Dose-dependent suppression of photosynthetic pigments in maize (Zea mays L.) seedlings exposed to aflatoxin B1

Introduction

Maize (Zea mays L.), commonly known as corn, constitutes a major pillar of agricultural production in Northern India, particularly in Bihar, where it contributes substantially to household food security and rural livelihoods (Kumar et al. 2014) [8] . Despite its agronomic importance, maize cultivation in Bihar is persistently challenged by the state's warm and humid conditions, which favour the proliferation of mycotoxin-producing fungi, especially Aspergillus flavus and A. parasiticus. These fungi frequently infest maize grains and lead to contamination with aflatoxins (Sinha and Sinha 1990; Reddy et al. 2010) [13,11] . Among the aflatoxins, aflatoxin B1 is regarded as the most potent and is widely reported as a recurrent contaminant of maize, raising concerns not only for consumer health but also for crop performance and physiological integrity (Pitt and Hocking 2009; Bhat and Vasanthi 2003) [10,3] . Importantly, aflatoxin B1 is not merely a post-harvest food safety issue; it can also exert direct phytotoxic effects during early plant development. Evidence suggests that exposure to aflatoxin B1 interferes with fundamental metabolic pathways, including the biosynthesis and stability of photosynthetic pigments such as chlorophylls and carotenoids molecules that are indispensable for photosynthetic function and seedling establishment (Mehan 1971; Kang 1970) [9,7] . Chlorophyll a and chlorophyll b are central to light harvesting, while carotenoids contribute both to accessory light absorption and to photoprotection through quenching of reactive oxygen species, thereby safeguarding the photosynthetic apparatus under stress (Taiz and Zeiger 2010) [14] . Previous investigations have documented pigment suppression under aflatoxin stress in crops such as Abelmoschus esculentum and mustard, indicating that toxininduced disruption of photosynthetic machinery may represent a broader phytotoxic mechanism (Kang 1970 ; Sinha et al. 1992) [7,12] . However, systematic quantification of these effects in maize seedlings remains relatively limited. Given Bihar's dependence on maize and its heightened vulnerability to aflatoxin contamination, clarifying the extent of pigment inhibition is essential for assessing potential risks to seedling vigour and, ultimately, yield stability. Therefore, the present study quantifies the influence of graded aflatoxin B1 exposure on chlorophyll and carotenoid contents in maize seedlings to better elucidate its physiological consequences and to support the development of mitigation strategies for aflatoxin-affected agroecosystems.

Materials and Methods

Maize seeds (Zea mays L.) of uniform quality were obtained from the Crop Section of Rajendra Agricultural University, Sabour, Bhagalpur, India, a hub of maize cultivation in Bihar. Aflatoxin B1 (Sigma-Aldrich, St. Louis, MO, USA) was prepared as a stock solution in 1 mL ethanol and diluted with sterile distilled water to concentrations of 100, 250, 500, 1000, and 2000 µg/L. A control treatment used distilled water alone to establish baseline pigment levels. Seeds were pre-soaked in 100 mL sterile distilled water for 1 hour to initiate imbibition, then immersed in 100 mL of aflatoxin B1 solutions (or water for controls) for 20 hours, ensuring toxin uptake. Each treatment comprised 100 seeds in triplicate (n=300 per concentration). Treated seeds were placed on moist blotting paper in petri dishes and incubated at 28 ± 2 °C in a seed germinator for 10 days, allowing seedling development under controlled conditions. Chlorophyll and carotenoid contents were measured in newly emerged leaves on day 10, following established protocols. For chlorophyll, 250 mg of fresh leaf tissue was homogenised in 5 mL of 80% acetone using a mortar and pestle. The extract was filtered through Whatman No. 1 paper in a Buchner funnel, with extraction repeated 2-3 times using additional 5 mL aliquots of 80% acetone. The mortar, pestle, and funnel were rinsed with 5 mL of 80% acetone, and the final volume was adjusted to 25 mL. Absorbance was recorded at 645 nm and 663 nm using a spectrophotometer (UV-Vis, Shimadzu) against an 80% acetone blank. Chlorophyll a and b contents (mg/100 mg fresh weight) were calculated using Arnon's equations (Arnon 20): mg chlorophyll a / 100 g tissue = Carotenoid content was determined at 480 nm using Davis's method (Davis 35) [5] , accounting for chlorophyll interference:

Results were expressed as mg/100 mg fresh weight (%). Measurements were replicated thrice, and data are presented as means ± standard error (SE). Observed data were analyzed using one-way analysis of variance (ANOVA), with a t-test (t-values: chlorophyll a = 13.974, chlorophyll b = 11.704, total chlorophyll = 13.737, carotenoids = 6.945; df = 4) and Pearson correlations (rvalues: chlorophyll a = 0.989, chlorophyll b = 0.986, total chlorophyll = 0.989, carotenoids = 0.961). Significance was assessed at p < 0.05.

Results and Discussion

Aflatoxin B1 significantly reduced chlorophyll a, chlorophyll b, and total chlorophyll contents in maize seedling leaves, with inhibition increasing with concentration (Table 1). Control seedlings exhibited 0.670 ± 0.0015% chlorophyll a, 0.280 ± 0.0070% chlorophyll b, and 0.950 ± 0.0085% total chlorophyll. At 2000 µg/L, these values dropped to 0.200 ± 0.0012% (70.15% inhibition), 0.100 ± 0.0045% (64.29% inhibition), and 0.298 ± 0.0057% (68.60% inhibition), respectively. Intermediate concentrations showed graded declines (e.g., 1000 µg/L: 44.78%, 30.36%, 40.63% inhibition for chlorophyll a, b, and total, respectively), with strong correlations (r = 0.989 for chlorophyll a and total chlorophyll, r = 0.986 for chlorophyll b). Carotenoid levels followed a similar trend, decreasing from 0.021 ± 0.0010% in controls to 0.005 ± 0.0009% at 2000 µg/L (76.19% inhibition). Intermediate reductions ranged from 9.52% (100 µg/L) to 61.90% (1000 µg/L), with a correlation of r = 0.961. All reductions were statistically significant (p < 0.05) [9, 7] . These findings align with reports of chlorophyll inhibition in Abelmoschus esculentum (Kang 1970) [7] and mustard (Sinha et al. 1992) [12] , suggesting a conserved phytotoxic mechanism across species. The slightly greater inhibition of chlorophyll a versus chlorophyll b may reflect differential sensitivities in biosynthetic pathways, with chlorophyll a being more susceptible to toxin-induced oxidative damage (Taiz and Zeiger 2010; Bennett and Klich 2003) [14,2] . Carotenoid depletion, peaking at 76.19%, exceeds chlorophyll losses, possibly due to their role as antioxidants, rendering them more vulnerable to aflatoxin-induced reactive oxygen species (Das et al. 2015; Pitt and Hocking 2009) [4,10] . This imbalance could impair photoprotection, exacerbating photosynthetic stress. The strong correlations (r > 0.96) underscore a robust dose-response relationship, consistent with aflatoxin's known metabolic interference (Reddy et al. 2010) [11] . Since maize is a dietary staple and aflatoxin contamination is prevalent in Bihar (Bhat and Vasanthi 2003; Kumar et al. 2014) [3,8] , these reductions threaten seedling vigor and yield potential by limiting their efficiency (Taiz and Zeiger 2010) [14] . Future research should investigate molecular targets, such as chloroplast enzyme activity or lipid peroxidation (Das et al. 2015) [4] , and explore mitigation strategies like resistant cultivars or biocontrol agents (Pitt and Hocking 2009) [10] . These efforts are critical to sustaining maize production in contaminated regions.

Conclusion

The present investigation establishes that aflatoxin B1 significantly compromises the photosynthetic pigment content of maize [8,11] . The greater inhibition of carotenoids compared to chlorophylls suggests heightened vulnerability of photoprotective mechanisms, potentially amplifying photooxidative damage under field conditions (Pitt and Hocking 2009) [10] . These observations align with prior studies documenting pigment suppression in crops like Abelmoschus esculentum and mustard (Kang 1970; Sinha et al. 1992) [7,12] , reinforcing aflatoxin B1's broad phytotoxic profile, though maize appears less severely affected than some dicots. The study's findings emphasize the necessity of addressing aflatoxin contamination to preserve maize's physiological integrity and yield potential. Future investigations should explore the toxin's specific biochemical targets such as enzymes in the chlorophyll biosynthesis pathway (e.g., magnesium chelatase) or carotenoid degradation pathways and assess the efficacy of mitigation approaches, including resistant germplasm, fungal biocontrol, or antioxidant treatments to bolster seedling resilience (Pitt and Hocking 2009; Das et al. 2015) [4, 10] . Such efforts are critical to mitigating the agricultural and economic risks posed by aflatoxin B1 in maizedependent regions, ensuring the sustainability of this vital crop.

References

1. Arnon DI. Copper enzymes in isolated chloroplasts: Polyphenoloxidase in Beta vulgaris. Plant Physiol. 1949; 24(1):1-15.
2. Bennett JW, Klich MA. Mycotoxins. Clin Microbiol Rev. 2003; 16(3):497-516.
3. Bhat RV, Vasanthi S. Mycotoxin contamination of foods and feeds in India. Food Saf. 2003; 8(1):10-18.
4. Das N, et al. Phytotoxic effects of aflatoxins on seed germination and seedling growth in crops. Plant Pathol J. 2015; 19(2):33-40.
5. Davis BH. Carotenoids: Chemistry and biology. New York: Academic Press; 1969. p. 30-45.
6. Jones RK, et al. Mycotoxins and their effects on plant growth. J Plant Pathol. 1985; 12(3):85-92.
7. Kang MS. Inhibition of chlorophyll synthesis by aflatoxin B1 in Abelmoschus esculentum. Indian Phytopathol. 1970; 23(2):40-47.
8. Kumar A, et al. Maize cultivation in northern India: Challenges and opportunities. Agric Rev. 2014; 35(1):13-20.
9. Mehan VK. Aflatoxin effects on seedling physiology. J Agric Sci. 1971; 29(3):145-152.
10. Pitt JI, Hocking AD. Fungi and food spoilage. 3rd Ed. New York: Springer; 2009.
11. Reddy KRN, et al. Aflatoxin contamination in crops: A global perspective. Crop Sci Rev. 2010; 15(2):43-50.
12. Sinha KK, et al. Aflatoxin B1 inhibition assay using mustard and gram seedlings. Mycotoxin Res. 1992; 8(2):75-80.
13. Sinha AK, Sinha RK. Mycotoxin contamination in Indian agriculture. Indian J Agric Sci. 1990;25(4):2128.
14. Taiz L, Zeiger E. Plant physiology. 5th ed. Sunderland (MA): Sinauer Associates; 2010.