Aflatoxin In Corn Evaluation Essay

1. Introduction

Corn production in the southern United States frequently encounters a period of drought and heat stress during flowering and kernel development. These weather conditions have been reported to be the major factors in increased aflatoxin contamination produced by the fungi Aspergillus flavus and A. parasiticus in corn and other economically important crops such as peanuts, cotton and tree nuts [1,2]. A. flavus infection and subsequent aflatoxin contamination is a serious issue in the southern US. Aflatoxins are toxic, highly carcinogenic secondary metabolites of these pathogens, which when produced during fungal infection of a susceptible crop in the field or after harvest, contaminate food and feed and threaten human and animal health [1,2].

Previous studies have shown that reduction in drought stress by irrigation reduces aflatoxin contamination, and drought stress tolerant corn varieties produce significantly less aflatoxin in the field in drought stress conditions [3,4,5]. Under conditions of drought and high temperatures, lowering soil temperature by irrigation was also found to reduce aflatoxin contamination [3,6]. In addition to drought and heat stress, other factors that produce stress on the plants such as inadequate plant nutrition, insect feeding on developing kernels, weed competition, excessive plant density, plant diseases, and other biotic and abiotic stresses facilitate the infection and production of aflatoxin by the fungus [2,7,8]. Several studies have shown that aflatoxin production is associated with oxidative stress, which is caused by abiotic and biotic stresses on plants [9,10,11,12,13,14]. Effects of drought, heat and other stress factors on physiological traits such as photosynthetic pigments, cell membrane thermostability, and maximum quantum efficiency of photosystem II are associated with oxidative stress [15,16,17,18,19,20,21]. The aim of this study was to evaluate the relationship between aflatoxin contamination and these stress response-related physiological traits in corn genotypes under drought and heat stress. Moisture and temperature conditions in the soil and the air were monitored to determine the extent of stress imposed on the plants.

2. Results

2.1. Environmental Moisture and Temperature Conditions

The 2010 growing season was hotter and drier than that of 2009, with temperatures of 36 °C and 33 °C averaged over the months of June, July and August for 2010 and 2009, respectively, and a growing season total precipitation of 266 mm and 666 mm for 2010 and 2009, respectively (Mississippi State University weather network). In addition to the weather information, data from the plot-level automated monitoring system was used to observe the level of drought and heat stress imposed on the corn plants. Soil moisture in the non-irrigated plots was about four times lower than in the irrigated plots, and air temperature in the canopy microclimate and soil temperature in each plot were 2 °C–5 °C higher in the non-irrigated plots than in the irrigated plots [22]. Differences were observed among the plots of each corn genotype in these temperature and moisture measurements. Details are presented in Kebede et al. [22]. These data were important in explaining the effect of the temperature and moisture conditions on the growth and development of the corn plants as well as the fungus.

2.2. Aflatoxin Contamination

Aflatoxin contamination in the corn kernels was higher in 2010 than in 2009 under both soil moisture treatments (Figure 1), with levels significantly higher (p < 0.046) in the non-irrigated than the irrigated plots in 2010. In 2009, aflatoxin was determined only on four of the corn genotypes (P31G70, P32B34, P33F87, and DKC63-42) due to glyphosate damage on P31B13, PI 639055 and PI 489361 plants. Significant differences were observed among the genotypes in aflatoxin contamination in both years. In 2009, DKC63-42 had more than a nine-fold aflatoxin contamination compared to the other three hybrids, while P32B34 had the least amount under both soil moisture treatments (Figure 1A). The aflatoxin levels in P32B34 and P31G70 in the irrigated treatments, and in P32B34 in the non-irrigated treatments, were below 20 parts per billion (ppb), which is the allowable level of aflatoxin set by the FDA in corn grain that is used for human consumption [23]. In 2010, the aflatoxin test included the genotypes which were not tested in 2009 (P31B13, PI 639055 and PI 489361). In the irrigated treatments, aflatoxin was around the 20 ppb threshold level for all genotypes except for two hybrids, P33F87 (68 ppb) and DKC63-42 (109 ppb), which were significantly higher (Figure 1B). The aflatoxin resistant line (PI 639055) and P31G70 had levels below the threshold, 16 and 14 ppb, respectively. However, under non-irrigated treatments, all genotypes had levels above 20 ppb including the aflatoxin resistant line. The resistant line had the lowest contamination (29 ppb) followed by P31G70 (33 ppb), but there was no significant difference between the two values. DKC63-42 and PI 489361 had the highest level of aflatoxin among all genotypes, 186 and 185 ppb, respectively, which were more than double the accumulation of aflatoxin in most of the other genotypes. Aflatoxin contamination was not significantly different among P33F87, P32B34, and P31B13 under non-irrigated conditions, with intermediate levels compared to the other genotypes.

Figure 1. Aflatoxin contamination in corn kernels under irrigated and non-irrigated conditions in (A) 2009 and (B) 2010. Different letters on top of bars show significant differences (p < 0.05). The dashed lines in the graph (20 parts per billion, ppb) show the allowable level of aflatoxin contamination by the US Food and Drug Administration (FDA) [23].

Figure 1. Aflatoxin contamination in corn kernels under irrigated and non-irrigated conditions in (A) 2009 and (B) 2010. Different letters on top of bars show significant differences (p < 0.05). The dashed lines in the graph (20 parts per billion, ppb) show the allowable level of aflatoxin contamination by the US Food and Drug Administration (FDA) [23].

2.3. Physiological Responses to Drought and Heat Stress

2.3.1. Leaf Water Potential and Canopy Temperature

Leaf water potential (Ψw) and canopy temperature (CT) were used to assess moisture deficit and heat stress effects on the plants. Leaf water potential was negatively correlated with CT (r = −0.4907; p < 0.0516) and positively correlated with soil water potential (r = 0.6550; p < 0.0080), and it was significantly lower in the non-irrigated than the irrigated plants in both years, as shown in Figure 2A. Leaf water potential values were lower than −1.5 MPa after the second sampling date (June 12) in both irrigated and non-irrigated plants, but it was significantly lower in the non-irrigated plots going down as low as −2.3 MPa. No significant differences were detected among the corn genotypes in Ψw due to large variability in values within each genotype. Canopy temperature in the non-irrigated plots was 2 °C–5 °C higher than in the irrigated plots [22]. Differences were observed in CT among the corn genotypes. Figure 2B shows CT among the genotypes under non-irrigated conditions in 2010. Among the hybrids, DKC63-42 had the highest CT, particularly after the middle of June, followed by P32B34. The two germplasm lines, PI 639055 and PI 489361, had higher CT. Hybrid P31G70 had the lowest CT among all the genotypes.

Figure 2. Leaf water potential (Ψw) and canopy temperature (CT) for hybrids P31G70, P32B34, DKC63-42 and inbred lines PI 639055 and PI 489361 under irrigated and non-irrigated treatments in the months of June and July, 2010; (A) mean values across genotypes for Ψw on seven sampling dates; (B) mean values for hourly measurements (hours between 13:00 and 17:00) of CT in the non-irrigated plots of each genotype in 2010.

Figure 2. Leaf water potential (Ψw) and canopy temperature (CT) for hybrids P31G70, P32B34, DKC63-42 and inbred lines PI 639055 and PI 489361 under irrigated and non-irrigated treatments in the months of June and July, 2010; (A) mean values across genotypes for Ψw on seven sampling dates; (B) mean values for hourly measurements (hours between 13:00 and 17:00) of CT in the non-irrigated plots of each genotype in 2010.

2.3.2. Photosynthetic Pigments

Changes in chlorophyll and carotenoid content followed a similar pattern to the changes in Ψw. Highly significant correlation was observed between Ψw and the photosynthetic pigments (chlorophyll, r = 0.8532, p < 0.0012; carotenoids, r = 0.8821, p < 0.0007). Irrigation started in early June and drought stress started showing effect on the samples taken on June 21 and continued through the end of July. Mean values for chlorophyll and carotenoid contents were significantly higher in irrigated plants compared to the non-irrigated plants on these sampling dates (Line graphs in Figure 3A–D). However, reductions in these pigments were also observed in samples from the irrigated plants. Air temperature was high during this period of time, with average maximum temperatures in the range of 36 °C–38 °C during June 13–29 [22]. An increase in these photosynthetic pigments in the July 8 sampling date was due to several rain showers at the end of June and beginning of July with slightly cooler temperatures. In addition, it could be due to the age of the leaves, older leaves gradually accumulating more chlorophyll. Chlorophyll a to b ratio (Chl a/b) (Figure 3E,F) and carotenoids to chlorophyll ratio (Carot/Chl) (Figure 3G,H) were significantly higher in the non-irrigated plants.

Figure 3. Photosynthetic pigment content in leaves of seven corn genotypes sampled in June and July, 2010, under irrigated and non-irrigated treatments. Values for individual genotypes are shown in bar graphs and mean values for each sampling date are shown in line graph on top of each bar graph (secondary axis): (A and B) chlorophyll (Chl); (C and D) carotenoids (Carot); (E and F) chlorophyll a to chlorophyll b ratio (Chl a/b); (G and H) carotenoids to chlorophyll ratio (Carot/Chl). Samples were taken from eight plants for each genotype.

Figure 3. Photosynthetic pigment content in leaves of seven corn genotypes sampled in June and July, 2010, under irrigated and non-irrigated treatments. Values for individual genotypes are shown in bar graphs and mean values for each sampling date are shown in line graph on top of each bar graph (secondary axis): (A and B) chlorophyll (Chl); (C and D) carotenoids (Carot); (E and F) chlorophyll a to chlorophyll b ratio (Chl a/b); (G and H) carotenoids to chlorophyll ratio (Carot/Chl). Samples were taken from eight plants for each genotype.

Statistically significant differences were observed among the genotypes in these photosynthetic pigments as shown in Figure 3 (Bar graph). The germplasm line PI 489361 had significantly higher chlorophyll and carotenoids among all the genotypes (p < 0.0001) under both soil moisture treatments, which could be attributed to its thicker leaf [22]. PI 639055 had significantly lower chlorophyll and carotenoids than the other genotypes in most of the sampling dates under non-irrigated conditions, but had higher Chl a/b ratios. Among the hybrids, DKC63-42 had lower chlorophyll under non-irrigated conditions, and lower content of carotenoids under irrigated conditions. DKC63-42 had higher Chl a/b than the other genotypes under both irrigated and non-irrigated treatments (Figure 3E,F). PI 489361 had the lowest Chl a/b ratio, but this has been observed under non-stress conditions in other experiments under greenhouse and field conditions, which indicates that this ratio is not related to the plant's response to the stress conditions. Carotenoids to chlorophyll ratios were not consistent for all genotypes across sampling dates under both soil moisture treatments, but PI 639055, PI 489361 and DKC63-42 had higher ratios on several of the sampling dates (Figure 3G,H).

2.3.3. Chlorophyll Fluorescence and Cell Membrane Thermostability

Maximum quantum efficiency of PS II (Fv/Fm), Chlorophyll content, and CMT were significantly reduced when heat stress was imposed on the corn plants in vivo at 38 °C/33 °C (day/night temperature) for seven days under greenhouse conditions (Figure 4A–C). Significant differences were observed among genotypes in these parameters. A gradual reduction was observed in Fv/Fm during the course of the seven-day heat treatment: P31G70 had the highest and PI 639055 had the lowest Fv/Fm throughout the treatment with significant differences among the genotypes (Figure 4A). The greatest reduction in chlorophyll was observed in PI 639055 by the end of the heat stress treatment (Figure 4B). Reduction in chlorophyll in PI 489361 was comparable to that of the commercial hybrids because of the higher content due to its thicker leaves as mentioned above, which also helped it to have a comparable Fv/Fm to that of the commercial hybrids. Cell membrane injury was significantly lower in the commercial hybrids than in the germplasm lines (Figure 4C). After five days of treatment, PI 639055 and PI 489361 had drastic reductions in CMT and had significantly lower CMT than the commercial hybrids. There were also differences among the hybrids: DKC63-42 had higher CMT on day 3 and day 5 than the other hybrids, however, P31G70 and P31B13 had a more gradual reduction in CMT and, by the end of the 7-day heat stress treatment, P31G70 had the highest CMT even though it was not significantly different from the other two genotypes. Similar results were observed in in vitro heat stress treatments by exposing leaf discs of field grown plants (which were not exposed to heat or drought stress) for 6 h at 40 °C (Figure 4D). In this treatment, the germplasm lines, particularly PI 639055, reached 50% relative injury in about 4 h, whereas it took about 6 h for the commercial hybrids to reach that level of injury.

2.3.4. Seed Composition

Abstract

Aflatoxins, produced by the fungus Aspergillus flavus, contaminate maize grain and threaten human food and feed safety. Plant resistance is considered the best strategy for reducing aflatoxin accumulation. Six maize germplasm lines, TZAR101–TZAR106, were released by the International Institute of Tropical Agriculture-Southern Regional Research Center (IITA-SRRC) maize breeding collaboration for use in African National Programs and U.S. maize breeding programs. The present investigation was conducted to evaluate aflatoxin reduction by these lines in a U.S. environment. As germplasm lines, resistance was demonstrated by the lines tested in 2010 and 2014 trials. In 2010, TZAR106 was among the lines with the lowest toxin accumulation, and in 2014, along with TZAR102, supported low aflatoxin. When evaluated as single cross hybrids in 2012, 2013 and 2014, several crosses involving IITA-SRRC lines accumulated low toxin. In 2012, TZAR103 × HBA1 was one of 4 lines with the lowest concentration of aflatoxin. In 2014, five IITA-SRRC hybrids were among the lowest with TZAR102 × Va35 and TZAR102 × LH132 being the two lowest. Results demonstrate significant aflatoxin reduction by IITA-SRRC lines in a U.S. aflatoxin-conducive environment (at Mississippi State University). Further testing in different locations and environments is needed to further evaluate the potential usefulness of these germplasm lines. View Full-Text

Keywords: aflatoxin; host resistance; breeding; field trialsaflatoxin; host resistance; breeding; field trials

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MDPI and ACS Style

Brown, R.L.; Williams, W.P.; Windham, G.L.; Menkir, A.; Chen, Z.-Y. Evaluation of African-Bred Maize Germplasm Lines for Resistance to Aflatoxin Accumulation. Agronomy2016, 6, 24.

AMA Style

Brown RL, Williams WP, Windham GL, Menkir A, Chen Z-Y. Evaluation of African-Bred Maize Germplasm Lines for Resistance to Aflatoxin Accumulation. Agronomy. 2016; 6(2):24.

Chicago/Turabian Style

Brown, Robert L.; Williams, W. P.; Windham, Gary L.; Menkir, Abebe; Chen, Zhi-Yuan. 2016. "Evaluation of African-Bred Maize Germplasm Lines for Resistance to Aflatoxin Accumulation." Agronomy 6, no. 2: 24.

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