03. Masri.cdr Vol.15, No.4, December 2021, p 135-138 DOI: 10.5454/mi.15.4.3 SHORT COMMUNICATION Anti Fungal Activity of Chitinolytic Bacteria Lysinibacillus fusiformis and Brevibacillus reuszeri against the Fungal Pathogens Rhizoctonia solani and Fusarium oxysporum * MASHURI MASRI , EKA SUKMAWATY, AND AS AWALIAH AMIR Department of Biology, Faculty of Science and Technology, Universitas Islam Negeri Alauddin, Jalan HM Yasin Limpo No. 36 Samata Kab. Gowa 90221, Sulawesi Selatan, Indonesia. Chitinolytic bacteria can produce chitinase, reported as a biocontrol agent against plants. This research aims to observe chitinolytic activity in inhibiting the growth of Rhizoctonia solani and Fusarium oxysporum. Anti fungal testing in dual culture test by growing each of the chitinolytic bacteria, Lysinibacillus fusiformis and Brevibacillus reuszeri, with the pathogenic fungi, F. oxysporum and R. solani, in petri dishes containing chitin agar media facing a distance of 3 cm. The results showed that chitinolytic bacterial isolates were capable inhibit the fungus by having the activity of each index inhibition of L. fusiformis isolates (30%), B. reuszeri (77%) against F. oxysporum, and R. solani fungi isolates (100%) for each chitinolytic bacterial isolate. Key words: anti fungal, chitinolytic bacteria, pathogenic fungi Bakteri kitinolitik dapat menghasilkan enzim kitinase dan dilaporkan sebagai agen biokontrol terhadap tanaman. Penelitian ini bertujuan untuk melihat aktivitas kitinolitik dalam menghambat pertumbuhan Rhizoctonia solani dan Fusarium oxysporum. Pengujian anti-cendawan menggunakan teknik kultur ganda dengan menumbuhkan masing-masing bakteri kitinolitik, Lysinibacillus fusiformis dan Brevibacillus reuszeri, dengan jamur patogen, F. oxysporum dan R. solani, dalam cawan petri yang berisi media agar mengandung kitin secara berhadapan dengan jarak 3 cm. Hasil penelitian menunjukkan bahwa isolat bakteri kitinolitik mampu menghambat cendawan dengan indeks penghambatan L. fusiformis (30%), B. reuszeri (77%) terhadap cendawan F. oxysporum dan R. solani (100%) untuk setiap isolat bakteri kitinolitik. Kata kunci: anti cendawan, bakteri kitinolitik, cendawan patogen MICROBIOLOGY INDONESIA Available online at http://jurnal.permi.or.id/index.php/mionline ISSN 1978-3477, eISSN 2087-8575 *Corresponding author: Phone: : +62-411-841879; Fax : +62- 411-8221400; email : mashuri.masri@uin-alauddin.ac.id two types of pathogens that often attack horticultural crops (Liu et al. 2017; Zhao et al. 2014). The main composition of fungal cell walls is chitin so that biological control strategies can be carried out by utilizing chitinolytic enzyme-producing bacteria. Chitinolytic bacteria are a group of bacteria that are capable of producing the chitinase enzyme (Asif et al. 2020; Moon et al. 2017; Wang D et al. 2018). The enzyme functions to catalyze the chitin degradation reaction by cutting the glycosidic bonds between N- residues. acetylglucosamine. Chitinolytic bacteria have strong antagonistic activity against fungal pathogens with hyperparasitism and antibiotic mechanisms (Pliego et al. 2011).The use of chitinolic bacteria as biological control agents is expected to degrade the cell walls of pathogenic fungi so that it can inhibit the growth of pathogenic fungi in horticultural crops. Two Chitinolic bacteria, namely Lysinibacillus fusiformis and Brevibacillus reuszeri, were tested for their antifungal abilities against F. oxysporum and R. solani. Horticultural crops have important potential in fulfilling nutrition and increasing farmers' income so as to support community welfare (Malhotra 2016; 'Kerutagi et al. 2019). However, in cultivation, there are obstacles in the form of plant diseases caused by fungal pathogens that can pose a risk of plant damage and decreased production (van Bruggen et al. 2016; Panth et al. 2020). This can cause economic losses in agriculture and the horticultural industry. Therefore, it is necessary to take preventive measures to control these pathogens such as chemical and biological control. Chemical control is a control that can be done quickly but has a bad long-term effect on the environment and can cause pathogen resistance. Therefore, biological control is the main solution in preventing fungal pathogens that destroy horticultural crops. Fusarium oxysporum and Rhizoctonia solani are The isolates of the fungus R. solani and F. oxysporum (Patimah 2018) were cultured on Potato Dextrose Agar (PDA) media and incubated for 7 days at room temperature. Chitinolytic bacteria isolates, namely L. fusiformis and B. reuszeri (Aditia 2016) were cultured on Nutrient agar (NA) media and incubated for 1x24 hours at 37 °C. The antifungal test of chitinolytic bacterial isolates was carried out using a multiple culture test. Previously isolated chitinolytic bacteria, R. solani and F. oxysporum, were scratched at a distance of 2 cm from the edge of the media. After the bacteria were 24 hours old, the bacteria were tested in the Dual Culture test against fungi using Chitin Agar Media. Chitin Agar is used to break down chitin compounds in bacteria. Pathogenic fungal mycelium with a diameter of 0.5 cm was placed in a petri dish with a distance of 2 cm from the edge of the media using a needle loop. Then the chitinolytic bacteria were scratched lengthwise at a distance of 3 cm from the mycelium of the pathogenic fungi, the cultures were incubated at room temperature for 7 days (Anesini and Perez 1993; Yurnaliza et al. 2012). This test was repeated 1 time. The inhibition index used in this study was carried out by observing the resulting inhibition zone on the 7 days calculated using the formula: R1: Growth radius towards the edge of the Petri R2: Growth radius towards antagonistic bacteria Based on the results of the anti-fungal activity test, it was found that L. fusiformis produced an inhibitory index of 100% against R. solani and 30% against F. oxysporum. Meanwhile, B. reuszeri produced an inhibitory index of 100% against R. solani and 77% against F. oxysporum (Table 1). The inhibition index of 100% meant that there was no growth of pathogenic fungi when the antagonist was tested with chitinolytic bacteria L. fusiformis (Fig 1a) and B. reuszeri (Fig 1c). Inhibition index <100% means that there is still growth of pathogenic fungi. This growth was indicated by the presence of hyphae formed on the surface of the growing medium when antagonist testing with chitinolytic bacteria was carried out (Fig 1b and 1d). The presence of an inhibitory index showed that chitinolytic bacteria L. fusiformis and B. reuszeri were able to inhibit the growth of pathogenic fungi. This is because chitinolytic bacteria produce chitinase enzymes that are able to degrade fungal cell walls. The main component of fungal cell walls is chitin. Chitin is a linear polymer composed of monomers, namely β- 1,4- N-acetyl-glucosamine (Cabib et al. 2001; Roncero 2002; Moon et al. 2017). In the presence of chitinase enzymes produced by chitinolytic bacteria, hydrolysis of chitin compounds occurs at -1,4-glycosidic bonds and produces oligosaccharides or N-acetyl- glucosamine monomers (Tronsmo and Harman 1993; Herdyastuti et al. 2010). The magnitude of the inhibitory index produced depends on how much N-acetyl-glucosamine Table 1 Activity data of chitinolytic bacteria against pathogenic fungi 136 MASRI ET AL. Microbiol Indones Chitinolytic Bacteria Pathogenic Fungi Inhibition Radius Inhibition Index R1 R2 Lysinibacillus fusiformis Rhisoctonia solani 0 0 100% Lysinibacillus fusiformis Fusarium oxyforum 3.6 2.2 30% Brevibacullus reuszeri Rhisoctonia solani 0 0 100% Brevibacullus reuszeri Fusarium oxyforum 3.6 0.8 77% Fig 1 Anti-fungal activity of chitinolytic bacteria. a. Lysinibacillus fusiformis against Rhisoctonia solani, b. Lysinibacillus fusiformis against Fusarium oxysporum, c. Brevibacullus reuszeri against Rhisoctonia solani, d. Brevibacullus reuszeri against Fusarium oxysporum. monomer is produced from the chitin hydrolysis process using the chitinase enzyme. The greater the number of N monomers produced, -acetyl-glucosamine the greater the inhibitory index that will be formed. Testing of the anti-fungal ability of chitinolytic bacteria was carried out simultaneously, but there were differences in the inhibitory index of each isolate of chitinolytic bacteria. This is due to the different species of chitinolytic bacteria and fungal pathogenic species used. Each chitinolytic bacteria certainly produces different chitinase enzymes so that it affects the anti- fungal activity which is characterized by the presence of an inhibition zone. In addition, each pathogenic fungus certainly has a specific defense mechanism so that it affects self-defense against foreign compounds that interfere with its existence. Based on Table 1, it can be seen that the larger the inhibition zone produced, the chitinase produced by chitinolytic bacteria is able to hydrolyze large amounts of chitin so that the growth of pathogenic fungi is inhibited. The fungus F. oxysporum is more resistant to chitinase because the cell wall composition of the fungus F. oxysporum in the outer layer contains glycoprotein compounds that protect the surface of the mycelium. The glycoprotein content in the cell wall is 50-60% of the total cell wall mass (Schoffelmeer et al. 1999; Yurnaliza et al. 2012). Chitinase is widely used as a biocontrol agent, especially for plants that are often infected with fungi, it is because chitin, which is the main component of fungal cell walls, can be degraded by the chitinase enzyme to produce an environmentally friendly product compared to chemicals (Asif et al. 2020). The chitinase enzyme's role is widely used as an effective antifungal against R. solani on transgenic cotton (Nicotiana tabacum L) (Broglie et al. 1991), Transgenic elite indica rice (Datta et al. 2001), or F. oxysporum on strawberries (Wang Y et al. 2003), F. oxysporum f. sp. Udum on pigeon pea (Bapat and Shah 2000), Bacillus thuringiensis can inhibit the growth of several fungal pathogens and fungal cell wall degradation (Hollensteiner et al. 2017). Many species of Bacilli are well known as plant growth-promoting bacteria (PGPB), biocontrol of pests and diseases. It was reported that species of B. brevis are effective antagonists for pathogenic fungi such as F. oxysporum f. sp. 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