr probably brought forth by irrigation or rainfall to control, suggesting that for yield and agronomic traits,
timing events during inoculations which may have increased genetics and management can have limitations in meeting
or decreased the amount of aflatoxin accumulation across dif- producer’s needs.
ferent environments. The full model for transformed aflatoxin
data explained 84% variation with only 16% variation remain-
ing as unexplained residual error which is relatively good for 3.3 Correlations
an aflatoxin experiment, perhaps because of the number of
replications tested and the large variations in environments Transformed aflatoxin was significant (p < .01) and nega-
and genotypes. tively correlated with grain yield at r = –.17 while Afppb
Repeatability was used over heritability due to the lack of correlated negatively with grain yield at r = –.12 (Figure 1).
family structure in the genotypes. Repeatability is typically These negative correlations showed that better-yielding mate-
high for flowering and height and lower for yield and aflatox- rial was less aflatoxin-accumulating overall, supporting the
ins (Farfan, et al., 2015; Wahl, et al., 2017). Across all envi- hypothesis that adaptation may be more important than resis-
ronments, repeatability for aflatoxin accumulation data was tance, per se, across diverse elite hybrids. Plant height and
88% using transformed data down to 72% on nontransformed EHT were significant (p < .01) and positively correlated with
raw data (Table 2). Repeatability for aflatoxin accumulation yield at r = .73 for PHT and r = .36 for EHT. This positive cor-
within individual environments was lower than for all other relation between plant height and grain yield is very similar to
important agronomic traits, as expected, but still moderate to that found by Farfan et al. (2013) (r = .61) across over 10,000
high. plots of elite commercial hybrids. Negative correlations
PEKAR ET AL. Crop Science 873
T A B L E 2 Variance components and percent total for traits measured in all years and locations combined for transformed aflatoxin value
(Aftrans), actual measured aflatoxin accumulation in parts per billion (Afppb), yield (T ha-1 ), plant height (PHT), ear height (EHT). Terms defined as
Location (Loc), Year (Year), Irrigation method (Irr), Inoculation method (Inoc), Genotype (Gen), Replication (Rep), Range (Range), and Row (Row)
TA B L E 2 (Continued)
TA B L E 2 (Continued)
2015 by 107% and in 2016 by 105%. In addition to the high majority of entries were of TAMU × TAMU hybrids. This
yield from this hybrid, it accumulated 62% less aflatoxin than bias will inherently skew the trend to favor the TAMU ×
the average checks in 2015, while in 2016 it accumulated TAMU hybrids, which have been selected for adaptation to
15% less aflatoxin than the average checks. This supports the the subtropical environment of central Texas that this testing
overall hypothesis that inbreds and hybrids bred in the central- was conducted in, for resistance to aflatoxin accumulation.
South Texas growing region can be better adapted, meaning
they may have both higher grain yield and lower aflatoxin AC K N OW L E D G M E N T S
accumulations than commercial hybrids bred elsewhere that The authors would like to thank Steven Anderson, David
have better overall adaptation (Ewing, et al., 2019). Rooney, and staff of the Texas A&M AgriLife Research and
Several hybrids did well in both dryland and irrigated Extension Center at Weslaco for their help with this research.
regimes (Table 3). Most notable were the hybrids consisting of This research was funded by USDA National Institute of Food
Bolivian material (Tx777 and Tx779); several hybrids tested and Agriculture Competitive Grant 2014-6804-21836, USDA
shared one of these parents, and again, the Tx781/Tx777 Hatch funds, USDA-ARS, Texas A&M AgriLife Research,
hybrid performed well in both dryland and irrigated regimes. the Aflatoxin Mitigation Center of Excellence, and Texas
In previous studies, LH195 consistently produced hybrids Corn Producers Board.
with comparable yield to local commercial checks in the
stressful Texas environments (Murray, et al., 2019). This tester
AU T H O R C O N T R I B U T I O N S
was used to make several of our hybrids for this study both as
Jacob J. Pekar: Data curation; Formal analysis; Writing – orig-
an expired plant cultivar protection line (Foley, 1991) and as
inal draft; Writing – review & editing. Seth C. Calder Mur-
modern-traited inbred. The expired plant cultivar protection
ray: Conceptualization; Funding acquisition; Methodology;
line has shown some differences, as both a line and in hybrid
Project administration; Supervision; Writing – original draft;
combination, from the commercially available inbred, though
Writing – review & editing. Thomas Isakeit: Methodology;
both look nearly identical.
Supervision; Writing – review & editing. Luke S. Pruter: Data
curation; Writing – review & editing. Nancy J. Wahl: Writ-
ing – review & editing. Michael J. Brewer: Conceptualization;
4 CONCLUSIONS Methodology; Writing – review & editing.
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