Antifungal activity of essential oil of Syzygium aromaticum on Rhynchosporium secalis

Scald caused by Rhynchosporium secalis is one of the most devastating barley foliar diseases worldwide. Morocco has not been spared from this scourge. Our research focused on the antifungal effects of clove essential oil. Essential oils are extracted from the flower buds of aromatic plants using hydrodistillation. According to this study, the obtained extraction rate (9.07%) is quite satisfactory, making the plant a valuable natural resource. The aroma essential oil was identified by gas chromatography, and its main component was eugenol with a content of more than 52%, followed by eugenol acetate (25.94%), caryophyllene (7.845%) and caryophyllene oxide (1.74%). Two R. secalis isolates were tested for antibacterial efficacy. For the two isolates (Rs1 and Rs2) at a low concentration of 0.4 µl/ml, complete inhibition was observed under the action of S. aromaticum essential oil. According to our results, the essential oil has an antifungal effect on Scald.


INTRODUCTION
Along with net blotch, scald is one of the most destructive fungal diseases of barley leaves, especially in cool temperate climates.The causative agent of the disease is R. secalis (Oudem) J.J., which also affects rye and other grasses (1,2).The disease causes severe yield loss and reduces barley seed quality (3).
The overuse of fungicides has led to the emergence of resistant strains.The resistance of R. secalis to MBCs (benzimidazoles and carbamates) and DMIs (sterol demethylation inhibitors) was reported by Torriani in 2004 (4).In addition to the variable efficacy of fungicides against Rhynchosporiosis (which varies according to geographical location) (5), the threat of this disease can also be addressed by biocontrol of medicinal plant essential oils.They have a wide range of applications in therapeutics, BioBacta cosmetics, agri-food, and more.Furthermore, the potential use of essential oils and their constituents as fungicides is enhanced by their biodegradability (6,7).
Following the results obtained by Essaouaadi et al 2021 (12), showing the superior efficacy of gifole extract against this pathogen, we opted to finalize the results by working on the essential oil of this plant against R.Secalis.This work aimed to extract essential oil from cloves and to characterize and evaluate the antifungal efficacy of this essential oil against R. secalis.

2-1-Materials
The medicinal plant used is Clove, a plant available on the market throughout the year.It was supplied by a herbalist in the Dakhla region during March.

2-2-Fungal material.
The two R. secalis isolates, Rs1 and Rs2, are from Khémisset and Gharb respectively.The choice of these two isolates was based on the one hand on the evaluation of their severity in the field and on the geographical distribution.

Extraction of essential oil
The essential oil of

Microbiological procedure
The minimum inhibitory concentration (MIC) of Essential oils and Tween 80 in concentrations of 0.4, 0.6, 0.8 and 1 l/ml.shake the mixture well before pouring it into Petri dishes.A control containing only PDA was also prepared.
Inoculation was performed by depositing fragments of the fungal culture approximately 6.5 mm in diameter taken from 7-day-old mycelial mats.
Incubate for 7 days at 22 °C with a 12 h photoperiod.
Each experiment was repeated three times.

Statistical analysis
GraphPad Prism 9 program was used and data was analyzed by Microsoft Excel and design tables and comparison.

1-Yield and chemical composition of S. aromaticum essential oil
The yield of S. aromaticum essential oil extracted from cloves by hydrodistillation on a laboratory scale was 9.07%.
The analysis of the essential oil of S. aromaticum by gas chromatography made it possible to identify thirty terpene compounds cited in the table in order of retention time (Table 1; Figure 1).These findings show that 94.886% of the elements of clove essential oil represent the total of the obtained constituent percentages.The primary ingredients in this oil are eugenol (52.515%), eugenol acetate (25.945), secondary ingredients like caryophyllene (7.845%), and minor ingredients like isoaromadendrene epoxide (1.93) and humulene (1.206%).

2-Antimicrobial activity of S. aromaticum essential oil against R. secalis
Table 2 summarizes the findings of the antifungal activity of S. aromaticum essential oil on the two isolates of R. secalis, the causative agent of Scald in barley (Rs1 and Rs2).This study of the antifungal activity of S.aromaticum essential oil showed that a concentration of 0.4ᶙl/ml was sufficient to completely inhibit the growth of the two R.secalis isolates Rs1 and Rs2 (Figure 2).and extraction technique all affect essential oil production differently.Eugenol, which makes up more than 52% of the S. aromaticum essential oil, serves as the primary constituent and is followed by Eugenol acetate (25.94%), caryophyllene oxide (1.74%), and caryophyllene (7.845%) (Table 1).At a modest concentration of 0.4 l/ml, the essential oil of S. aromaticum caused a complete suppression in both R. secalis isolates.This is in line with Eugénia P. (2009), who demonstrated the high fungicidal activity of eugenol and the essential oil of S. aromaticum against Candida albicans, dermophytes, and Aspergillus sp.This research reveals that Clove oil may be advantageous in the therapeutic management of cutaneous-mucosal candidiasis in particular, such as vulvovaginal candidiasis when compared to Fluconazole, a systemic antifungal medicine approved for the treatment of candidiasis (19).
The inactivation of fungal enzymes with the SH (Hydrogen Sulphide) group in their active site is the basis for the phenolic toxicity to phytopathogenic fungi [4].Phenolic terpenes also alter the permeability of the leakage of intracellular components by attaching to the amine and hydroxylamine groups of microbial membrane proteins (20).

CONCLUSION
The positive outcomes of our in vitro experiments allow us to conclude that the essential oil of S.
essential oils was determined by the method of Remmal et al. (1993) and Strani et al. (2001) (14,15).Since essential oils are not harmful to water and media, they are emulsified with a Tween 80 (0.05%) emulsifying solution to facilitate bacterial contact with the compound.In Erlenmeyer flasks containing 25 mL of autoclaved PDA medium (121 °C for 2 min) and cooled to 45 °C, prepare initial dilutions by adding 10, 15, 20, and 25 µL of aromatic essential oils to give final dilution.

Figure 2 :
Figure 2: Effect of the essential oil of S.aromaticum on the diametrical growth of isolate of R.secalis throughout 7 days.
These findings are comparable to those of Chaieb et al. (2007), who demonstrated that the primary constituents in the essential oil of S. aromaticum are eugenol and eugenol acetate (17).Additionally, Houari A.D.E.(2015) demonstrated that the components of S. aromaticum essential oil are almost identical to those discovered in our work, but in different proportions (18).
Our findings are in agreement with those of Matusinsky et al., (2015) who demonstrated the in vitro antifungal activity of five essential oils from five medicinal plants, namely Pimpinella anisu, Thymus vulgaris, Pelargonium odoratissimum, Rosamarinus officinalis and Foeniculum vulgare on fungal pathogens affecting Cereals, viz: Oculimacula yallundae, Microdochium nivale, Pyrenophora teres, Fusarium culmorum and Z.tritici on Wheat (21).Therefore, S. aromaticum is a more considerate of the environment and the health of users and consumers source of novel compounds in the quest for bioactive molecules against phytopathogenic fungi of natural origin.To utilize fewer chemical products, it can be incorporated into integrated pest management programs.

Table 2 : Antifungal activity of the essential oil of S.aromaticum against two isolates of R.secalis
-:Inhibition (-) and : Growth (+)