Antifungal Potency of Secondary Metabolites Produced by Endophytic Bacteria against Pathogenic Fungi Pyricularia oryzae Cav

Blast disease caused by Pyricularia oryzae Cav. is one of the most important plant diseases on rice. In Indonesia, P. oryzae attacks reached 19,629 ha of 12,883,578 ha of rice yields area in 2009 (Prayudi, 2009). Control strategies for blast must be carried out in order to maintain the quality and quantity of rice yields. The common effort to control blast is the use of fungicides (Srivastava et al., 2017). However, it is commonly known that the extensive use of fungicides has led to negative impacts, including the emergence of pathogens resistant population, accumulation of toxic residues, and negative effects on human health and environment (Sehajpal et al., 2009). One of the eco-friendly control techniques is the use of biopesticides. Biopesticide is defined as a formulation contains certain bioactive compounds that are potent antibiotics (Schumann & D’Arcy, 2012). Endophytic bacteria are known as producer of biologically active compounds. Secondary metabolites produced by endophytic bacteria are reported able to enhance the plant growth and to inhibit the plant diseases development (Ortiz-Castro et al., 2009). In addition, secondary metabolites from endophytic bacteria also have been reported to have the ability in producing growth regulators, providing nitrogen and phosphate to plants which contribute to stimulate and to enhance plant resistance (Ikeda et al., 2010). Widiantini et al. (2017) isolated several endophytic bacteria from healthy rice plant isolates which were able to inhibit the growth of several pathogenic fungi of rice including P. oryzae. Furthermore, Ainun (2018) reported the inhibitory mechanism shown by those isolates was related to its ability to produce chitinase and HCN. However, it is not known yet whether endophytic bacterial isolates produce secondary metabolites which can inhibit the ABSTRACT


INTRODUCTION
Blast disease caused by Pyricularia oryzae Cav. is one of the most important plant diseases on rice. In Indonesia, P. oryzae attacks reached 19,629 ha of 12,883,578 ha of rice yields area in 2009 (Prayudi, 2009). Control strategies for blast must be carried out in order to maintain the quality and quantity of rice yields. The common effort to control blast is the use of fungicides (Srivastava et al., 2017). However, it is commonly known that the extensive use of fungicides has led to negative impacts, including the emergence of pathogens resistant population, accumulation of toxic residues, and negative effects on human health and environment (Sehajpal et al., 2009).
One of the eco-friendly control techniques is the use of biopesticides. Biopesticide is defined as a formulation contains certain bioactive compounds that are potent antibiotics (Schumann & D'Arcy, 2012). Endophytic bacteria are known as producer of biologically active compounds. Secondary metabolites produced by endophytic bacteria are reported able to enhance the plant growth and to inhibit the plant diseases development (Ortiz-Castro et al., 2009). In addition, secondary metabolites from endophytic bacteria also have been reported to have the ability in producing growth regulators, providing nitrogen and phosphate to plants which contribute to stimulate and to enhance plant resistance (Ikeda et al., 2010). Widiantini et al. (2017) isolated several endophytic bacteria from healthy rice plant isolates which were able to inhibit the growth of several pathogenic fungi of rice including P. oryzae. Furthermore, Ainun (2018) reported the inhibitory mechanism shown by those isolates was related to its ability to produce chitinase and HCN. However, it is not known yet whether endophytic bacterial isolates produce secondary metabolites which can inhibit the growth of P. oryzae. This study reported the ability of endophytic bacteria in producing antifungal secondary metabolites against P. oryzae.

Extraction of Secondary Metabolites on ISP2 Liquid Media
Endophytic bacteria isolates were cultured on ISP2 liquid media. Two loops full of the endophytic bacteria isolates were transferred into Erlenmeyer flask containing 100 ml sterile liquid ISP2 media. Endophytic bacteria were incubated for 10 days at shaking incubator at 120 rpm in room temperature. Then, the optical density values were determined. The endophytic bacteria suspension was centrifuged for 30 minutes at 6000 rpm to separate the bacterial cells from the supernatant. The supernatant was extracted using two types of solvents, 100% methanol and mixture of ethyl acetate and methanol (4:1) (v/v). The supernatant was then evaporated using a rotary evaporator at 45ºC and pressure of 218 mbar until the solvent completely evaporated. The filtrate was re-suspended in 1 ml of sterile RO water and filtered through a 0.2 µm pore size of membrane filter.

Screening for Antifungal Activity of Secondary Metabolites against of P. oryzae
Antifungal activity of secondary metabolites against P. oryzae was tested using agar well diffusion method. Approximately 20 ml PDA media was poured into 9 cm diameter petri dishes. After the media set, a 6 mm diameter well was made using cork borer. As much as 30 µl of the filtrate was pipetted into the well. A 6 mm in diameter of plug of P. oryzae was placed 3 cm away from the well. Plates were incubated at room temperature.
The antifungal activity was observed by measuring the radial growth of P. oryzae colony. The effect of the filtrate against the mycelia of P. oryzae was also observed under the microscope. The percentage of inhibition in radial growth (PIRG) was calculated using the following formula: Description:

Thin Layer Chromatography (TLC) Analysis
The filtrates which prepared earlier were further analyzed by the TLC method. Each filtrate was blotted on Silica gel 60 F264 plate size 10 cm×5 cm (Whatman). Dichloromethane and methanol (9: 1) (v/v) was used as solvent (Dewi et al., 2017). TLC plates were removed from the chamber immediately before the solvent reached 1 cm from the top of the plates. TLC plates were sprayed with butanol, acetic acid, and water with (4: 1: 5) (v/v/v). Then the plate was visualized under the UV light. The appeared stains were marked using a pencil in which the Rf (Retention factor) value were calculated.

Analysis of High Performance Liquid Chromatography (HPLC)
The HPLC analysis was started with preparation of Os extract samples. The samples analyzed by HPLC were only 3 secondary metabolite extracts of Os1, Os3, and Os10. The selection of these samples was based on the antifungal activity of the extract against pathogenic fungi P. oryzae. Following the antifungal activity test, the metabolite extract which has high antifungal activity was the extract Os3 isolate, the moderate antifungal activity was the extract Os1 isolate, and no antifungal activity was the extract of Os10 isolate.
Chromatographic separation required 10 µl of each sample solution. The sample was injected into a 10 µl size vial. HPLC separation was done using C-18 column and the pump system used was a gradient system of milli Q water and methanol.

RESULTS AND DISCUSSION
The antifungal activity of secondary metabolites produced by endophytic bacteria extracted with two different solvents was tested against P. oryzae and ISSN 1410-1637 (print), ISSN 2548-4788 (online) PIRG = R1-R2 R1 ×100% R1 = radial colony of P. oryzae in the control treatment R2 = radial colony of P. oryzae in the treatment plate showed different results. Secondary metabolites extracted with ethyl acetate and methanol (4:1) showed a higher percentage of inhibition compared to the percent inhibition of secondary metabolites extracted with methanol only. The highest percentage inhibition on secondary metabolites from ethyl acetate extraction mixed with methanol reached 42%, while the highest inhibition percentage in secondary metabolites from methanol extraction only reached 38% (Table 1). When the filtrate was able to inhibit the growth of P. oryzae, smaller than control radial colony of P. oryzae was formed, leaving gap which called as inhibition zone. The effectiveness of the secondary metabolites in inhibiting the growth of pathogen detected by the size of the inhibition zone. The greater inhibition zone was formed, the more potent of the compound in inhibiting the growth of pathogens (Barry et al., 1979). Not all of the endophytic bacteria isolates were able to produce antifungal compound against P. oryzae. Five isolates (Os1, Os3, Os4, Os5 and Os6) demonstrated antifungal activity when extracted using both methanol and methanol-ethyl acetate solvent.
To determine which compound responsible for the antifungal activity against P. oryzae, TLC analysis was performed. The result demonstrated that the TLC system used in this study was unable to separate the compunds (Figure 1). The TLC analysis for all of the filtrates showed only one spot with the same Rf value although the antifungal activity of each secondary metabolites were different. There were no stain completely separated on the TLC plates.
Analysis of secondary metabolite compounds was further carried out using HPLC. Three samples were run for the HLPC analysis (Figure 2). Those were Os1 and Os3 showed high antifungal activity whereas Os10 has no antifungal activity. Chromatogram patterns produced by Os1 and Os3 filtrates demonstrated some similar peaks. At the secondary metabolites extracted by ethyl acetate : methanol, similar peaks at the same retention time of 3.5 minutes, 5.5 minutes, and 7.0 minutes were shown at Os1 and Os3 sample. Similar peaks were also detected from Os1 and Os3 samples extracted with methanol. Those peaks were come out at 1.1 min, 1.4 min, 1.8 min, and 3.5 min. Those similar peaks that found both in Os1 and Os3 samples were not found at the Os10 filtrate.
The antifungal testing through agar well diffusion found the potential of the secondary metabolites produced by the endophytic bacterial isolates to inhibit and affect the growth of P. oryzae. Mycelia of P. oryzae were unable to grow normally when treated with filtrates produced by Os1, Os3, Os4, Os5 and Os6 compared to untreated. Although the inhibition rate was not high (<50% inhibition), this could be optimized through modification of nutritional and cultural conditions. Varieties of carbon source and nitrogen as well as elevated temperature and pH for culture condition has been reported to be able to raise the amount of antifungal compound produces by Streptomyces CTF9 (Sajid et al., 2011). 3.20 a -3.67 d -TLC is one of cheap and fast method for identifying the antifungal compound. The TLC bioautography following the compounds separation can distinguish which compound is responsible for antifungal activity (Suleimana et al., 2009). However, in this study, attempt to separate the bioactive compounds using TLC was not successful. The solvent system used was not able to separate the compounds. Nevertheless, following the HPLC analysis, we found that several similar peaks were detected from filtrates produced by both Os1 and Os3 but not in Os10. It can be assumed that the compound represented by those peaks were the compounds that responsible for antifungal activity. The HPLC analysis is one of commonly used analytical tools for detecting various compounds in a crude mixture (Cieśla & Moaddel, 2016). However, it uses to identify the compound is limited to the availability of spectra in the database. Only compound within its spectra that available in the database that will be able to identified (Akpotu et al., 2017). In this study, the detected active compounds were unable to be identified as not listed yet in the spectra database. Therefore, we are unable to identify the type of compounds produced by the endophytic bacterial isolates. Further study to identify the compound needs to be done by employing relatively novel and hyphenated techniques such as LC-NMR or HPLC-NMR.

CONCLUSION
Several bacterial endophyte isolates isolated from healthy rice plant demonstrated potential ability to produce antifungal compounds which were able to inhibit the growth of P. oryzae. Compound responsible for the antifungal activity was detected by using HPLC analysis. However, its identification of the type of antifungal compounds has not been elucidated. Further study needs to be conducted to identify the respective compounds LITERATURE CITED