Seleksi dan Karakterisasi Bakteri Kitinolitik Asal Tanah yang Mampu Menghambat Pertumbuhan Fusarium proliferatum

Abstract

engendalian penyakit tanaman masih menjadi tantangan bagi patologi tanaman untuk pertanian yang berkelanjutan. Salah satu masalah utama di bidang pertanian adalah kerusakan yang disebabkan oleh fungi fitopatogen seperti Fusarium proliferatum. Fungi F. proliferatum menyerang berbagai tanaman termasuk sayuran dan buah-buahan, serta menginfeksi lebih dari 25 spesies tanaman. Pengendalian fungi fitopatogen secara konvensional telah dilakukan menggunakan pestisida sintetik, namun penerapannya dapat menyebabkan resistensi pada mikroorganisme dan membahayakan kesehatan manusia, hewan, dan lingkungan. Oleh karena itu alternatif untuk mengurangi pestisida sintesis dengan menggunakan bakteri kitinolitik asal tanah sebagai agen penghambat pada fungi fitopatogen. Bakteri kitinolitik mampu menghasilkan enzim kitinase yang dapat menghidrolisis ikatan ß-1,4-glikosidik antara residu N-asetilglukosamin yang terdiri dari rantai kitin. Kitin merupakan salah satu penyusun dinding sel fungi fitopatogen seperti F. proliferatum. Oleh karena itu, tujuan penelitian ini ialah menyeleksi bakteri kitinolitik asal tanah yang mampu menghasilkan enzim kitinase dan mengukur aktivitas enzim kitinase yang dihasilkan serta mengetahui potensinya dalam menghambat pertumbuhan fungi fitopatogen F. proliferatum. Penelitian ini dilakukan melalui beberapa metode, yaitu seleksi isolat yang memiliki aktivitas enzim kitinase secara kualitatif, aktivitas antagonistik terhadap F. proliferatum dan aktivitas patogenisitasnya terhadap hewan dan tumbuhan. Sebanyak 2 isolat terpilih dari 15 isolat hasil isolasi, dianalisis identitasnya berdasarkan gen 16S rRNA. Selanjutnya 1 isolat potensial Bacillus yang telah diidentifikasi digunakan sebagai isolat uji untuk pengukuran aktivitas enzim kitinase secara kuantitatif. Selain itu, pada isolat terpilih dilakukan pula deteksi gen penyandi enzim kitinase (chi gene). Enzim kitinase yang dihasilkan oleh bakteri, selanjutnya diendapkan menggunakan ammonium sulfat dan dilakukan pengukuran aktivitas pH dan suhu optimum enzim. Enzim kitinase hasil pengendapan dilakukan pengukuran aktivitas penghambatan terhadap F. proliferatum dengan metode food poisoning assay. Kerusakan pada miselia F. proliferatum hasil uji antagonisme diamati dengan menggunakan mikroskop cahaya dan Scanning Electron Microscope (SEM). Hasil seleksi dari kelima belas isolat bakteri pada media agar-agar kitin menunjukkan beberapa isolat memiliki aktivitas kitinolitik. Tujuh isolat menghasilkan zona bening di sekitar koloni bakteri setelah 48 hingga 60 jam inkubasi. Uji penghambatan langsung dengan menggunakan metode dual culture pada isolat terpilih TSU4 dan TSU5 menunjukkan persentase penghambatan masing-masing sebesar 22,4% dan 16,1%, sedangkan metode food poison dengan filtrat kultur menunjukkan penghambatan masing-masing sebesar 28,1% dan 23,9%. Isolat terpilih Bacillus cereus TSU4 dievaluasi secara kuantitatif aktivitas enzim kitinase. Aktivitas kitinase memiliki peningkatan pada jam ke-48 dengan nilai aktivitas spesifik enzim kitinase yaitu 9,58 U/mg. Gen chi yang menyandikan kitinase pada isolat B. cereus TSU4 mendukung produksi kitinase. Enzim dari B. cereus TSU4 dimurnikan menggunakan ammonium sulfat, menghasilkan aktivitas spesifik 15,66 U/mg dan peningkatan kemurnian hingga 4,82 kali. Enzim kitinase yang telah dimurnikan dari isolat B. cereus TSU4 optimum pada pH 6 dan suhu 30 oC. Selain itu, enzim kitinase yang telah dimurnikan menggunakan amonium sulfat 70% dapat menghambat pertumbuhan miselia F. proliferatum sebesar 33,82%. Penghambatan ini dikonfirmasi dengan mengamati kerusakan hifa F. proliferatum menggunakan scanning electron microscope (SEM). Penelitian ini menyimpulkan bahwa isolat B. cereus TSU4 penghasil enzim kitinase memiliki potensi sebagai agens penghambat terhadap fungi F. proliferatum.

Plant disease control remains a challenging for plant pathology in sustainable agriculture. One of the main problems in agriculture is caused by phytopathogenic fungi such as Fusarium proliferatum. F. proliferatum fungi infect various plants, including vegetables and fruits, as well as 25 plant species. Conventional control of phytopathogenic fungi has been carried out using synthetic pesticides, but their application can lead to resistance in microorganisms and pose a risk to human, animal, and environmental health. Therefore, an alternative to reducing synthetic pesticides is to use soil-derived chitinolytic bacteria as biocontrol agents against phytopathogenic fungi. Chitinolytic bacteria can produce chitinase enzymes that hydrolyze ß-1,4-glycosidic bonds between N acetylglucosamine residues that make up the chitin chain. Chitin is one of the components of the cell walls of phytopathogenic fungi such as F. proliferatum. Therefore, this study aims to select soil-derived chitinolytic bacteria capable of producing chitinase enzyme, to assess their chitinase activity, and to determine their potential in inhibiting the growth of the phytopathogenic fungus F. proliferatum. This study was conducted using several methods: selecting isolates with qualitative chitinase enzyme activity, antagonistic activity against F. proliferatum, and pathogenicity against animals and plants. Two selected isolates out of the 15 isolates obtained from the isolation process were analyzed for their identity based on the 16S rRNA gene. Furthermore, one identified Bacillus isolate was used as a test isolate for quantitative measurement of chitinase enzyme activity. Additionally, the selected isolates were also tested for the presence of the chitinase enzyme gene (chi gene). The chitinase enzyme produced by bacteria was then precipitated using ammonium sulfate, and the optimum pH and temperature of the enzyme were measured. The activity of the precipitated chitinase enzyme was measured in terms of its inhibitory effect on F. proliferatum using the food poisoning assay method. Damage to the mycelium of F. proliferatum resulting from the antagonism test was observed using a light microscope and a scanning electron microscope (SEM). The results of the selection of fifteen bacterial isolates on chitin agar medium showed that several isolates had chitinolytic activity. Seven isolates produced clear zones around the bacterial colonies after 48 to 60 hours of incubation. Direct inhibition tests using the dual culture method on selected isolates, TSU4 and TSU5, showed inhibition percentages of 22,4% and 16,1%, respectively. In comparison, the food poisoning method using culture filtrates showed inhibition of 28.1% and 23.9%, respectively. The selected Bacillus cereus TSU4 isolate was quantitatively evaluated for chitinase enzyme activity. Chitinase activity increased at 48 hours with a specific chitinase enzyme activity value of 9,58 U/mg. The chi gene encoding chitinase in the B. cereus TSU4 isolate supports chitinase production. The enzyme from B. cereus TSU4 was purified using ammonium sulfate, resulting in a specific activity of 15.66 U/mg and an increased purity of up to 4,82 times. The purified chitinase enzyme from the B. cereus TSU4 isolate was optimal at pH 6 and a temperature of 30 °C. In addition, the chitinase enzyme purified by 70% ammonium sulfate precipitation inhibited the growth of F. proliferatum mycelium by 33.82%. This inhibition was confirmed by observing the damage to F. proliferatum hyphae using a scanning electron microscope (SEM). This study concluded that the B. cereus TSU4 isolate, which produces a chitinase enzyme, has potential as an inhibitory agent against the fungus F. proliferatum