The Extracts of Cinnamon and Clove as Potential Biofungicides against Strawberry Grey Mould -

The Extracts of Cinnamon and Clove as Potential Biofungicides against Strawberry Grey Mould

Strawberry is one of the many plants infected by grey mould (Botrytis cinerea), and innovative methods of biocontrol against B. cinerea are under investigation. Clove (Syzygium aromaticum L.) and cinnamon (Cinnamomum cassia L.) accumulate natural compounds, such as eugenol and cinnamaldehyde, which provide antimicrobial and antifungal properties; thus, extracts of these plants could be possibly used as biofungicides. During this study, the inhibition of B. cinerea by clove and cinnamon extracts was evaluated in vitro on Petri plates and detached strawberry leaves. Clove extract consisted of 52.88% eugenol, and cinnamon consisted of 74.67% cinnamaldehyde. The extracts on detached strawberry leaves showed that 12 mL/L concentration of clove extract was effective in suppressing the grey mould infection. Clove and cinnamon extracts showed an equal ability to inhibit B. cinerea on Petri plates. However, the results of the detached strawberry leaves assay showed that clove extract was more effective as a biocontrol product.

Methods

The extracts of cinnamon bark (Cinnamomum cassia L.) and clove bud (Syzygium aromaticum L.) were chosen for the determination of antifungal activity against strawberry pathogen Botrytis cinerea at different concentrations. Dried cinnamon bark and clove bud were obtained and subcritical CO₂ extraction was performed while producing cinnamon and clove extract. Both extracts were kept at 4 °C until the experiments.

Antifungal activity against B. cinerea was determined by pouring clove and cinnamon extracts to PDA at concentrations of 200–2200 µL/L. Inoculation was performed with a 6-mm plug of 7-day-old single spore isolate of B. cinerea, fungal side down. Isolates were obtained from infected strawberry fruit and had previously been identified as B. cinerea. Petri dishes were incubated at 22 ± 2 °C in the dark for 7 days. The radial colony growth of the pathogen (plug excluded) was measured 2, 4 and 7 days after inoculation (DAI), and antifungal activity was expressed as the percentage inhibition of mycelial growth

The lowest concentration of cinnamon and clove extract with 100% inhibition was considered as minimal inhibitory concentration (MIC). Minimal fungicidal concentration (MFC) was determined after reinoculating B. cinerea treated with extracts from the margin of the colony on the fresh PDA. The lowest concentration with no visible radial colony growth of B. cinerea after 48 h was considered as MFC.

Detached strawberry leaves were soaked in 70% ethanol for 2 minutes and rinsed in sterile distilled water three times. Each leaf was placed in a Petri dish with filter paper and 5 mL of sterile distilled water. The mixtures for leaf treatment were prepared with sterile distilled water, 1% Tween 80 and cinnamon and clove extracts at 6 mL/L and 12 mL/L. Concentrations were chosen and modified according to the obtained MICs of clove and cinnamon extracts. Detached strawberry leaves were sprayed with prepared mixtures, then wounded with a sterile needle and a 9-mm plug of 7-day-old B. cinerea was placed on the wound. Four replications were carried out. Incubation was carried out at 22 ±2 °C in the dark for 7 days.

Results

In total, 98.86% of the compounds of cinnamon extract were identified and trans-cinnamaldehyde was determined to be the dominant compound. Meanwhile, based on 99.81% of the components identified in clove extract, eugenol, eugenol acetate and trans-caryophyllene were observed in the highest quantities. The antifungal activity of cinnamon and clove extracts was investigated on PDA (potato dextrose agar) under different concentrations. This extract did not demonstrate inhibition at 200 µL/L at 4 and 7 DAI (days after inoculation) and reached 20% at 400 µL/L at 4 DAI. However, the mycelial growth of the pathogen was fully inhibited from 600 µL/L to the full range of concentrations. The growth of B. cinerea up to 80% at 4 DAI and 60% at 7 DAI. Both tested extracts entirely suppressed the mycelial growth at 600–2200 µL/L. The determined MIC (minimal inhibitory concentration) of cinnamon and clove extracts was low and equal to 600 µL/L. The fungicidal activities of C. cassia and S. aromaticum extracts were investigated by measuring the radial colony growth of B. cinerea after reinoculation. Cinnamon extract demonstrated higher fungicidal activity than clove extract. The MFC (minimal fungicidal concentration) of the cinnamon extract was 600 µL/L, as no visible growth of B. cinerea was observed 48 h after inoculation, while the MFC of the clove extract was 1400 µL/L. The detached strawberry leaves assay revealed that the clove extract had a higher inhibitory effect of the infection of grey mould. Among all of the investigated treatments, only 12 mL/L concentration of clove extract significantly reduced the infection on strawberry leaves compared to control. In comparison, 6 mL/L of both tested extracts increased the spread of grey mould, while 12 mL/L concentration of the cinnamon extract exhibited a low antifungal effect on detached leaves.

Conclusion

In conclusion extracts of cinnamon and clove, containing characteristic compounds trans-cinnamaldehyde and eugenol, showed significant antifungal activity against B. cinerea in vitro. The results showed that the antifungal activity of clove extract was stronger than that of the cinnamon extract at lower concentrations. Infection of grey mould on detached strawberry leaves was suppressed by the application of clove oil at the highest investigated concentration. The cinnamon extract was not that effective at inhibiting the spread of grey mould on strawberry leaves. The use of natural antifungal agents is being increasingly encouraged now a day. Clove extract has high potential to be applied in the formulation of biopesticides for safer plant protection. Based on the in vitro results, further analysis of cinnamon extract is encouraged.

Reference:

Šernaitė, L., Rasiukevičiūtė, N. and Valiuškaitė, A., 2020. The Extracts of cinnamon and clove as potential biofungicides against strawberry grey mould. Plants, 9(5), p.613.