CONTACT : NI’MATUZAHROH nimatuzahroh@fst.unair.ac.id 95 Abstract Ralstonia solanacearum is a soil borne pathogen, which has a very wide host range and causes bacterial wilt. The use of biological agents, such as bacterial groups has been tried in several areas of potato plantations in Java. This study aims to obtain Bacillus isolates from the potato cropping area, Sumber Brantas, Bumiaji, Batu, which had the potential to suppress growth R. solanacearum. Bacillus was isolated from the soil rhizosphere of potato plants, while R. solanacearum was isolated from the base of potato stems showing bacterial wilt symptoms on tetrazolium chloride (TZC) selective medium. Bacillus spp. and R. solanacearum isolates were tested for hypersensitivity on the leaves of the KR- 15 tobacco plant. Isolates that cause necrosis symptoms in tobacco leaves can be ascertained to be pathogenic. This study succeeded in obtaining 13 Bacillus spp. isolates with different colony morphologies. Three isolates of Bacillus spp. were selected from the 13 isolates obtained, based on their ability to suppress the growth of R. solanacearum and are expected to be potential as biological agents. Based on genetic analysis, the 3 bacterial isolates were identified as Bacillus mycoides and Bacillus weihenstephanensis. ISSN : 2580-2410 eISSN : 2580-2119 Exploration Indigenous Bacillus Bumiaji-Malang Against Ralstonia solanacearum Causing Potato Bacterial Wilt Endang Triwahyu Prasetyawati1,2, Tini Surtiningsih3,4, Ni’matuzahroh3,4,5*, Purkan6, Silvia Kurnia Sari3,4, Ana Mariatul Khiftiyah3,4, 1Faculty of Agriculture, UPN “Veteran” Jawa Timur, Surabaya, Indonesia 2Faculty of Science and Technology, Universitas Airlangga, Surabaya, Indonesia 3Department of Biology, Faculty of Science and Technology, Universitas Airlangga, Surabaya, Indonesia 4University-Center of Excellence- Research Center for Bio-Molecule Engineering, Universitas Airlangga, Surabaya, Indonesia 5Faculty of Advanced Technology and Multidiscipline, Universitas Airlangga, Surabaya, Indonesia 6Department of Chemistry, Faculty of Science and Technology, Universitas Airlangga, Surabaya, Indonesia Introduction One of the factors that can affect plant productivity is pathogen attack. Plant pathogenic microbes (phytopathogens) can be bacteria, fungi or viruses. Some important phytopathogenic bacteria include Ralstonia solanacearum, Pseudomonas syringae pv. OPEN ACCESS International Journal of Applied Biology Keyword Bacillus spp; Bacterial wilt; Pesticide reduction; Potato plants; Ralstonia solanacearum Article History Received 20 June 2021 Accepted 03 July 2021 International Journal of Applied Biology is licensed under a Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. International Journal of Applied Biology, 5(1), 2021 96 glycines, Pseudomonas fluorescens, Erwinia sp., and Xanthomonas oryzae. Ralstonia solanacearum is a soil borne plant pathogen that is found in many subtropical and tropical areas which naturally infects roots and multiplies in xylem tissue (Hayward, 1991; Yabuuchi et al. 1995). Ralstonia solanacearum is one of the phytopathogens that attack potato plant. R. solanacearum causes bacterial wilt on potatoes. This pathogenic infection is reported to cause huge losses in various potato production centers and a threat to development target areas in Indonesia (Kuswinanti, et al., 2014). Sumber Brantas Village, Bumiaji District, Batu, is one of the centers for potato plantations in East Java. The use of chemicals by farmers in Sumber Brantas village to protect crops from pathogens is still being carried out. Control of soil borne pathogens such as R. solanacearum using chemicals formulated as pesticides, fungicides, bactericides and others, is still questionable about its safety. Excessive use of chemicals is harmful to both the biotic and abiotic environment. One of the efforts to overcome these problems is by using bacteria as biocontrol agents. The mechanisms for controlling pathogens by biocontrol agents include antibiosis, space and nutrition competition, microparasitism, cell wall degrading enzymes, resistance inducers, growth promoters, rhizosphere colonization and anti-quorum sensing (Lo, 1998; Yin et al, 2010; Junaid et al, 2013). Bacteria that have the ability as biocontrol agents can be isolated from soil in the plantation land (Rahman et al., 2012; Saha et al., 2012; Ali et al., 2020). Several groups of bacteria that are known to have potential as biocontrol agents are Agrobacterium, Pseudomonas and Bacillus (Fravel, 2005). The genus Bacillus has the ability to synthesize several compounds that are useful in agriculture and industry. Several secondary metabolites produced by several species and strains of Bacillus show antibacterial and antifungal activity against plant pathogens (Yu et al., 2002; Ongena & Jacques, 2008). Bacillus also has different enzymatic capabilities in producing enzymes, including in producing amylase, protease and lipase enzymes. Bacillus sp. can produce phytohormones that have the potential to develop sustainable agricultural systems. The phytohormones produced can affect plant growth, either directly or indirectly. Indirectly, the phytohormones from bacteria inhibit pathogenic activity in plants, while the direct influence of these phytohormones increases plant growth and can act as a facilitator in the absorption of some nutrients from the environment. The application of bacteria as a biocontrol agent, although it is environmentally friendly, but it’s not widely used. Potato farmers in Sumber Brantas Village are also not interested in using it, due to its effects that do not meet their expectations, such as slow response effect. Thus, exploration of bacteria as biocontrol agents must be carried out to obtain potential isolates to suppress growth of R. solanacearum which causes bacterial wilt on potato plants in Sumber Brantas Village. The purpose of this study was to obtain Bacillus spp. isolates from potato plantation in Sumber Brantas Village, Bumiaji District, Batu, which were able to suppress the growth of the R. solanacearum which causes bacterial wilt on potato plants, and also to identify Bacillus spp. isolates that were the most capable to suppress the growth of R. solanacearum. Materials and Methods Isolation of Ralstonia solanacearum Diseased plant samples were taken from the potato planting area in the Sumber International Journal of Applied Biology, 5(1), 2021 97 Brantas area, Batu, Malang. The base of the potato stems showing wilting symptoms, washed with sterile water then cut into pieces 5 mm x 5 mm in size then crushed with a mortar until smooth and added 1 ml of sterile distilled water. The bacterial suspension in the mortar was taken as much as 0.5 ml and then put in a test tube containing 4.5 ml of MgSO4.7H2O. Furthermore, it was diluted to reach 10-5 dilution based on the Kelman (1953) method. A total of 0.1 ml of the diluted suspension was cultured in a Petri dish containing sterile Triphenyl Tetrazolium Cloride (TTC / TZC) medium, and leveled with a sterile L- shaped glass rod and incubated at 30°C for 2 x 24 hours. The growing bacterial colonies were separated between virulent and non-virulent colonies on new sterile Kelman TZC solid media, and re-incubated according to the previous work step. Isolation of Bacillus spp. Soil samples from the rhizosphere of potato plantation were weighed 10 grams and put in an Erlenmeyer, were added 90 ml of sterile distilled water and shaken for 30 minutes at a speed of 150 rpm. Furthermore, the sample was heated in a water bath at 80 °C for 15 minutes, then dilutions were carried out in series until 10-5. Dilutions of 10-3, 10-4 and 10-5 were taken as much as 100 μl grown on Petri dishes containing Tryptic Soy Agar (TSA) medium (Singh et al., 2008). Petri dishes were incubated at a temperature of approximately 28 oC for 24 hours. Hypersensitivity Test Bacillus spp. isolates were propagated in TSA media, R. solanacearum isolates in TZC media for 48 hours. All colonies were harvested, each isolate was added in a tube of 2 ml of sterile distilled water. The suspension was then infiltrated into tobacco K-15 on the lower surface, incubated for 48 hours and observed for leaf necrosis. Bacillus spp. isolates that showed negative reactions (no leaf necrosis were observed) were used for further testing (Klement et al, 1990). Meanwhile, R. solanacearum isolates used for further research were those that showed a positive reaction (showing symptoms of necrosis). Antagonist Test The antagonist test was carried out in vitro. R. solanacearum was cultured on TZC medium, made a suspension at λ600nm = 2 or reached a population of about 2.0x108 CFU / mL, added to Yeast Pepton Glucose Agar (YPGA) medium and poured on a Petri dish. Ten µl of cells (OD600 = 3) Bacillus spp. was dropped on sterile filter paper with a diameter of 0.8 cm, and placed in a Petri dish that already contained R. solanacearum. Furthermore, the Petri dishes were incubated for 48 hours at 28 oC and observed for the formation of clear zones and measured the diameter. As a control filter paper was dripped with sterile water and each treatment was repeated three times. In Vivo Selection of Antagonistic Bacteria Isolates The in vivo test used the KR-15 variety of tobacco as a test plant. Prepare 24-hour- old Bacillus spp. isolates, 48-hour-old R. solanacearum isolates, and 2-month-old tobacco plants in polybags. Tobacco plants were placed in a plastic cover in the green house, the plants that were ready are sprayed with Bacillus spp. isolate, respectively. According to the treatment, there were three tobacco leaves in each treatment and left for 3 days. Furthermore, the tobacco plants, inoculated with R. solanacearum, were placed on the lower surface of the leaf veins. Plants remained in plastic cover, observed daily starting from International Journal of Applied Biology, 5(1), 2021 98 two days after treatment until three weeks after inoculation of R. solanacearum. The percentage of inhibition was calculated by the formula: 𝐵 𝐴 ×100% A : Leaf area (cm2) B : Necrosis area (cm2) Characterization and Identification of Bacteria Three bacterial isolates were selected to be identified, of the 13 isolates that showed the worst symptoms on the in planta test. Characterization was carried out through colony macroscopic, cell microscopic, physiological, and genetic observations. Colony macroscopic characterization was performed by culturing bacteria on nutrient agar for 24 hours. Microscopic characterization was conducted by Gram staining, while physiological characters were performed using KIT A, which include, catalase, oxidase, oxidative- fermentation requirements, hydrolysis of gelatin, starch, levan formation, Proskauer's Voges test, arginine, dehydrolase, motility, tolerance for bacterial growth at several temperatures, pH and HCl concentration, use and overhaul of carbon, citrate and nitrogen compounds (Lelliot and Stead, 1987; William et al., 1989, Chun and Vidaver, 2001). Identification was continued molecularly, by sequencing the 16S rRNA gene. Preparation before the sequencing process refers to the protocol kit (Genomic DNA Mini Kit, Geneaid), 16S rRNA was amplified by Polymerase Chain Reaction (PCR) technique using universal primers (27F 5 "-AGA GTT TGA TCC TGG CTCAG-3" and 1429R 5 "-GGT TAC CTT GTT ACG ACTT-3"). The PCR mixture was prepared in a 25 µl volume containing 1 µl F primer, 1 µl R primer, 12.5 µl mix (KAPA Taq Ready Mix, Kapa Biosystems, United States), 2.5 µl DNA extract, and 8 µl dH2O. Then the DNA amplification was carried out on a PCR (Swift Maxi Thermal Cyber) machine with an initial denaturation for 1 minute at a temperature of 95 oC. Then followed by 30 denaturation cycles at 95 oC for 1 minute, annealing at 55 oC for 1 minute and extension at 72oC for 1.5 minutes. Followed by an additional extension step at 72oC for 5 minutes. 2 for 5 minutes. PCR products were visualized by electrophoresis on 1% agarose gel which had been added with ethidium bromide and TBE buffer. The sequencing of PCR results was carried out by a sequencing service provider company. DNA sequence homology searches were executed using a DNA database (GenBank) using the BLAST program from the National Center for Biotechnology Information (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Results and Discussion Isolation of Bacteria The results of the isolation of R. solanacearum from potato plants, obtained 6 different isolates based on colony morphology. R. solanacearum belongs to a group of Gram-negative bacteria, the morphology of short rod-shaped cells, single cells. Colony morphology of R. solanacearum was irregularly rounded, milky white, the texture of the colony was slimy / shiny, the edges of the colony were uneven, the elevation was convex. According to Ray et al (2013) Ralstonia sp. can grow on Sucrose Peptone Agar (SPA) media, with irregularly shaped colonies, slightly convex elevation, slimy, creamy milk color, shiny surface, and in terms of physiology Ralstonia sp. in the form of a prosthetic with a size of https://blast.ncbi.nlm.nih.gov/Blast.cgi International Journal of Applied Biology, 5(1), 2021 99 0.5-0.7 μm. Based on these characteristics which were confirmed by the characteristics listed in Bergey's Manual of Determinative Bacteriology and as reported by Ray et al (2013), the bacteria isolated in this study were identified as the Ralstonia genus. Meanwhile, Bacillus spp., based on differences in colony morphology, there were 13 isolates of Bacillus spp. on Nutrient Agar medium. In general, the colonies have a characteristic whitish cream color and a round and irregular colony. The edges of the isolates were flat and some were irregular. This characterization showed 13 isolates including the genus Bacillus sp. Hypersensitivity Test Hypersensitivity test results on tobacco plant leaves showed that three isolates of R. solanacearum were able to cause necrose symptoms, namely isolates Rs-3, Rs-4 and Rs-1, each of which had an incubation period of 4 days after incubation (DAI), 8 DAI and 11 DAI (Figure 1). From these results selected Rs-3 which has a faster incubation period. According to Agrios (2005), environmental conditions that support the growth of pathogens will accelerate the incubation period of the disease, so it will be faster to infect plants. Meanwhile, Bacillus spp. none of the 13 isolates showed symptoms of necrosis, therefore all of them have the opportunity to be used for further research. Antagonist Test The in vitro antagonist test results of 13 Bacillus spp. isolates against R. solanacearum showed similar results, namely that no isolate was able to produce an inhibition zone. The absence of a zone of inhibition from Bacillus spp. isolates does not mean that the bacteria were unable to suppress the development of R. solanacearum. The inability of Bacillus spp. isolates to form a zone of inhibition in vitro, presumably due to the condition and nutritional content of the media used, allows bacteria to not produce antibiotic compounds. Saputra (2015) stated that Bacillus did not show bactericidal against R. solanacearum. These results are also in line with research by Zicca et al., (2020) who reported that B. oleronius, B. licheniformis, and B. megaterium did not show a zone of inhibition when grown together with Xylella fastidiosa in the same media. The mechanism of inhibition of antagonistic bacteria can also be by competition for space and nutrition, releasing degrading enzymes or through induced resistance mechanisms (Lo, 1998). Figure 1. Hypersensitivity test results for R. solanacearum bacteria isolates in Tobacco KR- 15, symptoms of necrosis caused by isolate (A) Rs-6, (B) Rs-4, (C) Rs-1. International Journal of Applied Biology, 5(1), 2021 100 Solanacearum suppression testing in greenhouse The results showed that the ability of Bacillus spp. to suppress the growth of R. solanacearum was different. This is indicated by the percentage area of necrosis symptoms that appear varies (Figure 2). This difference was due to the different abilities of the thirteen Bacillus spp. isolates in suppressing R. solanacearum. According to Choudhary and Johri (2008) biological control of Bacillus spp. through the mechanism of antibiosis, secretion of lysing enzymes and inducers of systemic resistance. Figure 2. Potential of Bacillus isolates in suppressing necrotic symptoms in plants caused by R. solanacearum Identification of Bacillus spp. Three Bacillus spp. isolates which had the strongest ability to suppress the R. solanacearum growth, Ba-1, Ba-2 and Ba-11, were selected for further identification. Table 1. Macroscopic colony and microscopic characters of selected bacteria Isolate Codes Microscopic and Macroscopic Characteristics of Bacillus Isolates Surface Edge Shape Color Gram Endospores Isolate Ba-1 (figures 3a and 4a) Flat Flat Round White + + Isolate Ba-2 (figures 3b and 4b) Flat Irregular Round White + + Isolate Ba-11 (figures 3c and 4 d) Slimy Flat Round White + + Figure 3. Morphological colony of Bacillus spp. which had the strongest ability to suppress the growth of R. solanacearum. Isolate colonies (A) Ba-1, (B) Ba-2, (C) Ba-11 International Journal of Applied Biology, 5(1), 2021 101 The round colony shape and white colony color generally indicate that the bacteria belong to the genus Bacillus sp. According to Corbin (2004), the colony of Bacillus sp. has the general characteristics of having a whitish cream color and a round and irregular colony shape, flat and uneven colony edges. According to Hatmanti (2000), the bacteria Bacillus spp. colony has various kinds of flat and uneven edges, the surface is rough and not slimy, there are even tend to be dry and powdery, the colonies are large and not shiny. Figure 4. Morphology cell of Bacillus spp. (A) Ba-1, (B) Ba-2, (C) Ba-11. The three isolates showed a rod-shaped form of bacterial cells, Gram positive, forming endospores. According to Sofyan et al. (2009), Bacillus sp. is a Gram-positive bacteria with a short rod to a single rod with a single arrangement. Table 2. Physiological and Biochemical Test with Kit A. No. Characteristics Bacillus Isolates Code 1 2 11 1. Oxidase + + + 2. Motilitas + + + 3. Nitrate + + + 4. Lysine - - - 5. Ornithine - - - 6. H2S - - - 7. Glucose - - - 8. Mannitol - - - 9. Xylose - - - 10. ONPG - - - 11. Indole - - - 12. Urease - - - 13. VP - - - 14. Citrate - - - 15. TDA - - - 16. Gram Test Positif Positif Positif 17. Shape Rod Rod Rod 18. Endospore Present Present Present International Journal of Applied Biology, 5(1), 2021 102 Table 3. Molecular identification of bacterial isolates No Code Isolates Results Identification (%) 1 Ba-1 Bacillus weihenstephanensis 99% 2 Ba-2 Bacillus weihenstephanensis 99% 3 Ba-11 Bacillus mycoides 99% The homology of Ba-1 and Ba-2 isolates based on the 16S rRNA gene from the BLAST results showed that these bacteria were related to several Bacillus weihenstephanensis. Meanwhile, Ba-11 isolate is related to Bacillus mycoides (Table 3). Based on the 16 s rRNA sequence of Bacillus weihenstephanensis, one group with B. cereus, B. mycoides, B. anthracis, B. pseudomycoides and B. cytotoxicus, because they both have the cspA, glpF, gmk, purH, and tpA genes (Habazar et al., 2018). In a further development between B. mycoides and B. weihenstephanensis it is stated that they are not different species. B. mycoides grows in the temperature range 10-15 °C to 35-40 °C, while B. weihenstephanensis grows in a temperature range of 7 - 43 °C (Soufiane et al., 2013). According to Habazar (2018), research on B. weihenstephanensis as a biological agent is still very limited. B. weihenstephanensis is known to have the potential to inhibit the phytopathogenic growth of Verticillium (Hollensteiner et al., 2017) and as an insecticidal agent in Schistocerca gregaria (Mashtoly et al., 2019). Genomic analysis conducted by Hollensteiner et al. 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