Penapisan Cendawan Laut sebagai Agens Pengendalian Hayati Colletotrichum acutatum pada Tanaman Cabai | Screening of Marine Fungal as Biological Control Agent of Colletotrichum acutatum on Chili Plants

Gejala antraknosa pada cabai berupa bercak coklat konsentris kehitaman pada permukaan buah dan lama kelamaan meluas menjadi busuk lunak atau mengering. Penyakit antraknosa sangat merugikan karena menurunkan hasil produksi. Berbagai pengendalian telah dilakukan oleh petani cabai, namun hasilnya belum optimal. Cendawan laut merupakan salah satu jenis mikrob yang memiliki jalur metabolik yang spesifik dan telah banyak digunakan sebagai sumber obat-obatan pada manusia. Banyak senyawa-senyawa metabolit yang dihasilkan oleh cendawan laut seperti benzopyranone, stemphyperylenol, pleosporalone A dan B, (-)-cercosporamide, dan sesquiterpenes yang memiliki aktivitas anticendawan. Oleh karena itu, cendawan laut berpotensi juga dijadikan sebagai agens pengendalian hayati pada tanaman. Penelitian ini bertujuan untuk mendapatkan cendawan laut yang berpotensi menghambat pertumbuhan C. acutatum dan menekan penyakit antraknosa pada cabai, serta mengidentifikasi isolat cendawan laut yang potensial sebagai agens pengendalian hayati. Penelitian dilaksanakan pada bulan Agustus 2019 sampai Januari 2022 di Laboratorium Mikologi Tumbuhan, Departemen Proteksi Tanaman, Fakultas Pertanian dan Laboratorium Mikrobiologi Hasil Perairan, Departemen Teknologi Hasil Perairan, Fakultas Perikanan dan Ilmu Kelautan, serta kegiatan uji in planta dilaksanakan di kebun percobaan Cikabayan, Institut Pertanian Bogor. Sebanyak 27 isolat cendawan laut dari koleksi Laboratorium Mikrobiologi Hasil Perairan, Institut Pertanian Bogor, diuji pada penelitian ini. Cendawan tersebut diisolasi dari tanaman pesisir pinggir pantai (daun magrove dan terong pungo), kayu apung, alga, dan rumput laut. Sebelum digunakan, isolat tersebut diremajakan dengan menggunakan media agar-agar dekstrosa kentang (ADK) yang dimodifikasi dengan penambahan NaCl. Penapisan potensi agens hayati dari koleksi cendawan tersebut dimulai dengan uji antagonisme 27 isolat koleksi tersebut dengan metode kultur biakan ganda (dual culture) pada media ADK. Penapisan selanjutnya dilakukan terhadap 15 isolat terpilih dari pengujian sebelumnya untuk mengetahui potensi produksi senyawa organik volatil dalam menekan pertumbuhan patogen. Pengujian ini menggunakan media ADK dengan konsentrasi 25, 50, 75, dan 100% dilakukan dengan cara menumbuhkan isolat cendawan patogen dan cendawan laut calon agens antagonis pada cawan yang berbeda dengan diameter yang sama, lalu keduanya saling ditangkupkan dan pertemuan kedua cawan ditutup dengan plastik perekat. Pertumbuhan patogen akan terhambat bila calon agens hayati tersebut menghasilkan senyawa volatil yang toksik bagi patogen. Dari 15 isolat terpilih tersebut selanjutnya juga diuji kultur filtratnya dalam menghambat pertumbuhan koloni dengan metode peracunan media. Konsentrasi kultur filtrat yang digunakan dalam pengujian ini adalah 0.25, 0.5, 0.75, dan 1%. Dari 15 isolat yang diuji dalam kemampuan produksi senyawa organik volatil dan kemampuan kultur filtratnya menghambat pertumbuhan koloni C. acutatum terpilih 6 isolat terbaik untuk diuji kemampuan filtratnya dalam menghambat perkecambahan konidia C. acutatum dan keamanan hayatinya. Dari hasil pengujian ini, kemudian dipilih 4 isolat untuk diuji kemampuannya dalam menghasilkan enzim kitinolitik dan menghambat perkembangan penyakit antraknosa pada buah cabai. Semua isolat yang telah diuji kemampuan menghambat perkembangan penyakit antraknosa pada buah cabai tersebut diidentifikasi, baik secara morfologi maupun molekuler. Satu diantara isolat tersebut yang memiliki kemampuan menghasilkan senyawa volatil dideteksi jenis senyawa yang dihasilkan dengan metode gas chromatography mass spectrometry (GC-MS). Dari 27 isolat yang diuji kemampuan menghambat pertumbuhan koloni C. acutatum diperoleh 8 isolat yang menunjukkan penghambatan relatif lebih dari 40%. Selanjutnya dari 15 isolat terpilih termasuk yang menunjukkan penghambatan lebih dari 40% tersebut, ternyata dapat menghasilkan senyawa volatil yang mampu menghambat pertumbuhan koloni C. acutatum. Penghambatan relatif yang ditunjukkan sangat bervariasi mulai dari 4.18 sampai 25.39% tergantung konsentrasi media yang digunakan. Kultur filtrat dari isolat yang terpilih memiliki kemampuan menghambat pertumbuhan koloni C. acutatum dengan tingkat penghambatan relatif yang bervariasi antara 8.64% dan 16.48% tergantung konsentrasi filtrat yang digunakan. Enam isolat terpilih, yaitu A4st1, A4st3, 4145, B3st2, GN322, dan Z521 kultur filtratnya mampu menghambat perkecambahan konidia. Perkecambahan konidia terendah ditunjukkan oleh isolat B3st2 yang berarti memiliki kemampuan tertinggi dalam menghambat perkecambahan konidia C. acutatum. Dari keenam isolat tersebut hanya 4 isolat, yaitu 4145, B3st2, GN322, dan Z521 tidak menyebabkan hemolisis yang ditandai dengan tidak terbentuknya zona bening pada media uji, sehingga isolat tersebut aman digunakan sebagai agens pengendalian hayati karena terbukti tidak berpotensi sebagai patogen terhadap mamalia. Dalam uji secara in planta pada buah cabai terhadap 4 isolat terpilih yang aman secara hayati tersebut diperoleh isolat B3st2 dan Z521 yang mampu menunjukkan insidensi penyakit terendah, yaitu masing-masing 20% dibandingkan dengan kontrol (75%). Perlakuan dengan kedua isolat tersebut juga menunjukkan diameter gejala antraknosa yang lebih rendah, yaitu 1.60 dan 0.94 mm, dibandingkan kontrol (5.18 mm). Selain itu, dalam uji aktivitas kitinolitik menunjukkan hasil bahwa keempat isolat cendawan laut tersebut mampu menghasilkan enzim kitinase. Satu isolat yang terpilih berdasar semua uji tersebut, yaitu B3st2, dapat memproduksi senyawa phenyl acetaldehyde, phenylethyl alcohol, 1-methylethyl, dan cyclohexanone berdasarkan uji GC-MS. Diantara senyawasenyawa yang dihasilkan tersebut, phenyl acetaldehyde dan cyclohexanone diketahui bersifat anticendawan. Identifikasi secara morfologi isolat 4145 memiliki ciri-ciri struktur miselium seperti beludru, warna koloni putih tampak bagian atas dan ungu bagian bawah, hifa hialin, septat, dan mikrokonidia aseptat, ciri-ciri tersebut diduga merupakan anggota genus Fusarium. Isolat B3st2memiliki ciri struktur miselium seperti kapas, warna koloni putih kehijauan tampak bagian atas dan kuning kehijauan bagian bawah, konidiofor hialin, dan konidia berbentuk bulat dan diduga merupakan genus Trichoderma. Isolat GN322 memiliki ciri struktur hifa seperti kapas, warna koloni putih tampak bagian atas dan putih kekuningan bagian bawah, hifa septat, makrokonidia panjang, mikrokonidia berbentuk oval, dari ciri tersebut diduga Fusarium. Isolat Z521 memiliki ciri struktur hifa seperti kapas, warna koloni putih tampak bagian atas dan bawah, hifa septat, dan mikrokonidia, dari ciri tersebut diduga Fusarium. Penyejajaran sikuen nukleotida cendawan hasil amplifikasi dengan database di GenBank, keempat isolat tersebut terkonfirmasi sebagai Fusarium proliferatum 4145, Trichoderma harzianum B3st2, fungal endofit GN322, dan Fusarium solani Z521.

Anthracnose is an important disease in red chili plants in Indonesia, which is caused by Colletotrichum acutatum. Symptom of anthracnose in chili is concentric blackish brown spots on the surface of the fruit, which gradually expand to become soft rot or dry out. Anthracnose disease is very detrimental because it reduces production yields. Various controls have been carried out by chili farmers, but the results have not been optimal. Marine fungal is a type of microbe having specific metabolic pathways and has been widely used as a source of drugs in humans. Many metabolic compounds produced by marine fungal such as benzopyranone, stemphyperylenol, pleosporalone A and B, (-)-cercosporamide, and sesquiterpenes, have antifungal activity. Therefore, marine fungal has the potential as a biological control agent in plants. This study aims to obtain marine fungal having the potential to inhibit the growth of C. acutatum, to suppress anthracnose disease in chilies, as well as to identify potential marine fungal isolates as biological control agents. The research was carried out from August 2019 to January 2022 at the Plant Mycology Laboratory, Department of Plant Protection, Faculty of Agriculture and Aquatic Product Microbiology Laboratory, Department of Aquatic Products Technology, Faculty of Fisheries and Marine Sciences. In addition, the in planta test activity was carried out in the Cikabayan experimental garden, Bogor Agricultural University. A total of 27 isolates of marine fungi from the collection of the Laboratory of Aquatic Microbiology, Bogor Agricultural University were tested in this study. The fungus was isolated from coastal plants (magrove leaves and eggplant pungo), driftwood, algae, and seaweed. Prior to use, the isolate was rejuvenated using potato dextrose agar (PDA) medium modified with the addition of NaCl. Screening of potential biological agents from the fungal collection was started by testing the antagonism of 27 isolates from the collection using the dual culture method on PDA media. Further screening was carried out on 15 selected isolates from previous tests to determine the potential for production of volatile organic compounds in suppressing the growth of pathogens. This test using PDA media with concentrations of 25, 50, 75, and 100% was carried out by growing isolates of pathogenic fungi and marine fungus candidates for antagonist agents in different plates with the same diameter, then both cupped together and sealed with adhesive plastic. . Pathogen growth will be inhibited if the candidate for biological agents produces volatile compounds that are toxic to the pathogen. Of the 15 selected isolates, were then tested their filtrate culture for inhibiting colony growth of C. acutatum using the media poisoning method. The filtrate culture concentrations used in this test were 0.25, 0.5, 0.75, and 1%. Among the 15 isolates tested for their ability to produce volatile organic compounds and their filtrate culture ability to inhibit the growth of C. acutatum colonies, 6 best selected isolates were further tested for their filtrate ability to inhibit the germination of C. acutatum conidia and their biosafety. From the results of this test, the best 4 isolates were then tested for their ability to produce chitinolytic enzymes and inhibit the development of anthracnose disease in chilies. All isolates that have been tested for their ability to inhibit the development of anthracnose disease on chili peppers were identified, both morphologically and molecularly. One of these isolates which has the ability to produce volatile compounds was detected the type of compounds produced using the gas chromatography mass spectrometry (GC-MS) method. Out of the 27 isolates tested for the ability to inhibit the growth of C. acutatum colonies, 8 isolates showed a relative inhibition of more than 40%. Furthermore, from 15 isolates were selected. including those that showed inhibition of more than 40%, turned out to be able to produce volatile compounds that were able to inhibit the growth of C. acutatum colonies. The relative inhibition shown varied from 4.18 to 25.39% depending on the concentration of the medium used. The filtrate culture of the selected isolates had the ability to inhibit the growth of C. acutatum colonies with a relative inhibition level that varied between 8.64% and 16.48% depending on the concentration of the filtrate used. The filtrate culture of six selected isolates, namely A4st1, A4st3, 4145, B3st2, GN322, dan Z521 were able to inhibit conidia germination. The lowest conidia germination was shown by isolate B3st2, which means it had the highest ability to inhibit the germination of C. acutatum conidia. Among of the six isolates, only 4 isolates, namely 4145, B3st2, GN322, and Z521 did not cause hemolysis which was indicated by the absence of a clear zone on the test media, so that these isolates were safe to be used as biological control agents since there were no potential indication as pathogens for mammals. In the in planta test on chili pepper fruits for 4 selected isolates that were biologically safe, 2 isolates, namely B3st2 and Z521, resulted the lowest disease incidensce, each was 20%, compared to the control (75%). The treatment with these two isolates also showed a lower diameter of anthracnose symptoms, namely 1.60 and 0.94 mm, compared to the control (5.18 mm). In addition, the chitinolytic activity test showed that the four selected marine fungus isolates were able to produce chitinase enzymes. One isolate that was selected based on all those above tests, namely B3st2, could produce phenyl acetaldehyde, phenylethyl alcohol, 1- methylethyl, and cyclohexanone compounds based on the GC-MS test. Among the compounds produced, phenyl acetaldehyde and cyclohexanone are known to be antifungal. Morphological identification of isolate 4145 revealed that the isolate had characteristics of mycelium structure such as the presence of velvet, the color of white colonies appeared on the top and purple on the bottom, hyaline hyphae, septat, and aseptat microconidia. These characteristics are owned by fungal belonging to the genus Fusarium. B3st2 isolate was characterized by a cotton-like mycelium structure, greenish-white colonies on the top and greenish-yellow on the bottom, hyaline conidiophores, and round conidia. Thus, the isolate was thought to be of the genus Trichoderma. GN322 isolate had hyphae structure characteristics like cotton, white colonies appeared on the top and yellowish white on the bottom, septat hyphae, long macroconidia, oval-shaped microconidia. Based on the characteristics, the isolate was suspected to be Fusarium. Z521 isolate had hyphae structure characteristics like cotton, white colonies appeared on the top and bottom, septate hyphae, and produced microconidia on conidiophore from these characteristics, it was suspected as Fusarium. Alignment of the amplified fungal nucleotide sequences with the database at GenBank, the four isolates were confirmed as Fusarium proliferatum 4145, Trichoderma harzianum B3st2, fungal endophytic GN322, and Fusarium solani Z521.