Potensi Aktinomiset Rizosfer Tanaman Liliaceae sebagai Agens Pengendali Hayati Penyakit Moler (Fusarium oxysporum f. sp. cepae) pada Bawang Merah

Abstract

Penyakit moler merupakan salah satu penyakit penting pada tanaman bawang merah yang disebabkan oleh Fusarium oxysporum f. sp. cepae. Patogen ini merupakan patogen tular tanah, yang dapat bertahan di tanah meskipun tidak ada inang. Pengendalian F. oxysporum f. sp. cepae dengan menggunakan fungisida sintetik merupakan pengendalian yang paling diminati oleh petani karena lebih praktis diaplikasikan dan mudah didapatkan. Namun, aplikasi fungisida sintetik ternyata belum efektif dalam mengendalikan penyakit moler. Selain itu, penggunaan fungisida sintetik yang tidak bijaksana dapat menyebabkan resistensi patogen. Keberhasilan aktinomiset dalam mengendalikan penyakit tular tanah telah banyak dilaporkan. Namun, pemanfaatan aktinomiset untuk mengendalikan penyakit moler belum banyak dilakukan. Aktinomiset merupakan penghasil antibiotik terbesar yang potensial dimanfaatkan dalam pengendalian penyakit tanaman. Penggunaan aktinomiset yang diisolasi dari tanaman Liliaceae diharapkan dapat menekan penyakit moler sekaligus memacu pertumbuhan tanaman. Penelitian ini bertujuan untuk mengevaluasi dan menyeleksi isolat-isolat aktinomiset yang dapat menekan pertumbuhan F. oxysporum f. sp. cepae sekaligus memacu pertumbuhan tanaman bawang merah. Penelitian terdiri dari empat tahap, yaitu: isolasi, seleksi, karakterisasi dan identifikasi isolat aktinomiset yang potensial. Isolat yang berhasil diisolasi diuji keamanan hayatinya melalui uji hipersensitivitas, uji patogenisitas dan uji hemolisis. Isolat yang lolos seleksi pada ketiga uji tersebut selanjutnya diuji kemampuan antagonismenya terhadap F. oxysporum f. sp. cepae melalui uji dual culture. Isolat yang memberikan penghambatan tertinggi diuji keefektifannya di rumah kaca. Isolat tersebut juga dikarakterisasi kemampuan fisiologinya dalam menghasilkan enzim kitinase, melarutkan fosfat, memproduksi IAA dan menambat nitrogen. Isolat-isolat potensial diidentifikasi secara molekuler menggunakan primer spesifik aktinomiset 27F dan 16Sact1114R. Isolat terbaik yang mampu menekan F. oxysporum f. sp. cepae baik secara in vitro maupun in vivo dianalisis kandungan senyawa bioaktifnya dengan menggunakan Gas Chromatography- Mass Spectrometry (GC-MS). Sebanyak 43 isolat aktinomiset berhasil diisolasi dari tanaman Liliaceae dan 14 isolat diantaranya tidak bersifat patogen pada tumbuhan dan mamalia. Pada pengujian in vitro, 14 isolat tersebut menghasilkan penghambatan terhadap F. oxysporum f. sp. cepae sebesar 3,67-53,67%. Sebanyak 13 isolat mampu memproduksi enzim kitinase dengan indeks kitinolitik sebesar 0,31-1,38. Berdasarkan hasil uji in vitro, sebanyak lima isolat yaitu: ABF 42, ABF 59, ACF 45, AEF 35 dan AEF 45 potensial untuk diuji secara in vivo.

Pada pengujian pemacuan pertumbuhan menggunakan metode ragdoll seed germination, isolat ABF 42, ABF 59, AEF 45 dapat meningkatkan daya berkecambah. Isolat ABF 42, ACF 45, AEF 35, AEF 45 dapat meningkatkan panjang hipokotil. Isolat ACF 45 dan AEF 45 dapat meningkatkan bobot basah bawang merah. Berdasarkan hasil pengujian di rumah kaca, perlakuan lima isolat potensial dapat menurunkan kejadian penyakit moler baik pada varietas Bauji maupun varietas Tuk Tuk. Penekanan penyakit tertinggi pada varietas Bauji ditunjukkan oleh perlakuan isolat ABF 59 dan ABF 42 yang diisolasi dari tanaman Zephyrantes rosea. Pada varietas Tuk Tuk penekanan penyakit tertinggi ditunjukkan oleh isolat AEF 45 yang diisolasi dari tanaman Hymenocallis littoralis. Isolat ABF 42, ABF 59 dan AEF 45 juga dapat meningkatkan pertumbuhan tanaman bawang merah. Lima isolat potensial memiliki karakter fisiologi dapat melarutkan fosfat dan menghasilkan IAA. Indeks pelarutan fosfat tertinggi ditunjukkan oleh isolat ACF 45 sebesar 2,06. Produksi IAA tertinggi ditunjukkan oleh isolat AEF 45 sebesar 82,88 ppm. Isolat ABF 42, ACF 45, dan AEF 45 dapat menambat nitrogen. Isolat ABF 59 merupakan isolat yang memberikan penghambatan yang tinggi terhadap F. oxysporum f. sp. cepae pada uji in vitro maupun in vivo. Berdasarkan hasil analisis GC-MS, sebanyak 50 senyawa diperoleh dari ekstrak kasar isolat ABF 59. Delapan senyawa dominan memiliki bioaktivitas sebagai antimikroba, antifungi, insektisida, dan herbisida. Berdasarkan sekuens gen 16S rRNA, lima isolat memiliki identitas berturut-turut teridentifikasi sebagai: Streptomyces rameus ABF 42, S. lydicus ABF 59, S. panaciradicis ACF 45, S. seoulensis AEF 35, dan S. fuscichromogenes AEF 45.

Fusarium basal rot is one of the major plant diseases of shallots in Indonesia caused by Fusarium oxysporum f. sp. cepae. This pathogen is soil-borne pathogen, which can survive in the soil even in the absence of the host. Chemical fungicides are the main methods used to control the disease because it is more practical to be applied and easier to be obtained. However, the unwise application of synthetic fungicides can cause pathogen resistance and will ultimately harmfull for the farmers and environment. The success of actinomycetes in controlling soil-borne diseases has been widely reported. However, the use of actinomycetes to control fusarium basal rot disease has not been carried out. Actinomycetes are the largest producer of antibiotics that have the potential to be used in controlling plant diseases. The use of actinomycetes isolated from Liliaceae plants is expected to suppress fusarium basal rot disease and stimulate plant growth. Actinomycetes have potential as biological control agents for F. oxysporum f. sp. cepae because of their ability to produce a number of bioactive compounds. This study aims to evaluate the potencies of actinomycete isolates to inhibit the growth of F. oxysporum f. sp. cepae in vitro, to control the basal rot disease in vivo, and potentially promote the growth of shallots. The study was divided into four stages: i.e. isolation, selection, characterization of potential isolates, and identification of potential actinomycetes isolates. The isolated actinomycetes were tested for their biosafety through hypersensitivity test, pathogenicity test, and hemolysis test. Isolates that did not react in the three tests were selected for their antagonism ability against F. oxysporum f. sp. cepae through dual culture test. The isolates which gave the highest inhibition were tested for their effectiveness in the greenhouse. The isolates were also characterized for their physiological ability to produce chitinase enzymes, dissolve phosphate, produce IAA and fixation of nitrogen. These isolates were also identified molecularly using actinomycete-specific primers 27F and 16Sact1114R. The best isolate which capable of suppressing F. oxysporum f. sp. cepae in vitro and in vivo was analyzed for bioactive compounds' content using Gas Chromatography-Mass Spectrometry (GC-MS). Forty-three isolates were successfully isolated from Liliaceae plants, 14 of which were not pathogenic to plant and mammalian. Those fourteen isolates were able to inhibit the growth of F. oxysporum f. sp. cepae by 3,67%-53,67%, and 13 isolates were able to produce the chitinase enzyme with their lysis index of 0,31- 1,38. Based on the results of in vitro tests, five isolates, namely: ABF 42, ABF 59, ACF 45, AEF 35 and AEF 45 were potential to be tested in vivo. In the growth promotion test using the ragdoll seed germination method, isolates ABF 42, ABF 59, AEF 45 could increase seed germination. Isolates ABF 42, ACF 45, AEF 35, AEF 45 can increase the hypocotyl length. ACF 45 and

AEF 45 isolates can increase the wet weight of shallots. Based on testing in a greenhouse, the treatment of five potential isolates could reduce disease incidence in both the Bauji and Tuk Tuk varieties. The highest disease suppression in the Bauji variety was shown by ABF 59 and ABF 42 treatment respectively. Both isolates were isolated from Zephyrantes rosea plant. The highest disease suppression was shown by AEF 45 treatment, which was isolated from the Hymenocallis littoralis plant on Tuk Tuk variety. ABF 42, ABF 59 and AEF 45 isolates can also increase the growth of shallot plants. From physiological characters, five potential isolates have the ability to dissolve phosphate and produce IAA. The highest phosphate solubilization index indicated by ACF 45 isolate, which was 2.06. AEF 45 isolates showed the highest IAA production 82,88 ppm. ABF 59 isolate was an isolate that gave high inhibition to F. oxysporum f. sp. cepae in both in vitro and in vivo tests. Based on GC-MS analysis, 50 compounds were obtained from the crude extract of isolate ABF 59. Eight dominant compounds have bioactivity as antimicrobial, antifungal, insecticide, and herbicide. Based on the 16S rRNA gene sequence, five isolates were identified as: Streptomyces rameus ABF 42, S. lydicus ABF 59, S. panaciradicis ACF 45, S. seoulensis AEF 35, and S. fuscichromogenes AEF 45.