Phytochemical Constituents and Antimicrobial Activity of Crude Extract of Scent Leaf (Ocimum Gratissimum) on Fungal Phytopathogens

Plant diseases are estimated to cause 5-10% loss of the world’s major crops annualy, resulting in economic losses up to $100billion. The fungal pathogens account for 70-80%and significantly affect crop growth and yield (Li et al 2017). In recent years, the prevalence of fungal plant diseases has increased due to large scale agricultural practices that reduce crop quality and increase plant susceptibility to infection (Marin-Menguiano et al. 2019), posing a major challenge for sustainable agriculture. These diseases are often caused by the interaction of multiple pathogens rather than one single organism (Chatterjee et al., 2016). Since ancient times, humans have relied on medicinal. Plants to prevent, manage or treat many diseases (Chaachouay et al., 2022). Traditional medicines have witnessed a rising popularity over conventional drugs due to the effectiveness of the bioactive compounds present in the them, as well as the ease of access and affordability (Ijioma et al., 2021). As stated by WHO (2019), in recent years, there has been a significant increase in research on medicinal plants and their bioactive compounds, highlighting their potential for combating and managing plant pathogenic diseases. Ocimum gratissimum L. (Lamiaceae), referred to as ‘scent leaf ‘, ‘tea bush” or ‘fever plant ‘is a medicinal plant with antimicrobial properties and potential use as a natural fungicide. In Nigeria, it is known by local names such as Nchuanwu,” “Shinji,” “Achigbu” (Igbo), “Efirin” (Yoruba), “Ihiri eziza” (Bini), “Dai doya tagida” (Hausa), or “Ntion” (Efik). This medicinal plant belongs to the Lamiaceae family and can be found in South America, Asia and Africa (Ugbogu et al., 2021). It used as a vegetable, spice or seasoning in cooking. Studies have shown that O. Gratissimum contains bioactive compounds including flavonoids, polyphenols, and essential oils which exhibit a range of beneficial properties (Irondi et al., 2016). The phytochemicals present in this plant are associated with the ability to inhibit the growth of fungal pathogens. Therefore, this study aimed to evaluate the effectiveness of O. Gratissimum leaf extract against fungal phytopathogens

MATERIALS AND METHOD

Collection and Preparation

Leaves of Ocimum gratissimum were collected from Irewolede Area, Ogbomosho, Ogbomosho south local government area of Oyo state, Nigeria.  The leaves were thoroughly washed under running water, air dried for weeks, ground into a fine powder, and sieved. The powdered material was stored in a dry, cool place until further use.,

Plant Extraction Procedure

A total of 150 g of O. Gratissimum leaf powder was macerated in 450 mL of methanol for 48 hours. The solution was filtered through sterile muslin cloth, and the filtrate was allowed to settle in a dry, cool place to concentrate by evaporation. The resulting crude extract, which formed a colloidal substance, was stored at –4°C for subsequent phytochemical and antimicrobial analyses.

Phytochemical Screening of Plant Extract

Qualitative phytochemical analysis of the methanolic crude extract was performed using the methodology reported by Oladeji et al. (2020).

Preparation of Different Concentrations of the Crude Extracts

Solutions of crude extract were prepared at varying concentrations (25-500mg/ml)) following the procedure described by Oladeji et al. (2019).

Antifungal Susceptibility Analysis of the Methanolic Extract of O. Gratissimum

Potato Dextrose Agar (PDA) was prepared according to manufacturer instructions and aseptically inoculated with fungal suspensions. After inoculation, excess inoculum was removed. Sterile Whatman No. 1 filter paper discs (6 mm diameter) were oven-sterilized at 120°C for 2 hours. The discs were then impregnated with the plant extract solutions of different concentrations, while separate discs impregnated with methanol served as controls. The discs were placed on the surface of the inoculated PDA plates. Plates were incubated at 25°C for 2–3 days. Antifungal activity was assessed by measuring the zones of inhibition (mm) around each disc (Oladeji et al., 2019).

Pathogenicity Test

The fresh healthy oranges were double sterilized in sterile distilled water. The fruits were sterilized using 70% ethanol and washed with sterile water. Fruits were placed on sterile paper towels and dried under laminar airflow hood for 12 mins. Holes were created in the healthy oranges with the help of a sterile 10 mm cork borer. Test tubes containing the fungal isolates (pure culture) were inoculated in the cork borer hole. Finally, after inoculation, the inoculated oranges were sealed with sterile blue seal Vaseline to prevent contamination and the oranges were labeled accordingly. A control test without any isolate was conducted and sealed using the sterile blue seal Vaseline. After inoculation of each of the test isolates into their respective healthy oranges, the oranges were incubated at 25 °C in the humid chamber for 5 days. The fruits were observed daily for rotting characteristics like softening, color, and odor. The fruits were opened on the fifth day to expose internal portions of the fruits which were examined for rot. Circumference and diameter of the rotten and intact fruit of the rotten area and intact fruit were taken using transparent scale and recorded.

Statistical Analysis

The data obtained from the methanolic crude extract at different concentrations were analysed using Graph pad Prism 5. A two-way ANOVA test was carried out to check for the significance of the extract at different concentrations. Comparisons were made between the crude extract and the control as well as the extracts overall antimicrobial activity and that of the standard Nystatin. Values of P < 0.05; P < 0.01 and P < 0.001 were considered statistically significant and used to reject the null hypothesis. Graph pad Prism 5 was used in the analysis of methanolic crude extract at different concentrations. Two-way ANOVA test was used in the determination of the significance level of the crude extract at different concentrations. Statistical comparison was made between the methanolic crude extract and the control, and also the overall antimicrobial activities of the extracts with nystatin. P < 0.05; P < 0.01 and P < 0.001 was used to reject the null hypothesis

RESULTS AND DISCUSSION

The qualitative phytochemical screening of Ocimum gratissimum showed the presence and absence of various bioactive compounds as presented in Table 1 below.

Table 1: Phytochemical analysis of Ocimum gratissimum crude extract

KEYS: ‘-, ++, and +++’ indicates absent, present, very present.

As shown in the table above, the methanolic crude extract contained anthraquinones, tannins, phenolic content and alkaloids and flavonoid while saponins, phlobatannins, quinones, reducing sugars, terpenoids and steroids were not detected. The graphs (Figure 1-6) below show the antifungal susceptibility test (AFST) results showing the level of significance of Ocimum gratissimum crude extracts compared with the control across different phytofungal isolates. Values marked with (*, **, and ***) indicate results significantly higher than the control at P <0.05; P < 0.01 and P < 0.001. Respectively;(ns) indicate no significant difference.

Figure 1: AFST of Ocimum gratissimum extract against Aspergillus flavus