Kemampuan pembintilan dan penambatan nitrogen isolat-isolat bakteri gram positif

SETIATI, Catarina Retno Herrani, Ir. Donny Widianto, Ph.D

2009 | Tesis | S2 Bioteknologi

Legume Nodulating Bacteria (LNB) yang secara umum dikenal sebagai rhizobia memiliki kemampuan bersimbiosis dengan legum, menambat N2 udara menjadi NH4. Hingga saat ini belum pernah ditemukan adanya LNB Gram positif yang mampu membentuk bintil akar dan menambat N2 simbiotik pada legum. Berdasarkan hasil isolasi rhizobia dari 7 sampel tanah agroekosistem di Sumber Jaya-Lampung diperoleh 50 isolat-isolat bakteri yang diantaranya masuk pada golongan bakteri Gram positif. Penelitian ini bertujuan untuk melakukan karakterisasi isolat LNB yang masuk pada golongan Gram positif dalam membentuk bintil dan menambat nitrogen, analisa keragaman gen nifH serta identifikasi berdasarkan sekuen gen 16S rRNA. Sebanyak 50 isolat bakteri diuji kemampuan pembintilannya pada Macroptilium atropurpureum DC. Aktivitas nitrogenase ditentukan dengan Acetylene Reduction Assay. Isolat-isolat yang menunjukkan aktivitas nitrogenase tinggi diuji sifat Gramnya dan diuji kembali kemampuan pembintilan serta aktivitas nitrogenasenya pada Vigna sinensis, Vigna sp., Vigna unguiculata L., Phaseolus radiatus L., Phaseolus vulgaris L., dan Glycine max. Keragaman gen nifH ditentukan dengan teknik PCR-RFLP dan pola polimorfismenya dianalisis menggunakan metode Unweight Pair Group with Mathematical Average (UPGMA) dengan program NTSYS. Analisis hasil sekuensing gen 16S rRNA dibandingkan dengan data yang ada di Gene Bank menggunakan program BLAST. Dari 50 isolat yang diuji diperoleh 2 isolat (UGM2 dan UGM3) yang merupakan bakteri Gram positif. Isolat UGM2 dan UGM3 mampu membentuk bintil akar dan menambat N2 pada Macroptilium atropurpureum DC., Vigna sinensis, Vigna sp., Vigna unguiculata L., dan Phaseolus radiatus L., namun tidak mampu membentuk bintil akar dan menambat N2 pada Phaseolus vulgaris L. dan Glycine max. Isolat UGM2 menunjukkan aktivitas nitrogenase tertinggi pada Vigna sinensis, sedangkan isolat UGM3 pada Vigna sp. Isolat UGM3 memiliki tingkat kemiripan gen nifH yang tinggi dengan isolat-isolat Rhizobia Gram negatif, sedangkan isolat UGM2 tingkat kemiripannya lebih rendah. Hasil analisis sekuensing 16S rRNA menunjukkan isolat UGM2 memiliki kemiripan 99% dengan Bacillus cereus strain MK30 (Accession:EU272862) dan isolat UGM3 memiliki kemiripan 99% dengan Bacillus amyloliquefaciens strain JY-863 (Accession:EU981825).

Legume Nodulating Bacteria (LNB), known collectively as rhizobia have ability to form symbiosis with legume, fix and reducing atmospheric nitrogen (N2) to biologically available ammonium (NH4). Until recently, there is not Gram positive LNB that can form nodule and fix N2 symbiotic with legume. Based on isolation product from 7 soil samples from Sumber Jaya-Lampung was obtained isolates expected gram positive that can form nodule and fix N2 symbiotic with legume. The aim of this research was to characterize Gram positive isolates based on their ability to form nodule, fix nitrogen, analize nifH gene diversity and identification of 16S rRNA gene sequence. Fifty (50) isolates were tested their ability to nodulate Macroptilium atropurpureum DC. Nitrogenase activity was measures by using Acetylene Reduction Assay. They classified as having high nitrogenase activity were tested with Gram staining and subsequently tested their ability to nodulate Vigna sinensis, Vigna sp., Vigna unguiculata L., Phaseolus radiatus L., Phaseolus vulgaris L., and Glycine max as well as their nitrogenase activity. Their nifH diversity was determined by PCR-RFLP technique and the polymorphism patterns obtained was analyze by using Unweight Pair Group with Mathematical Average (UPGMA) with NTSYS programme. The 16S rRNA gene partial sequences were compared with those from Gene Bank using BLAST programme. This research obtained 2 Gram positive isolates (UGM2 and UGM3) from 50 isolates. Gram positive isolates UGM2 and UGM3 performed ability to nodulate and fixed N2 on Macroptilium atropurpureum DC., Vigna sinensis, Vigna sp., Vigna unguiculata L., dan Phaseolus radiatus L., but they could not nodulated and fixed N2 on Phaseolus vulgaris L. dan Glycine max. UGM2 isolate showed high level nitrogenase activity at Vigna sinensis , whereas UGM3 isolate at Vigna sp. UGM3 isolate had high level similarity of nifH gene with Gram negative Rhizobia isolates, whereas UGM2 had lower level similarity of nifH gene. The result of 16S rRNA gene sequencing showed that UGM2 had 99% similarity with Bacillus cereus strain MK30 (Accession:EU272862) and UGM3 had 99% similarity with Bacillus amyloliquefaciens strain JY-863 (Accession:EU981825).

Kata Kunci : Bakteri pembentuk bintil,Isolat bakteri gram positif,Gen nifH,Aktivitas nitrogenase,legume nodulating bacteria, gram-positive bacteria isolate, nifH gene, nitrogenase activity