Ekologi Sianobakter Penambat N2 di Persawahan Organik melalui Pendekatan Molekuler

Abstract

Lahan persawahan menopang kehidupan beragam mikroorganisme, termasuk sianobakter penambat N2. Melalui metode kultur, penelitian ekologi sianobakter persawahan telah banyak diteliti di beberapa negara, seperti Banglades, Iran, India, dan Thailand. Berbagai penelitian tersebut menggambarkan keragaman sianobakter sebagai salah satu komponen penting komunitas mikrob tanah sawah beserta peran dan manfaatnya dalam mendukung pertumbuhan padi. Indonesia memiliki ekosistem sawah yang sangat beragam, namun penelitian ekologi sianobakter persawahan di Indonesia masih sulit didapatkan. Perkembangan ilmu dan teknologi dalam bidang molekuler dengan metode Denaturing Gradient Gel Electrophoresis (DGGE) kini memungkinkan penelitian diversitas, komposisi, dan peran mikrob tanah secara lebih akurat, mendekati sistem yang terjadi di alam. Penelitian ini ditujukan untuk memberikan informasi mengenai keragaman, struktur komunitas dan dinamika populasi sianobakter penambat N2 selama fase pertumbuhan padi. Selain itu, untuk mengkaji kesesuaian hasil yang diperoleh dari metode kultur dengan metode DGGE. Kajian ini penting dilakukan karena secara umum metode kultur masih diandalkan pada banyak laboratorium di berbagai negara. Penelitian ini juga ditujukan untuk menjawab pertanyaan mengenai peran sianobakter sebagai alternatif sumber nitrogen di lahan persawahan untuk mengurangi konsumsi pupuk nitrogen kimia. Sampel tanah yang diteliti adalah sampel tanah persawahan yang berasal dari lahan persawahan organik yang ada di Desa Serang Mekar, Kecamatan Ciparay, Kabupaten Bandung Selatan, Provinsi Jawa Barat. Pengambilan sampel tanah dilakukan pada bulan Januari-Maret 2018, meliputi dua fase vegetatif (40 dan 60 hari setelah tanam (hst)) dan dua fase generatif (80 dan 110 hst). Tanah sedalam 0-5 cm diambil dari setiap titik pengambilan sampel yang telah ditetapkan. Analisis sampel tanah meliputi enumerasi koloni sianobakter penambat N2, identifikasi isolat secara morfologi dan molekuler (menggunakan marka gen 16S rRNA dan nifH), kelimpahan dan frekuensi relatif spesies, rekonstruksi pohon filogeni isolat, dan interaksi antara sianobakter dan padi. Berdasarkan metode kultur ditemukan 23 morfospesies sianobakter penambat N2 di sawah organik. Sianobakter berfilamen lurus (Ordo Nostocales) lebih banyak daripada sianobakter filamen bercabang (Ordo Stigonematales). Kelimpahan sianobakter penambat N2 di sawah mengalami perubahan dari satu fase tumbuh padi ke fase lain dan mencapai puncak pada hari ke-80 setelah tanam (194 x 106 cfu/g). Pada setiap fase tumbuh padi, terdapat satu populasi yang mendominasi lahan persawahan. Di sisi lain terjadi penurunan jumlah spesies. Spesies di awal padi tumbuh (40 hst) berjumlah 17, kemudian menurun seiring pertumbuhan padi, yaitu 15 (60 hst), 13 (80 hst), dan 7 spesies (110 hst). Frekuensi kehadiran spesies bervariasi, antara 6.25% sampai dengan 93.75%. Jumlah spesies dengan frekuensi kehadiran rendah lebih banyak dibandingkan dengan spesies lain yang memiliki frekuensi kehadiran tinggi. Populasi CSO2 paling sering ditemukan (93.75%), diikuti oleh CSO7 (87.5%), CSO6 (75%), dan CSO3 (50%). Metode DGGE berhasil memberikan informasi yang lebih luas terkait komunitas bakteri penambat N2 persawahan. Sejumlah 28 Operational Taxonomic Unit (OTU) bakteri penambat N2 ditemukan. Berdasarkan 11 OTU yang berhasil disekuen, komunitas bakteri penambat N2 yang ditemukan di sawah organik diwakili oleh filum Proteobakteri (45%), Sianobakter (45%), dan Firmicutes (10%). Rekonstruksi pohon filogeni gen nifH memperlihatkan bahwa hubungan kekerabatan sianobakter penambat N2 lebih dekat dengan filum Firmicutes (bakteri gram positif) dibandingkan dengan filum Proteobakteri (bakteri gram negatif). Komunitas proteobakteri yang ditemukan memiliki hubungan kekerabatan dengan Azoarcus olearius, Methylocystis bryophila, dan Bradyrhizobium valentinum. Satu OTU dari filum Firmicutes berkerabat dengan Heliobacterium modesticaldum. Temuan ini menarik untuk diteliti lebih lanjut karena gen-gen nifH dan nifD H. modesticaldum diduga sebagai suatu percabangan baru (new lineage) dalam evolusi gen-gen penyintesa enzim nitrogenase. Komunitas metagenom sianobakter penambat N2 didominasi oleh populasi berfilamen lurus (Ordo Nostocales) dan berada dalam cabang yang sama dengan isolat-isolat yang berhasil dikultur dalam penelitian ini, yaitu strain CSO3, CSO4, dan CSO21. Berdasarkan rekonstruksi pohon filogeni gen 16S rDNA strain CSO3 memiliki hubungan kekerabatan dekat dengan Desmonostoc spp. (bootstrap 94 dan 87), sedangkan CSO21 berada satu cabang dengan Roholtiella mojaviensis (bootstrap 60). Strain CSO4 teridentifikasi sebagai Aliinostoc morphoplasticum (bootstrap 100). Keberadaan Desmonostoc, Roholtiella, dan A. morphoplasticum sebagai salah satu populasi sianobakter penambat N2 di lahan sawah merupakan informasi yang belum pernah dilaporkan dalam penelitian-penelitian terdahulu dan menjadi kebaruan penelitian ini (new record). Kelimpahan sianobakter di lapisan tanah yang bersifat anoksik dan minim cahaya (2-5 cm) lebih tinggi dibandingkan dengan lapisan tanah yang lebih luar (0-1 cm). Hal tersebut menarik untuk dipahami mengingat sebagian besar spesies sianobakter bersifat fotoautotrof sehingga seharusnya lebih banyak ditemukan pada permukaan tanah. Fenomena ini menunjukkan bahwa sianobakter mampu hidup pada lapisan tanah yang lebih dalam. Fakultatif sianobakter mampu memanfaatkan sumber cadangan karbonnya yang disintesis selama proses fotoasimilasi. Adaptasi lain adalah dengan meningkatkan volume membran tilakoid dengan membentuk lipatan-lipatan yang lebih banyak sehingga mampu mempertahankan energi (ATP) yang diperlukan untuk metabolisme sel. Sianobakter juga dapat menghasilkan lendir (mucilagenous sheath) untuk melindungi sel dari kekeringan dan memastikan kecukupan oksigen dalam mikrohabitatnya. Inokulasi strain sianobakter tidak berpengaruh terhadap tinggi padi, jumlah anakan, dan biomassa, tetapi berpengaruh terhadap kadar nitrogen tanaman dan jumlah akar lateral. Padi mengandalkan ketersediaan nitrogen dalam jaringan selama masa pematangan biji, sehingga kandungan nitrogen tanaman sangat penting. Serapan nitrogen tanaman yang diberi inokulan sianobakter lebih tinggi dibandingkan dengan kontrol. Hal ini menyiratkan bahwa ketersediaan nitrogen pada perlakuan yang diberi inokulan sianobakter lebih tinggi daripada yang tidak diberi inokulan. Tanaman padi yang diberi inokulan sianobakter memiliki jumlah akar lateral (per cm akar) lebih banyak daripada kontrol. Memiliki akar lateral yang banyak menjadi keuntungan tersendiri bagi tanaman padi karena dapat menyerap lebih banyak nutrien.

Rice field is one of ecosystems that inhabit many microorganisms, including the nitrogen-fixing (N2-fixing) cyanobacteria. The ability of N2-fixing cyanobacteria to reduced dinitrogen (N2) into amonium and their abundance in rice field are considered as advantages for paddy plants. Through exploration, isolation, and culturing, the diversity, function, and potential use of N2-fixing cyanobacteria are extensively studied in some countries, like Bangladesh, Iran, India, and Thailand. On the other hand, such research in Indonesia is very limited, amid the reality that this country is rich with rice field ecosystems. The development of molecular techniques opens a new opportunity to carry out the ecological study of cyanobacteria in their natural habitat more accurately. One of the techniques is Denaturing Gradient Gel Electrophoresis (DGGE). The present study is parted into three sections: 1). Community analyses of N2-fixing cyanobacteria communities through culture-based method; 2). Metagenomic data analyses of N2-fixing cyanobacteria using DGGE method; 3). Interaction between selected cyanobacteria and paddy plants cultured under hydroponic system. The first and second sections of the study were aimed to explain the diversity, community structure, and population dynamic of N2-fixing cyanobacteria during rice growth. Many research of rice field cyanobacteria community on many countries are still depended on culture-based technique, so that it is of important to understand how the data resulted from culture-based technique (section 1) correspond to DGGE data (section 2). The third section of this study was aimed to analyze the potency of N2-fixing cyanobacteria as nitrogen source for paddy plant. Soil samples used in this study were collected from organic rice field, belongs to Sarinah Organic Foundation, which was located at Serang Mekar Village, Ciparay District, West Java Province, Indonesia. Samples were collected on January-March 2018, following the vegetative (40 and 60 days after planting/dap) and generative phase of rice growth (80 and 110 dap). Soils from 0-5 cm depth layers were taken using plastic cylinder and brought into laboratory, while kept on a cooler box. Data analyses of N2-fixing cyanobacteria from soil samples including colony enumeration, isolation and purification of growth colonies, morphological and molecular identification (using 16S rRNA and nifH genes), community structure (relative abundance and frequency of species), phylogenetic tree reconstruction, and application of selected strains of cyanobacteria to paddy growth under hydroponic system. The abundance of N2-fixing cyanobacteria was changing from time to time following the rice growth. At each rice phase, a specific population dominated the rice field. Population reached peak at 80 dap (194 x 106 cfu/g). On the other hand, the number of species was decreased from 17 (at 40 dap) into 15 (60 dap), 13 (80 dap), and finally 7 (110 dap). The straight-filamentous cyanobacteria (Ordo Nostocales) was found to be more numerous than branched-filamentous (Ordo Stigonematales). The relative frequency of species was varied between 6.25% and 93.75%. The number of species with low relative frequency was greater than those with high frequency. Strain of CSO2 was always found at soil during vegetative and generative phase so that its relative frequency was high (93.75%). The strain of CSO7 was also frequently found (87.5%), followed by CSO6 (75%), and CSO3 (50%). Based on relative frequency and population abundance, strains of CSO2 and CSO7 were dominant populations on the rice field. Phylogenetic tree reconstruction based on 16S rRNA gene showed that strain of CSO2 was closed to Halotia wernerae, while CSO7 to Hapalosiphon welwetschii and Westiellopsis prolifica. Strain of CSO6 was on the same branch with Nostoc ellipsosporum (bootstrap 93), while CSO3 with Desmonostoc spp (94). One strain was found to be identic with sequence of Aliinostoc morphoplasticum (bootstrap 100), which was strain of CSO4. The occurrence of A. morphoplasticum in rice field has never been reported before so that this is a new record of A. morphoplasticum. The performance of DNA bands from DGGE gels showed 28 Operational Taxonomic Unit (OTUs) of N2-fixing bacteria. However, only 11 OTU had been successfully sequenced. Community of N2-fixing bacteria found in rice field consisted of 3 Phylums of Proteobacteria (45%), Cyanobacteria (45%), and Firmicutes (10%). Two OTUs belonged to alfa-Proteobacteria, three OTUs to beta-Proteobacteria, five OTUs to Cyanobacteria, dan one OTU to Firmicutes. Phylogenetic tree reconstruction of nifH gene showed that the proteobacteria community found in rice field had relationship with Azoarcus olearius, Methylocystis bryophila, and Bradyrhizobium valentinum. One OTU from Phylum Firmicutes was closed to Heliobacterium modesticaldum. The tree also showed that relationship between Cyanobacteria-Firmicutes was closer than Cyanobacteria-Proteobacteria. Population dynamic of N2-fixing cyanobacteria, based on DGGE, showed that species was rich at vegetative phase then decrease at generative phase. There was no difference between populations found at 0-1 cm and 2-5 cm depth. However, populations were abundance at 2-5 cm compared to 0-1 cm depth. The adaptation of cyanobacteria at low light condition had been tested on laboratory and it was related to a mechanism of multiplying thylakoid membrane to gain much ATP. Another strategy of cyanobacteria living on deep soil layer was production of mucilaginous sheat that could keep oxygen from their microhabitat. Application of three cyanobacteria strains into paddy cultivation system concluded that cyanobacteria alone could not substitute the exogenous nitrogen addition for paddy growth, an evidence which was well-documented from literature’s studies. Cyanobacteria had significant influence on the nitrogen concentration of paddy, but not to the plant height, number of tillers, and biomass. Nitrogen deposit as implied through nitrogen concentration is important for paddy when entering the phase of seed maturation. Nitrogen uptake of the plants inoculated with cyanobacteria was higher than control, indicating that nitrogen availability in cyanobacteria-treatments were higher too. The paddy inoculated with cyanobacteria had more lateral root than control. Hence, the result suggested that cyanobacteria influenced paddy growth through nitrogen deposition and formation of lateral root