Profil Mikrobiota Endofit Sayur Konsumsi Segar Daun Pohpohan (Pilea melastomoides (Poir.) Wedd.) Berbasis Analisis Metagenomik.

Abstract

Penelitian terbaru menunjukkan bahwa sayuran segar merupakan habitat utama komunitas bakteri endofit. Di Indonesia, beberapa tanaman tropis dikonsumsi segar tanpa melalui proses memasak, sehingga dapat menimbulkan kekhawatiran terhadap kesehatan manusia dan kesadaran keamanan pangan. Pilea melastomoides merupakan makanan etnik tradisional yang dikenal dengan Traditional and Etnic Food (TEF) dan dimanfaatkan oleh masyarakat Sunda di Indonesia sebagai sayuran untuk dikonsumsi segar. Tanaman ini dapat tumbuh secara liar dan dibudidayakan dengan standar pertanian, sehingga menimbulkan pertanyaan mengenai komunitas bakteri endofit diantara kedua jenis sistem tanam tersebut. Oleh karena itu, penelitian ini bertujuan untuk menyelidiki profil komunitas bakteri pada P. melastomoides liar dan budidaya berdasarkan pendekatan metagenomik dengan menargetkan gen 16S rRNA. Sampel daun P. melastomoides diperoleh dari kawasan hutan Taman Nasional Gunung Halimun Salak, Kabupaten Bogor, Jawa Barat. Teknik pengambilan sampel dilakukan secara cluster random sampling yang terbagi menjadi tiga petak. Metode pengumpulan sampel dilakukan menggunakan sample pooling pada masing-masing petak, yaitu dengan menggabungkan atau mencampur 10 sampel daun hasil ekstraksi P. melastomoides budidaya dan liar, masing-masing kedalam satu tabung (pool) untuk dilakukan pengujian sebagai sampel tunggal. Selanjutnya, sampel hasil ekstraksi DNA diamplifikasi melalui teknik PCR dengan target daerah amplifikasi V3-V4 menggunakan dua primer universal bakteri yaitu primer 341F dan primer 806R. Konstruksi library (perpustakaan) dilakukan untuk mempersiapkan sampel metagenom sebelum dilakukan sekuensing meliputi proses fragmentasi DNA, end conversion, adapter ligation, cleanup of adapter-ligated, library amplification, dan library quantitation. Kemudian perpustakaan metagenom diurutkan pada platform pair-end sequencing Illumina NovaSeq 6000 (2×250 bp). Data mentah hasil sekuensing (RawPE) dilakukan analisis bioinformatika. Analisis data mengungkapkan sampel daun pohpohan budidaya mempunyai keanekaragaman bakteri endofit tertinggi, ditunjukkan melalui indeks keanekaragaman dengan kekayaan jenis berdasarkan indeks Chao1 (0,0244), indeks Shannon (0,0291), dan kemerataan jenis berdasarkan indeks Pielou (0,0282). Filum yang paling melimpah adalah filum Firmicutes dan Proteobacteria. Beberapa bakteri endofit spesifik pada tanaman budidaya teridentifikasi sebagai Limosilactobacillus (5,38%), Escherichia/Shigella (4,96%), Bacillus (4,76%), Salmonella (3,57%), Staphylococcus (3,57%). Sebaliknya, Escherichia/Shigella (15,59%), Bacillus (10,48%), Limosilactobacillus (9,00%), Salmonela (7,78%), Listeria (5,94%), merupakan genus dominan pada tanaman liar. Analisis lebih lanjut perlu dilakukan konfirmasi untuk memastikan virulensi patogen potensial pada tanaman P. melastomoides liar maupun budidaya.

Kata kunci: mikrobioma, endofit, 16S rRNA, next-generation sequencing, Firmicutes

Recent studies exhibited that fresh vegetables are the major habitat for endophytic bacterial communities. In Indonesia, some of the tropical plants are consumed fresh without a cooking process, which may concern human health and food security awareness. Pilea melastomoides is a Traditional and Ethnic Food plant used by the Sundanese people in Indonesia as a vegetable for fresh consumption. This plant can be grown wild and cultivated in a standard plantation, thus raising the question of the endophytic bacterial communities between these two types of planting systems. Therefore, our research aimed to investigate the bacterial community profile of wild and cultivated P. melastomoides based on a metagenomic approach targeting the 16S rRNA gene. Samples of P. melastomoides leaves were obtained from the forest area of Mount Halimun Salak National Park, Bogor Regency, West Java. The sampling technique was carried out using cluster random sampling, divided into three plots. The sample collection method was carried out using sample pooling in each plot, namely by combining or mixing ten leaf samples extracted from cultivated and wild P. melastomoides into one tube (pool) for testing as a single sample. Next, the samples resulting from DNA extraction were amplified using the PCR technique targeting the V3-V4 amplification region using two universal bacterial primers, primer 341F and primer 806R. Library construction is carried out to prepare metagenome samples before sequencing, including the processes of DNA fragmentation, end conversion, adapter ligation, cleanup of adapter-ligated, library amplification, and library quantitation. Then, the metagenome library was sequenced on an Illumina NovaSeq 6000 paired-end sequencing platform (2×250 bp). The raw data from sequencing results (RawPE) was subjected to bioinformatics analysis. Data analysis revealed that leaf samples collected on cultivated plants had the highest diversity, as indicated by the diversity indices of species richness based on Chao1 (0,0244), Shannon (0,0291), and species evenness based on Pielou (0,0282). The most abundant phylum were phyla of Firmicutes and Proteobacteria. Some specific endophytic bacteria in cultivated plants were identified as Limosilactobacillus (5.38%), Escherichia/Shigella (4.96%), Bacillus (4.76%), Salmonella (3,57%), Staphylococcus (3.57%). In contrast, some genera of pathogens such as Escherichia/Shigella (15.59%), Bacillus (10.48%), Limosilactobacillus (9.00%), Salmonella (7.78%), Listeria (5.94%), are the dominant genus in wild plants. Further analysis should be carried out to confirm the virulence of those potential pathogens both in wild and cultivated P. melastomoides plants.

Keywords: microbiome, endophyte, 16S rRNA, next-generation sequencing, Firmicutes.