Rosyida, Risya and Dr. Ir. Mintarto Martosudiro,, MS. and Dr. Anton Muhibuddin,, SP., MP. (2023) Analisis Enzim Kitinase Trichoderma sp. dalam Mendegradasi Fusarium oxysporum. Magister thesis, Universitas Brawijaya.
Abstract
RINGKASAN Risya Rosyida. 216040701111001. Analisis Enzim Kitinase Trichoderma sp. dalam Mendegradasi Fusarium oxysporum. Di bawah bimbingan Dr. Ir. Mintarto Martosudiro, MS. sebagai Pembimbing Utama dan Dr. Anton Muhibuddin, SP., MP. sebagai Pembimbing Pendamping. Penyakit layu fusarium pada cabai yang disebabkan oleh Fusarium oxysporum merupakan salah satu faktor pembatas penting untuk mendapatkan hasil panen cabai yang optimal. Pengendalian penyakit yang sudah dilakukan yaitu aplikasi fungisida yang mempunyai dampak negatif terhadap lingkungan. Penggunaan fungisida yang tidak dikelola dengan baik menyebabkan residu fungisida pada buah cabai dan dapat membahayakan kesehatan. Adanya residu fungisida dikarenakan senyawa benomil fungisida tidak terhidrolisis secara sempurna. Diperlukan alternatif pengendalian hayati untuk mengatasi ketergantungan dengan bahan kimia, salah satunya pemanfaatan enzim yang bisa mendegradasi patogen dari strain Trichoderma sp. Kemampuan Trichoderma sp. dalam menghasilkan enzim pendegradasi dinding sel (kitinolitik, selulolitik, dan glukanolitik) telah menjadi perhatian para peneliti. Mekanisme pengendalian cendawan antagonis terhadap F. oxysporum diawali dengan dinding hifa F. oxysporum mengalami degradasi, menyebabkan nutrisi didalam hifa patogen terserap sehingga menyebabkan hifa patogen menjadi pipih dan nutrisi dalam hifa akan keluar, dan terjadi kematian sel-sel hifa. Tujuan dari penelitian ini adalah untuk menganalisis enzim kitinase Trichoderma sp. pada pH media yang berbeda terhadap pengendalian F. oxysporum untuk mendapatkan mikroba yang potensial. Penelitian dilakukan dari Februari - Juni 2023 dengan rincian penelitian pendahuluan dilakukan mulai Februari - Maret 2023 dan penelitian utama dilakukan mulai Februari - Juni 2023. Seluruh kegiatan dilaksanakan di laboratorium penyakit tumbuhan. Pada penelitian pendahuluan, pembuatan koloidal kitin, pembuatan media PDA dan MGMK, peremajaan isolat, dan pembuatan reagen 3,5-dinitrosalicylic acid (DNS). Pada penelitian utama, skrining isolat Trichoderma sp. dengan metode dual culture, uji kitinase isolat Trichoderma sp. dengan reagen bromocresol purple, identifikasi molekuler isolat Trichoderma sp. yang terpilih, produksi enzim kitinase dan karakterisasi pada kondisi pH yang berbeda menggunakan media produksi enzim kitinase, uji aktivitas enzim kitinase, dan pengamatan penghambatan pertumbuhan miselium (perkecambahan sporanya) dari Fusarium oxysporum. Data dianalisis dengan menggunakan metode deskriptif dengan menampilkan gambar berupa dokumentasi dan grafik. Pengumpulan data untuk analisis menggunakan Microsoft Excel. Berdasarkan hasil uji patogenesitas, isolat F. oxysporum yang ditemukan dilapang menunjukkan gejala yang mirip dengan isolat yang di re-isolasi. Kenampakan makroskopis dan mikroskopis patogen juga sama. Kemudian skrining lima isolat Trichoderma sp., rata-rata persentase daya hambat kelima isolat Trichoderma sp. mengalami peningkatan. Daya hambat tertinggi pada 7 hsi yaitu isolat UBPK6 sebesar 76,71%. Mekanisme Trichoderma sp. yang ditemukan selama pengamatan yaitu mikoparasit, antibiosis, kompetisi. Selain itu, hasil uji reagen bromocresol purple kelima isolat Trichoderma sp. menunjukkan bahwa isolat tersebut dapat memproduksi enzim kitinase. Uji reagen bromocresol purple ditunjukkan pada perubahan warna pada media. Perubahan warna ditunjukkan dari kuning menjadi ungu pada pemberian reagen bromocresol purple, disebabkan vi adanya perubahan pH akibat reaksi enzim dari pH rendah menuju tinggi. Hasil daya hambat dan uji bromocresol yang tertinggi yaitu pada isolat UBPK6. Isolat tersebut yang nantinya digunakan sebagai perlakuan aktivitas enzim kitinase dan pengamatan daya hambat (ekstrak kasar kitinase). Identifikasi isolat UBPK6 dilakukan secara molekuler berdasarkan analisis genetika dengan menggunakan primer ITS1 dan ITS4. Hasil klasifikasi menggunakan BLAST disimpulkan bahwa sampel yang dianalisis adalah Trichoderma asperellum karena satu klade dengan sekuen-sekuen jamur T. asperellum. Isolat T. asperellum diukur aktivitas enzim kitinasenya, hasil pengujian inkubasi enzim kitinase menunjukkan masa inkubasi dari hari ke-1 sampai ke-7 berbeda-beda. Hasil yang diperoleh menunjukkan bahwa masa inkubasi optimum untuk produksi enzim adalah hari ke-4 dengan nilai aktivitas enzim sebesar 4,05 U/mL. Hal ini menunjukkan bahwa waktu tersebut merupakan waktu yang tepat untuk dilakukan pemanenan enzim. Selanjutnya pengaruh pH terhadap aktivitas enzim kitinase jamur T. asperellum. Hasil uji aktivitas enzim kitinase dengan spektrofotometer UV-Vis menunjukkan bahwa nilai aktivitas optimum dihasilkan pada perlakuan media pH 5 suhu ruangan (yaitu ±27oC) dengan nilai aktivitas enzim sebesar 3,4 U/mL dan menurun setelahnya, aktivitas enzim dengan nilai terendah pada pH 9 sebesar 2,65 U/mL. Kemudian yang terakhir, kemampuan daya hambat ekstrak kasar kitinase jamur T. asperellum terhadap patogen F. oxysporum in vitro. Perlakuan pH 5 paling baik dalam menghambat pertumbuhan patogen dengan nilai daya hambat sebesar 60,63% pada pengamatan ke-7 setelah inkubasi. Pada pH 9 mempunyai kemampuan paling rendah dalam menghambat patogen F. oxysporum sebesar 37,50%. Begitu pula dengan pengamatan penghambatan perkecambahan spora F. oxysporum (pada pH 5). Perlakuan pH 5 mempunyai kandungan enzim paling tinggi sehingga bisa mendegradasi perkecambahan spora patogen lebih tinggi. Semakin tinggi kandungan enzim kitinase maka semakin tinggi juga kemampuannya dalam mendegradasi kerusakan perkecambahan spora patogen, sehingga menyebabkan pertumbuhan patogen terhambat.
English Abstract
SUMMARY Risya Rosyida. 216040701111001. Chitinase Enzyme Analysis of Trichoderma sp. in Degrading Fusarium oxysporum. Supervised by Dr. Ir. Mintarto Martosudiro, MS. and Dr. Anton Muhibuddin, SP., MP. Fusarium wilt disease in chili caused by Fusarium oxysporum is one of the important limiting factors to obtain optimal chili yields. Disease control that has been carried out is the application of fungicides which have a negative impact on the environment. The use of fungicides that are not managed properly causes fungicide residues on chilies and can endanger health. The presence of fungicide residues was due to the fact that the benomyl fungicide compound was not completely hydrolyzed. Alternative biological control is needed to overcome dependence on chemicals, one of which is the use of enzymes that can degrade pathogens from Trichoderma sp. The ability of Trichoderma sp. in producing cell wall degrading enzymes (chitinolytic, cellulolytic, and glucanolytic) has become the concern of researchers. The mechanism for controlling antagonistic fungi against F. oxysporum begins with the hyphae walls of F. oxysporum degrading, causing the nutrients in the pathogenic hyphae to be absorbed causing the pathogenic hyphae to become flat and the nutrients in the hyphae will come out, and the hyphae cells die. The purpose of this study was to analyze the chitinase enzyme Trichoderma sp. at different pH media against F. oxysporum control to get potential microbes. The research was conducted from February - June 2023 with details of preliminary research conducted from February - March 2023 and main research conducted from February - June 2023. All activities are carried out in the plant disease laboratory. In the preliminary research, the preparation of colloidal chitin, the preparation of PDA and MGMK media, the rejuvenation of isolates, and the preparation of 3,5-dinitrosalicylic acid (DNS) reagent. In the main study, screening of Trichoderma sp. isolates using the dual culture method, chitinase test of Trichoderma sp. isolates with bromocresol purple reagent, molecular identification of Trichoderma sp. selected, chitinase enzyme production and characterization at different pH conditions using chitinase enzyme production media, chitinase enzyme activity test, and observation of inhibition of mycelium growth (spore germination) from Fusarium oxysporum. Data were analyzed using descriptive methods by displaying images in the form of documentation and graphics. Data collection for analysis using Microsoft Excel. Based on the results of the pathogenicity test, F. oxysporum isolates found in the field showed similar symptoms to the re-isolated isolates. The macroscopic and microscopic appearance of the pathogen is also the same. Then screening of the five Trichoderma sp. isolates, the average percentage of inhibition of the five Trichoderma sp. isolates increased. The highest inhibition at 7 hsi was UBPK6 isolate of 76.71%. Mechanism of Trichoderma sp. found during the observation, namely mycoparasites, antibiosis, competition. In addition, the results of the bromocresol purple reagent test for the five isolates of Trichoderma sp. showed that these isolates can produce chitinase enzymes. The bromocresol purple reagent test is shown on the color change on the media. The color change was shown from yellow to purple in the administration of bromocresol purple reagent, viii due to a change in pH due to enzyme reactions from low to high pH. The results of the highest inhibition and bromocresol tests were on UBPK6 isolates. The isolates were later used as a treatment for chitinase enzyme activity and observation of inhibition (crude chitinase extract). Identification of UBPK6 isolates was carried out molecularly based on genetic analysis using ITS1 and ITS4 primers. . The results of the classification using BLAST concluded that the sample being analyzed was Trichoderma asperellum because it is in the same clade as the sequences of the fungus T. asperellum. Chitinase enzyme activity was measured for T. asperellum isolates, the results of the chitinase enzyme incubation test showed that the incubation period from day 1 to day 7 was different. The results obtained indicated that the optimum incubation period for enzyme production was the 4th day with an enzyme activity value of 4.05 U/mL. This shows that this time is the right time for harvesting enzymes. Furthermore, the effect of pH on the chitinase enzyme activity of T. asperellum fungus. The results of the chitinase enzyme activity test with a UV-Vis spectrophotometer showed that the optimum activity value was produced in the pH 5 media treatment at room temperature (ie ±27oC) with an enzyme activity value of 3.4 U/mL and decreased thereafter, the lowest enzyme activity at pH 9 was 2.65U/mL. Then finally, the inhibitory ability of the chitinase crude extract of the fungus T. asperellum against the pathogen F. oxysporum in vitro. The pH 5 treatment was the best in inhibiting the growth of pathogens with an inhibition value of 60.63% on the 7th observation after incubation. At pH 9 it has the lowest ability to inhibit the pathogen F. oxysporum by 37.50%. Likewise with the observation of inhibition of germination of F. oxysporum spores (at pH 5). The pH 5 treatment had the highest enzyme content so that it could degrade the germination of pathogenic spores. The higher the content of the chitinase enzyme, the higher its ability to degrade damage to germination of pathogenic spores, thus causing the growth of the pathogen to be inhibited.
| Item Type: | Thesis (Magister) |
|---|---|
| Identification Number: | 042304 |
| Divisions: | S2/S3 > Magister Ilmu Tanaman, Fakultas Pertanian |
| Depositing User: | Unnamed user with username saputro |
| Date Deposited: | 10 Jan 2024 04:05 |
| Last Modified: | 10 Jan 2024 04:05 |
| URI: | http://repository.ub.ac.id/id/eprint/207268 |
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