Investigating the antifungal efficacy of moringa leaf extracts against Fusarium oxysporum, a causal agent of fusarium dry rot.

Abstract

Fungal diseases are amongst the most-destructive potato pathogens worldwide. Potato (Solanum tuberosum L.) is one of the most-common and -widely consumed crops in South Africa (SA). Population growth in SA is continuously putting pressure on the potato production, which has subsequently accelerated mono-cropping and chemical use, particularly in an attempt to control fungal diseases. There are currently no fusarium dry rot- resistant cultivars and no commercial biological control agents available to reduce occurrence of this disease. In an attempt to reduce agricultural pollution, and effectively control fusarium dry rot, plant extracts have been investigated for their antifungal properties. Moringa oleifera Lam. (moringa) is a tree well-known for its wide phytochemical composition and its antifungal abilities have been documented, but not on potato dry rot. This study was, therefore, carried out to investigate the antifungal potential of moringa leaf extracts against Fusarium oxysporum, the causal agent of fusarium dry rot of potato. Various potato cultivars were used, based on seasonal availability. Since the moringa plant is containing high amounts of secondary metabolites, in this study, first, the phytochemical composition of Moringa leaf excracts (MLEs) and the effect of MLE treatment on the phenolic concentration of tubers were analysed. The Moringa leaf powder was extracted with either (30%, 50% or 80%) of acetone, ethyl acetate, methanol and the other moringa powder eas extracted with distilled water. Various MLEs were tested for their capability to improve tuber quality post- harvest by assessing potato quality parameters, such as percentage mass loss and firmness. An in vitro assay was carried out to evaluate the moringa leaf extracts’ (MLEs’) inhibition activity on F. oxysporum using a disc diffusion method. The ability of MLEs to prevent fusarium dry rot development and to delay disease progression was also investigated in in vivo assays. Phytochemical analysis of MLEs revealed the presence of tannins, phenolics, flavonoids and glycosides. Tannins were, however, absent in ethyl acetate MLEs. Treatment with MLEs enhanced the concentration of free and bound phenolics in ‘BP1’ and ‘Mondial’ potatoes. Tubers coated with methanol-MLE had the highest concentration of both, free and bound phenolics. Treatment with MLEs slowed down average percentage mass loss (seemingly water loss) in both cultivars. Treatment with 70% ethyl acetate MLE, however, accelerated this water loss in both cultivars, but particularly in ‘Mondial’. This average reduction in mass was slightly less than the ii average reduction in mass of control tubers. Moringa treatment also preserved the healthy appearance of tuber skin and tuber firmness more so than the control. Macroscopic characteristics of F. oxysporum were pinkish colonies and a dark pigmentation, as observed on the PDA plates. The conodogenous cell was long and branched and the macroconidia had three to five septations. The fungus was found to be pathogenic on both cultivars used in this experiment, as tubers had dark depressions, typical for fusarium dry rot, and a white mycelium on the tuber surface. The efficacy of MLEs as antifungal agents was tested using a disc diffusion method. Three solvents were used to produce MLE stock solutions, namely acetone, ethyl acetate, ethanol, at three concentrations (30%, 50% and 70%). These solvent solutions were each further diluted to yield 2% and 4% concentrations, yielding eight MLE treatments. The dilutions of the MLEs produced with lower solvent concentrations (30% and 50%) had 100% fungal inhibition activity, while dilutions of MLEs extracted with the higher solvent concentration (70%) inhibited fungal development to a lesser degree. The 2% and 4% dilutions of 70% acetone, 70% ethyl acetate, and 70% methanol; resulted in 95.0%, 86.3% and 90.3% inhibition, respectively. Water-MLE, on the other hand, inhibited the fungus only by 87.5%. These results indicate that the extraction solvent and its concentration influence the antifungal efficiency of MLEs. In vivo assays demonstrated that MLEs were able to prevent disease development to a certain extent. Solvent type and concentration were influential in preventing fusarium dry rot development, as tubers treated with either 30% and 50% of acetone-MLE and methanol-MLE concentrations had the smallest average lesion diameter (3.7 mm and 3.4 mm for ‘Valor’ and ‘Mondial’, respectively). The 70% Ethyl acetate-MLE, on the other hand, was ineffective in controlling fusarium dry rot in both cultivars, resulting in an average lesion diameter of 14 mm; this was not significantly different from the control, which had an average lesion diameter of 15 mm (P>0.05) in ‘Valor’ tubers. In ‘Mondial’, 70% ethyl acetate-MLE treatment resulted in an average lesion diameter of 11 mm. Further, tubers of both cultivars treated with 50% and 70% ethyl acetate-MLE were prone to secondary infections by bacterial soft rot. The ability of MLE to delay and slow fusarium dry rot development is an indication of its antifungal potential. Response to MLE treatment was found to be cultivar-dependent, as following MLE treatment, ‘Mondial’ was more tolerant to fusarium dry rot than ‘Valor’. Solvent type and concentration were also found to influence MLE antifungal activity. Tubers treated with MLEs extracted with higher solvent concentrations (70%) as well as those treated with ethyl acetate MLEs

were less tolerant to fusarium dry rot. This research, therefore, demonstrates that lower organic solvent (30% and 50%) concentrations should be used, when preparing antifungal extracts of moringa leaf powder.