Biocontrol of Fusarium Species Utilizing Indigenous Rooibos and Honeybush Extracts

Abstract

Mycotoxins produced by several Fusarium species have a significant effect on reducing maize yield and grain quality and have led to food safety concerns. The antifungal activities of rooibos (Aspalathus linearis) and honeybush (Cyclopia species) tea extracts reduced the growth of plant pathogen Botrytis cinerea, but their efficacy against Fusarium spp. is unknown. In this study, we examined the effects of fermented and unfermented rooibos (A. linearis) and honeybush (Cyclopia subternata) aqueous extracts as well as green tea (Camellia sinensis) against 10 Fusarium species. Conidial viability was assessed by fluorescence microscopy dyes, ATP production was determined using the BacTiter-Glo assay, the mode of action was analyzed by scanning electron microscopy (SEM), and quantification of polyphenols was done using high-performance liquid chromatography with diode array detection (HPLC-DAD). Fermented rooibos extract demonstrated the highest antifungal activity (P < 0.0001) against Fusarium verticillioides MRC 826-E, Fusarium subglutinans MRC 8553, Fusarium proliferatum MRC 8549, and Fusarium globosum MRC 6647, with only 9.53%, 9.26%, 11.0%, and 12.7% ATP production, respectively, followed by antifungal activity of the fermented C. subternata extract against F. subglutinans MRC 8553, F. subglutinans MRC 8554, F. proliferatum MRC 8550, and F. verticillioides MRC 826-E with 3.79%, 6.04%, 6.04%, and 8.40% ATP production, respectively. Extract-treated conidia examined by SEM exhibited disruption of conidial hyphae and collapsed spores. Overall, the fermented rooibos and C. subternata extracts showed higher antifungal activity against the Fusarium species than the unfermented extracts. IMPORTANCE In maize subsistence farming areas in South Africa, daily consumption of maize contaminated by high level of mycotoxins contributes to long-term health effects such as immune deficiency and cancer. Biocontrol methods that are safe and cost-effective are critical to addressing this public health problem. Plant extracts known as biocides or green pesticides are alternatives to chemical pesticides due to their safety and eco-friendly properties. In South Africa, rooibos (Aspalathus linearis) and honeybush (Cyclopia species) contain polyphenols with significant antioxidant and antimicrobial properties. These indigenous herbal teas are widely available and consumed in South Africa and have potential as an innovative approach to reduce mycotoxin levels and, subsequently, human and animal exposure to these toxins. This study evaluates the efficacy of the antifungal activities of several aqueous extracts prepared from fermented and unfermented rooibos (A. linearis), honeybush (Cyclopia subternata), and green tea (Camellia sinensis) on 10 Fusarium strains.

Keywords: Fusarium species; antifungal activity; honeybush; rooibos.

FIG 1

Natural log (Ln) of ATP production of 10 Fusarium strains treated with tea extracts, with fermented rooibos showing the most effective antifungal activity, followed by fermented C. subternata, then unfermented rooibos, then unfermented C. subternata.

FIG 2

(A) Tukey-Kramer comparison plot of the unfermented and fermented rooibos combined and compared to unfermented and fermented C. subternata, indicating a significantly (P < 0.0001) higher growth inhibition rate for rooibos at the lower concentrations (0.62 to 5 mg/mL) for all Fusarium strains combined. (B) The comparison plot between the fermented and unfermented tea extracts (rooibos and honeybush) against all the Fusarium strains was also highly significant (P < 0.0001), showing the fermented tea extract to be the most active extract against conidial growth inhibition.

FIG 3

Fermented and unfermented tea extracts inhibit conidial germination of several Fusarium strains. Fusarium strain conidia were grown in a liquid medium without (control) or with 20 mg/mL tea extract and were incubated at 26°C for 24 h before microscopic observation at a magnification of ×400. The conidial germination rate was calculated by randomly counting 50 conidia from each of three independent experiments and then plotted using GraphPad Prism 5.01 software (GraphPad Software, Inc., La Jolla, CA). *, P ≤ 0.05, and **, P ≤ 0.01, for comparison between control and tea extracts for each species; #, P ≤ 0.05, and ##, P ≤ 0.01, for comparison between fermented and unfermented tea extracts for each species (e.g., fermented C. subternata versus unfermented C. subternata).

FIG 4

Formation of conidial germ tube inhibited with 20 mg/mL tea extract compared with the control. Microscopic observation was done at a magnification of ×400. Images were taken using the Zeiss MoticamPro 282A camera, with LED-Module/ex 640 nm detected in the 840/15 bandpass filter. Germination was defined as the presence of a thin germ tube longer than its respective conidium.

FIG 5

Scanning electron microscopy (SEM) observations of fungal hyphal morphological changes when exposed to teas. F. verticillioides MRC 826-E and F. graminearum MRC-4927 cells were incubated in a liquid medium without (control) or with 20 mg/mL tea extract and then submitted to SEM. After 96 h, untreated hyphae and conidia with regular cell walls are visible, as indicated by the arrows in the control samples. After treatment, both hyphae and conidia suffered from malformed structure swelling; arrows in the treated samples indicate ruptured hyphae and deformed conidia with flattening of cell membrane.