BIODIVERSITAS ISSN: 1412-033X Volume 22, Number 3, March 2021 E-ISSN: 2085-4722 Pages: 1291-1295 DOI: 10.13057/biodiv/d220327 Extracellular protease from Bacillus subtilis B315 with antagonistic activity against bacterial wilt pathogen (Ralstonia solanacearum) of chili NUR PRIHATININGSIH 1, , ARI ASNANI 2 , HERU ADI DJATMIKO 1 1 Faculty of Agriculture, Universitas Jenderal Soedirman. Jl. Dr. Suparno, Purwokerto Utara, Banyumas 53123, Central Java, Indonesia. Tel./fax. +62-281-638791,  email: nur.prihatiningsih@unsoed.ac.id 2 Departmeny of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Jenderal Soedirman. Jl. Dr. Suparno, Purwokerto Utara, Banyumas 53123, Central Java, Indonesia Manuscript received: 22 January 2020. Revision accepted: 14 February 2021. Abstract. Prihatiningsih N, Asnani A, Djatmiko HA. 2021. Extracellular protease from Bacillus subtilis B315 with antagonistic activity against bacterial wilt pathogen (Ralstonia solanacearum) of chili. Biodiversitas 22: 1291-1295. Antagonistic bacteria isolated from a healthy potato are potential biocontrol agents against a number of bacterial pathogens. The capability of antagonistic bacteria to produce extracellular protease could be considered as an indicator of their antagonistic capacity. The objective of this research was to study the optimum activity of protease produced by Bacillus subtilis B315 to control Ralstonia solanacearum causing wilt of chili. In this research were optimum incubation time, pH and crude extract protease concentration of B. subtilis B315 examined for maximum production of extracellular protease. The results showed that B. subtilis B315 was capable of producing protease at optimum incubation time of 30 min, pH 9, and crude extract protease concentration 0.50% with an activity of 1.3185 U/mL. Protease-producing capability of B. subtilis B315 was shown by the formation of a clear zone around the colony of B. subtilis B315 grown on skim-milk agar medium. B. subtilis B315 was capable of inhibiting the growth of R. solanacearum in vitro as indicated by 32 mm clear zone. Furthermore, in chili application of B subtilis showed that this bacteria was capable of controlling chili bacterial wilt caused by R. solanacearum with effectiveness of 60.89%. The prospective of B. subtilis B315 could be formulated as biopesticide. Keywords: Bacillus subtilis B315, bacterial wilt, biocontrol, chili, protease activity INTRODUCTION Bacterial wilt caused by Ralstonia solanacearum is an important disease in plants that may include tomato, chili, eggplant, bananas, potatoes, ginger, and mulberries. In chili and tomato, bacterial wilt could reduce yields up to 80% (Vanitha et al. 2009). Currently, biological control using antagonistic bacteria has been widely studied, as it is more environmentally friendly and supports sustainable agriculture. Antagonistic bacteria could be explored from habitats of which the pathogen naturally lives. Bacillus subtilis B315 isolated from a healthy potato rhizosphere has been reported to be capable of controlling bacterial wilt in potatoes with an effectiveness of 64.9% (Prihatiningsih 2013), and 74.6% in tomato, chili, and eggplant (Prihatiningsih and Djatmiko 2016). The potential of bacteria to be antagonist is shown by its ability to produce secondary metabolites (antibiotics and other compounds) and enzymes. Protease is a product of microbes that could function to control plant pathogens, either fungi or bacteria. Bacillus sp. and other bacteria such as: B. sterothermophilus, B. mojavensis, B. megaterium and B. subtilis (Shumi and Anwar 2004) secrete extracellular enzymes including proteases. Saha et al. (2012) found two new strains of B. subtilis from eggplant rhizosphere that have the potential to be biocontrol agents which is shown by their capabilities of producing secondary metabolites, such as siderophore, and hydrolytic enzymes, such as chitinase, protease, lipase and amylase. B subtilis was also reported to be able to produce IAA (Indole Acetic Acid) ranging from 57.56-79.33 ppm (Prihatiningsih et al. 2020). B. subtilis has the ability to secrete amylase, protease, pullulanase, chitinase, xylanase, and lipase (Morikawa 2006; Lestari et al. 2017). Protease is a hydrolytic enzyme that hydrolyses extracellular proteins and increases the ability of cells to absorb useful hydrolysis products. B. subtilis has important bacteria because of its capability to secrete several hydrolytic enzymes, including protease, into the culture medium (Almas et al. 2009). Antagonistic Bacillus sp. significantly colonize rice plants and secretes various biocontrol determinants like protease of 1.1±5.5 U/mg of soil or U/mL of hydroponic solution. Bacillus spp. strains KFP-5 and KFP-17 produce various quantities of siderophore, protease and glucanase in absence as well as in presence of rice blast pathogen Pyricularia oryzae. The protease production is ranging about 20.9-29.3 U/mL and 11.6-24 U/mL in KFP-5 and KFP-17, respectively (Rais et al. 2017). Bacillus subtilis B315 has important potential to produce protease because this enzyme supports the crops for enhanced resistance to disease. Nevertheless, enzymatic treatment was secreted by B. subtilis B315 as antagonist bacteria resulted in degradation of the EPS (extracellular polymeric substances) components and significant eradication of the biofilm of bacteria pathogens. One of the hydrolytic enzymes can penetrate to biofilm, the use of protease can degrade pathogenic bacteria biofilms.