In Vitro Antimicrobial Activity of Zodia (Evodia suaveolens) Leaf Extract on Pathogenic Agents Dragon Fruit Plant

Key word: Antimicrobial Evodia suaveolens Pathogenic Agents The use of antimicrobials from plant extracts has not been used optimally to control pathogenic agents in dragon fruit plants. The purpose of this research was to determine the antimicrobial activity of zodia (Evodia suaveolens) leaf extracts on pathogenic agents of dragon fruit plants in vitro. The research method is laboratory research with Completely Randomized Design (CRD). The antimicrobial concentrations of zodia (E. suaveolens) leaf extract used six types including 50%, 60%, 70%, 80%, 90%, and 100% with four replications. The research sample was the leaf of zodia (E. suaveolens), Pseudomonas aeruginosa and Fusarium oxysporum strain Malang. Test the antimicrobial activity of zodia (E. suaveolens) leaf extracts on the growth of P. aeruginosa and F. oxysporum using the disc-diffusion method and wells method. The research instrument was used the observation sheet of the diameter of inhibition zone indicated by the clear zone. Diameter of inhibition zone data were analyzed using the One Way ANOVA test. The results showed that the antimicrobial activity of zodia (E. suaveolens) leaf extract significantly inhibited the growth of P. aeruginosa and F. oxysporum (P <0.05). These results recommend zodia (E. suaveolens) leaf extract as an antimicrobial agent for dragon fruit plant pathogens. Article history: Received: 14/07/2020 Revised: 20/08/2020 Accepted: 20/08/2020


Introduction
Dragon fruit is a plant group of the Cactaceae family with the genus Hylocereus and also known as pitaya fruit is round, juicy, and nutritious (Jamilah, Shu, Kharidaah, Dzulkifly, & Noranizan, 2011;Idris et al., 2013). Swastika, Yuliani and Saputra (2012) explained that dragon fruit plants in Indonesia consist of several species, such as Hylocereus undus, Hylocereus polyrhizus, Hylocereus megalanthus and Hylocereus costaricensis. An important benefit of dragon fruit for health is to facilitate the digestion process because it has lots of fiber, reduces body fat levels, prevents cancer because it contains antioxidants, and stimulates tissue formation because it is rich in vitamins and minerals (Swastika et al., 2012;(Noor, Yufita, & Zulfalina, 2016).
In addition to the benefits above, dragon fruit has excellent sales prospects because prices in the market are high, stable and demand is high (Setiawan & Soelistyo, 2017;Sudarjat, Suminar, Qanit, & Mubarok, 2019;Paundrianagari, Setyowati, & Qonita, 2019). However, the superiority of the dragon fruit was not followed by good dragon fruit cultivation techniques by farmers. Therefore, many dragon fruit plants are attacked by diseases (Jumjunidang, D., & Yanda, 2016).
Dragon fruit diseases found in Indonesia include stem and fruit cancers, anthracnose, and soft rot. Stem and fruit cancers are caused by Neocystalidium sp., Alternaria sp., and Pestaliopsis sp. Anthracnose is caused by Colletotrichum gloesporoides. Stem and fruit soft rot caused by Fusarium sp., Schlerotium sp., Alternaria sp., and Pseudomonas sp. (Riska et al, 2016;Swastika et al., 2012).
One of the plants that can be recommended as an antimicrobial is zodia (E. suaveolens) because the results of isolation and identification of active compounds find essential oils that can be used as antimicrobials (Maryuni, 2008 Handayani and Nurcahyanti (2015) used the maceration method and water distillation to obtain antimicrobial compounds. Fajri and Agustien (2015) used the disc diffusion method for antimicrobial tests on Escherichia coli and Staphylococcus aureus. Rahmawati, Samsumaharto, & Iryanto (2015) used the maceration method to obtain antimicrobial compounds and delusional methods in test tubes.
Research on inhibiting the growth of microbes that cause soft rot in dragon fruit plants with plant extracts has not been done. Research Wibowo et al. (2011) and Riska et al. (2016) focused on collecting important diseases that attack dragon fruit plants in three centers of dragon fruit plants on the island of Java and in Indonesia. The use of zodia (E. suaveolens) leaf extract as an antimicrobial has not been studied. Although there is one result of research related to the antibacterial extract of zodia leaves against P. aeruginosa (Rahmawati, Samsumaharto, & Iryanto W, 2015). However, the study wanted to prove the comparison of zodia leaf extract with n-hexane, chloroform, and water fractions using the tube dilution method and recommended that the highest concentration of zodia leaf extract and be effective in inhibiting bacterial growth by 50%. However, this research is different from this research. This research uses the discdiffusion method and wells method with concentration that starts from 50% to 100%. Isolation and identification of zodia leaf antibacterial compounds was carried out (Maryuni, 2008;Handayani and Nurcahyanti, 2015;Rahmawati, Samsumaharto, & Iryanto, 2015). However, the active compounds contained in zodia leaf extracts have not been used to inhibit the growth of fungal pathogens such as F. oxysporum. Thus, the purpose of this study was to determine the antimicrobial activity of zodia (E. suaveolens) leaf extract on pathogenic agents of dragon fruit plants in vitro.

Materials and Methods
The research sample used fresh zodia (E. suaveolens) leaf and used all types of leaves both young and old, P. aeruginosa and F. oxysporum. The solvent used to extract zodia (E. suaveolens) leaves is 95% alcohol. Growth media for P. aeruginosa are Nutrient Agar (NA) and F. oxysporum is Potato Dextrose Agar (PDA). The tools used in research such as Petri dishes for the culture of bacteria and fungi. Oven for sterilizing utensils made of glass such as Petri dishes. Autoclave for sterilization of bacteria and fungi medium. Evaporator to obtain extracts of zodia leaf antimicrobial compounds. Calipers to measure the inhibition zone for bacterial and fungal growth. An ose needle to plant bacteria and fungi onto the plate medium. Laminar Air Flow (LAF) for sterile bacterial and fungal planting sites. Electric vortex to homogenize zodia leaf extract. Drill the cork to make a hole in the culture medium. Watman paper for disc paper material.
This research use a laboratory experimental method. The research design used Completely Randomized Design (CRD) with six concentrations of zodia (E. suaveolens) leaf extracts including 50%, 60%, 70%, 80%, 80%, 90%, and 100% and each consisted of four times repeated. The design of this study can be seen in Table 1. The independent variable of the study was zodia (E. suaveolens) leaf extract and the dependent variable was the zone of growth inhibition of P. aeruginosa and F. oxysporum. The research procedure is as follows.
The research instrument used was the observation sheet of P. aeruginosa and F. oxysporum growth diameter of inhibition zone. Inhibitory zones are indicated by clear zones and measured using calipers. The calipers are placed in the clear zone horizontally and vertically. Furthermore, the results of the measurement of the clear zone are averaged so that the expected inhibition zone is obtained. Diameter of inhibition zone data were analyzed using the One Way ANOVA test through SPSS software.

Results and Discussion
The average diameter of P. aeruginosa growth inhibition zones is presented in Table  1. The average diameter of F. oxysporum growth inhibition zones can be seen in Table  2. Research data on the antimicrobial activity of zodia (E. suaveolens) leaf extract is stated to be normally distributed (sig > 0, 05) and homogeneous (sig > 0.05). The results of the One Way ANOVA test for diameter of inhibition zone of antimicrobial activity of zodia (E. suaveolens) leaf extracts against P. aeruginosa growth are presented in Table 3 and F. oxysporum can be seen in Table 4. The diameter of inhibition zone P. aeruginosa growth in several concentrations presented in Figure 1.  Based on Table 1 and Table 2 shows that the higher the concentration of zodia (E. suaveolens) leaf extract, the greater the inhibitory power on the growth of P. aeruginosa and F. oxysporum. The high concentration of zodia (E. suaveolens) leaf extract contains many active compounds, while the concentration of zodia (E. suaveolens) leaf extract contains few active compounds. Therefore, 100% concentration of zodia (E. suaveolens) leaf extract able to provide the highest inhibition on the growth of P. aeruginosa and F. oxysporum. The statement above corresponds to the statement Qomar, Budiyanto, Sukarsono, Wahyuni, & Husamah (2018) and Ali, Salih, & Daffalla (2020) that the higher the concentration of the extract, the more active compounds it contains so that it can produce high microbial growth inhibition. Asadi (2016) and Fikselova et al. (2014) added that antimicrobial activity was more commonly found at higher extract concentrations. Tables 3 and 4 show that the significance value was 0,000 and smaller than 0.050 (P <0.050). This means that there is a significant inhibitory effect of the antimicrobial activity of zodia (E. suaveolens) leaf extract on the growth of P. aeruginosa and F. oxysporum.

Figure 1. The Diameter of Inhibition Zone P. aeruginosa Growth in Several Concentrations of Zodia (E. suaveolens) Leaf Extract
Significant inhibitory effects of the antimicrobial activity of zodia extracts (E. suaveolens) are caused by the content of active compounds, such as essential oils, flavonoids, alkaloids, saponins, and tannins. The active compound contained in zodia (E. suaveolens) leaf extract acts as an antimicrobial. This statement is in line with the results of the study Rahmawati, Samsumaharto, & Iryanto (2015) states that zodia leaf extracts with n-hexane, chloroform, and water fractions contain active compounds including essential oils, flavonoids, alkaloids, saponins, and tannins that function as antibacterial agents to inhibit the growth of P. aeruginosa. Handayani and Nurcahyanti (2015) added that they had isolated essential oils that function as antimicrobial from zodia leaf using the water distillation method. Fajri and Agustien (2015) from their research have proven that the antimicrobial compounds from zodia leaf can inhibit the growth of Escherichia coli and Staphylococcus aureus.
Maryuni (2008)  Damage to cell walls and microbial cell membranes may cause the active compound extracts of zodia (E. suaveolens) leaf into the cell and affect the metabolic activities of bacterial cells. The metabolism of microbial cells becomes "error" resulting in death. Besides, damage to cell walls and microbial cell membrane makes molecules and compounds in the cytoplasm of cells out into the environment so that bacterial cells do not have the power to grow because all the molecules and enzymes needed for growth are lost.
Karta & Burhannuddin (2017) Yanti, Samingan, & Mudatsir (2016) states that antimicrobial active compounds contained in certain plant parts can increase cell membrane permeability and cell membrane damage. This causes interference with the metabolism of microbial cells so that they die. Konaté, Yomalan, Sytar, & Brestic (2015) and Alam, Forid, Roney, Aluwi, & Huq (2020) explains that flavonoids and tannins have important antimicrobial activity because flavonoids can dissolve the constituents of cell walls while tannins can inactivate microbial cell enzymes and proteins.

Conclusion
The antimicrobial activity of zodia (E. suaveolens) leaf extract can significantly inhibit the growth of P. aeruginosa and F. oxysporum (P <0.05). The findings of this study recommend that zodia (E. suaveolens) leaf extract can be used as an antimicrobial. Furthermore, the antimicrobial of zodia (E. suaveolens) leaf extract can be applied in direct research in the field.