Unraveling the potential of selected South African indigenous plants as biocontrol agents against aflatoxin-producing Aspergillus species

Abstract

Aspergillus flavus and A. parasiticus are saprophytic storage fungi, which primarily invade maize (Zea mays) and other cereal and legume crops. Contamination of stored grains with these fungal species reduces the quality of food and feed, which leads to yield loss. Moreover, A. flavus and A. parasiticus present a serious threat to human and animal health due to the production of aflatoxin B1 (AFB1), which is carcinogenic and is associated with stunting among children. Current control methods may be effective; however, due to the adverse residual effects associated with the use of synthetic chemical fungicides, alternate methods of pest control such as biodegradable agents are being researched. Plant extracts have been tested to be less toxic and environmentally friendlier compared to synthetic chemical fungicides. The goal of this study was to evaluate three plants, namely, Erythrophleum lasianthum, Heteropyxis natalensis and Warburgia salutaris for their antifungal activities and as seed treatments against known AFB1-producing A. flavus and A. parasiticus isolates in vitro. The extracts were screened using the broth microdilution method to obtain the minimum inhibitory concentration (MIC) values, of which the acetone, ethanol and water extracts of W. salutaris (0.117 mg/mL), E. lasianthum (0.234 mg/mL) and H. natalensis (0.469 mg/mL), respectively, showed noteworthy antifungal activity (≤ 1 mg/mL) against A. flavus. In contrast, the acetone extract of W. salutaris and the ethanol and water extracts of H. natalensis showed noteworthy antifungal activity (≤ 1 mg/mL) against A. parasiticus with MIC values of 0.117, 0.938 and 0.469 mg/mL, respectively. Following the antifungal screening, E. lasianthum (ethanol), H. natalensis (water) and W. salutaris (acetone) extracts were evaluated for their potential as AFB1 reducers at 15, 30 and 75 mg/mL extract concentrations. Using spectrophotometry, the extract which reduced AFB1 at 75 mg/mL the best was that made from the acetone extract of W. salutaris when compared to all the other tested extracts. Warburgia salutaris (75 mg/mL) was able to reduce AFB1 by 49.27 % in liquid media while AFB1 was least reduced by E. lasianthum (at 15 mg/ml), which was only able to reduce AFB1 production by 12.82 %. Furthermore, the selected five extracts, E. lasianthum (acetone), H. natalensis (acetone, ethanol and water) and W. salutaris (acetone) which showed good antifungal activity (against A. flavus and A. parasiticus) were screened for their potential toxicity on HeLa and HepG2 cell lines. All the extracts showed moderate to no cytotoxicity (> 50 μg/mL) on both cell lines with the exception of H. natalensis (water) and W. salutaris (acetone) extracts, which were toxic (≤ 50 μg/mL) to the HepG2 cell line, at the highest tested concentration (400 μg/mL). Bioassay-guided fractionation of the ethanolic extract of H. natalensis indicated that the liquid-liquid partition butanol fraction was the most active against A. flavus and A. parasiticus. The butanol liquid-liquid partitioned fraction was further subjected to isolation and identification using Column chromatography and GC-MS, revealing five major compounds as potential fungal growth inhibitors present in the butanol fraction of H. natalensis. Artificially inoculated maize seeds with A. flavus were treated with the three plant extracts, E. lasianthum (ethanol), H. natalensis (water) and W. salutaris (acetone), at 50 and 100 mg/mL and evaluated for their potential as seed treatments. The treated seeds were plated onto Potato Dextrose Agar (PDA) prior to storage, and three and six months after treatment. The extracts had no observable negative effects on seed germination. The extracts showed no significant fungal growth inhibition on treated maize seeds plated out prior to storage. However, after three months of storage, H. natalensis (water) and W. salutaris (acetone) at 100 mg/mL showed significant growth inhibition on maize treated seeds with fungal growth inhibition percentages of 65.00 and 70.50 %, respectively. After six months in storage, E. lasianthum (100 mg/mL) and H. natalensis (50 and 100 mg/mL) exhibited intermediate fungal growth inhibition of 48, 47 and 55 %, respectively. Therefore, these plants indicated the potential to be used as biological control agents against fungal contaminants of grains such as A. flavus in storage.