Eksplorasi Bakteri Rhizosfer Tanaman Kopi (Coffea Spp.) Di Agroforestri Ub Forest Kabupaten Malang Sebagai Agen Plant Growth Promoting Rhizobacteria (Pgpr)

Yuliatin, Ervinda (2020) Eksplorasi Bakteri Rhizosfer Tanaman Kopi (Coffea Spp.) Di Agroforestri Ub Forest Kabupaten Malang Sebagai Agen Plant Growth Promoting Rhizobacteria (Pgpr). Magister thesis, Universitas Brawijaya.

Abstract

Kopi (Coffea sp.) berperan penting dalam sektor perekonomian secara global. Universitas Brawijaya Forest (UB Forest) mengelola dan mengembangkan kopi sebagai produk utaman. Produksi kopi dari tahun ke tahun mengalami penurunan disebabkan oleh faktor kesuburan tanah dan cuaca yang kurang mendukung. Intensifikasi produksi kopi di UB Forest dilakukan dengan memanfaatkan pupuk organik sebagai penyubur tanah perkebunan kopi namun memerlukan proses yang cukup lama untuk mengembalikan kesuburan tanah. Alternatif untuk mengatasi permasalahan ini yaitu dengan memanfaatkan mikrobia tanah rhizosfer tanaman kopi sebagai agen biofertilizer penghasil hormon IAA, pemfiksasi N2, dan pelarut fosfat untuk mendukung pertumbuhan tanaman. Aktivitas bakteri tersebut kemudian dapat dimanfaatkan sebagai pelengkap nutrisi pupuk organik. Oleh karena itu isolasi, analisis keragaman, uji potensi, dan identifikasi bakteri tanah rhizosfer yang berperan sebagai agen Plant Growth Promoting Rhizobacteria (PGPR) perlu diteliti guna mendukung sistem perkebunan kopi berkelanjutan, produktivitas tinggi, dan ramah lingkungan. Tujuan penelitian ini untuk menganalisis pengaruh parameter lingkungan terhadap bakteri rhizosfer, mempelajari struktur komposisi bakteri rhizosfer, potensi bakteri rhizosfer sebagai kandidat PGPR dalam menambat nitrogen, melarutkan fosfat, memproduksi Indole Acetic Acid (IAA), dan mengidentifikasi isolat yang berpotensi tertinggi sebagai agen PGPR berdasarkan sekuen 16S rDNA. Penelitian ini meliputi analisis fisikokimia tanah, analisis metagenomik 16S rDNA bakteri tanah, isolasi bakteri rhizosfer kandidat PGPR, uji kemampuan isolat bakteri rhizosfer sebagai agen PGPR, dan identifikasi isolat terpilih. Isolatisolat PGPR diisolasi menggunakan metode seri pengenceran secara pour plate dalam media TSA, Nfb dan Pikovskaya, secara berurutan untuk penghasil IAA, penambat nitrogen, dan pelarut fosfat. Densitas setiap isolat kandidat PGPR dienumerasi secara TPC dan dianalisis indeks diversitas bakteri kandidat PGPR dari setiap sampel. Data parameter lingkungan, diversitas dan densitas isolat kandidat PGPR dianalisis multivariat (PCA) untuk menentukan hubungan parameter lingkungan dan densitas isolat kandidat PGPR. Setiap isolat kandidat PGPR diuji potensinya sebagai penghasil IAA, penambat nitrogen, dan pelarut fosfat. Konsentrasi hormon IAA diukur secara kolorimetri dengan uji Salkowski, konsentrasi amonium secara kolorimetri diukur dengan Sera Ammonia Test Kit dan Reagen Nessler, sedangkan konsentrasi fosfat diukur secara kolorimetri dengan Reagen Amoniummolibdat dan asam Klorostan. Potensi setiap isolat kandidat PGPR dalam menghasilkan IAA, menambat nitrogen, dan melarutkan fosfat diuji menurut percobaan Rancangan Acak Lengkap (RAL) dengan tiga kali ulangan. Data dianalisis ragam menggunakan program Windows SPSS software v. 20. Isolat PGPR unggul diidentifikasi berdasarkan similaritas sekuen 16S rDNA hasil amplifikasi dengan primer 27f (5’-GAG AGT TTG CTG GCT CAG- 3’) dan 1429r (5’-CTA CGG CTA TGT TAC GA-3’). Berdasarkan analisis korelasi Pearson dan multivariat bahwa parameter lingkungan seperti rasio C/N dan bahan organik yang tinggi memengaruhi peningkatan densitas dan indeks diversitas bakteri kandidat PGPR sedangkan faktor N-total, diameter pohon dan ix ketinggian tempat yang rendah berdampak pada peningkatan densitas bakteri kandidat PGPR. Faktor P-tersedia yang minimal dapat menstimulasi peningkatan densitas bakteri penghasil IAA dan penambat nitrogen. Namun, pH tanah yang semakin meningkat hanya meningkatkan densitas bakteri pelarut fosfat di rhizosfer tanah kopi. Berdasarkan analisis metagenomik 16S rDNA bakteri tanah, struktur komunitas bakteri rhizosfer tanaman kopi Robusta dan Arabika masing-masing memiliki kelimpahan Filum Proteobacteria (49,27% dan 39%), Kelas Alphaproteobacteria (30,1% dan 42,9%), Ordo Rhizobiales (26,8% dan 17,0%), Famili Xanthobacteraceae (12,5% dan 7,5%), Genus Bradyrhizobium (3,1% dan 6,1%) dan Spesies Bradyrhizobium elkanii (62,5% dan 75,0%). Indeks diversitas bakteri rhizosfer tanaman kopi Arabika dan Robusta masing-masing 0,990 dan 0,995 dikategorikan sebagai diversitas yang tinggi. Berdasarkan konstruksi filogenetik, isolat bakteri PGPR potensial S1.6.3.2 (Robusta) dan W2.4.4.1 (Arabika) secara berurutan menghasilkan IAA 104,6 μg/mL dan 88,12 μg/mL. Isolat bakteri potensial penambat nitrogen adalah W1.2 (Arabika) dan S3.4.2 (Robusta) secara berurutan mampu menghasilkan amonium 21,54 μg/mL dan 2,07 μg/mL. Isolat bakteri W3.5 (Arabika) dan S1.3.1 (Robusta) mampu melarutkan fosfat masing-masing 4,5 μg/mL dan 2,3 μg/mL. Isolat terpilih sebagai kandidat PGPR S1.6.3.2 dan S1.3.1 teridentifikasi Bacillus subtilis DSM 10T, isolat W1.2 merupakan Bacillus methylotropicus SY2, isolat S3.4.2 sebagai Bacillus wiedmannii FSL W8-0169, isolat W3.5 sebagai Pseudomonas putida S18, sedangkan isolat W2.4.4.1 sebagai Bacillus sp. W2.4.4.1. Hasil identifikasi isolat hanya ditemukan genus Pseudomonas sebesar 2-2,5% dan genus Bacillus sebesar 0,6-1,2% berdasarkan analisis metagenomik 16S rDNA tanah. Informasi jenis bakteri yang melimpah berdasarkan metode isolasi dan analisis metagenomik dapat dijadikan acuan untuk mengoleksi berbagai jenis bakteri PGPR dari rhizosfer tanah kopi Robusta dan Arabika guna memperoleh kandidat bakteri PGPR terbaik yang dapat dikembangkan menjadi biofertilizer tanaman kopi di UB Forest.

English Abstract

Coffee (Coffea sp.) has an important role in the economic sector in global countries. As the fourth rate of coffee production in the world, Indonesia, especially in East Java. Universitas Brawijaya Forest (UB Forest) manage and develop the coffee plantation as primary product. The coffee production in UB Forest decreased because of climate change and soil fertility. Organic fertilizer was used to improve soil fertility, however the coffee production was not increase significantly. For solving this problem, rhizospheric soil bacteria such as Indole Acetic Acid (IAA)-producing bacteria, nitrogen-fixing bacteria and phosphate-solubilizing bacteria could be applied to support plant growth. Therefore, isolation of bacteria, analysis of bacterial diversity, metagenomic analysis, potency assay and identification of selected rhizosphere bacteria are necessary to investigate the indigenous Plant Growth Promoting Rhizobacteria (PGPR) candidates which is support biofertilizer information, sustainable coffee agroforestry (high productivity), and eco-friendly environment of the coffee plantation production. The aims of this study were to analyze the correlation between environmental factors and density also diversity index of rhizospheric bacteria of coffee plantation as PGPR candidates, to investigate the structure composition of rhizospheric bacteria, to performe the potency of rhizospheric bacteria for producing IAA, fixing nitrogen, and solubilizing phosphate, and to identify the selected rhizospheric bacteria as PGPR candidates based on 16S rDNA sequencing. The steps of research were soil physicochemical analysis, 16S rDNA metagenomic analysing soil bacteria, isolation of rhizospheric bacteria, screening and identifying of selected rhizospheric bacteria. The PGPR was isolated using serial dilution and poured plate method on TSA, Nfb Agar, and Pikovskaya Agar medium for producing IAA, fixing nitrogen, and solubilizing phosphate respectively. The density of PGPR isolate candidates was enumerated by TPC and diversity index of PGPR isolate candidates was measured based on the morphological characteristics of bacteria. The environmental factors, density and diversity of PGPR isolate candidates were performed by multivariate analysis using PCA to determine their relationships. Each of PGPR isolates was screened its potency for IAA-producing, nitrogen-fixing and phosphate-solubilizing activity. IAA hormone consentration was measured by colorimetri method using Salkowski reagent, Ammonium was quatified by colorimetri method using Sera Ammonia Test Kit and Nessler Reagent, and Phosphate solubilizing were screened by Ammoniummolibdat Reagen and Chlorostand acid. These treatment were performed using completely randomized design (CRD) experiment with triplicates and the data analyzed using SPSS software v.20. The highest concentration of IAA-production, nitrogen-fixing and phosphate-solubilizing from PGPR isolates on both of rhizospheric coffee plantation of each screening were identified using the similarity of 16S rDNA sequencing using amplification primers such as 27f (5’-GAG AGT TTG CTG GCT CAG-3’) and 1429r (5’-CTA CGG CTA TGT TAC GA-3’). The results showed that based on Person Correlation and multivariate analysis, environmental factors such as C/N ratio and organic matter increased the density of PGPR bacteria. Otherwise, the minimum number of total-N, diameter of tree, water holding capacity xi and altitude factors increased PGPR density. Other factors, phosphate-availability in small quantity decreased the density of IAA-producing and nitrogen-fixing bacteria and the higher of soil pH incresed the density of phosphate solubilizing bacteria. Whereas, metagenomic soil analysis showed that the structure composition of rhizospheric bateria on Robusta and Arabica coffee soil performed the Phylum was Proteobacteria (49,27% dan 39%), the Class was Alphaproteobacteria (30,1% dan 42,9%), the Ordo was Rhizobiales (26,8% dan 17,0%), the Family was Xanthobacteraceae (12,5% dan 7,5%), the Genus was Bradyrhizobium (3,1% dan 6,1%) dan the Species was Bradyrhizobium elkanii (62,5% dan 75,0%) respectively. The diversity index of rhizospheric bacteria were 0,990 (Arabica) dan 0,995 (Robusta), showed the high diversity index on both rhizospheric coffee. The potency of S1.6.3.2 (Robusta) and W2.4.4.1 (Arabica) produced IAA-hormone 104.6 μg/mL and 88.12 μg/mL, W3.5 (Arabica) and S1.3.1 (Robusta) were able to dissolve phosphate 4.5 μg/mL and 2.3 μg/mL, while W1.2 (Arabica) and S3.4.2 (Robusta) produced the ammonia up to 21.54 μg/mL and 2.07 μg/mL respectively. The PGPR selected isolates S1.6.3.2 and S1.3.1 were identified as Bacillus subtilis DSM 10T. Isolate W1.2, S3.4.2, W3.5, and W2.4.4.1 were Bacillus methylotropicus SY2, Bacillus wiedmannii FSL W8-0169, Pseudomonas putida S18, and Bacillus sp, respectively. Based on 16S rDNA metagenomic soil analysis result, the identification found population of Pseudomonas and Bacillus 2-2.5% and 0.6-1.2% respectively. It was small quantity rather than Bradirhizobium elkanii abundant almost 60-75% in the rhizospheric soil. Further, both methods culturing and metagenomic analysis can be helpful for exploring and collecting the PGPR bacteria from rhizospheric soil Robusta and Arabica coffee. It will be useful to develop the biofertilizer for improving the coffee plantation production in UB Forest.

Other obstract

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Item Type: Thesis (Magister)
Identification Number: 0420090011
Uncontrolled Keywords: -
Subjects: 500 Natural sciences and mathematics > 583 Magnoliopsida (Dicotyledons) > 583.9 Asteridae > 583.93 Gentianales
Divisions: Fakultas Matematika dan Ilmu Pengetahuan Alam > Biologi
Depositing User: ismanto
Date Deposited: 25 Feb 2021 14:05
Last Modified: 11 Aug 2022 02:05
URI: http://repository.ub.ac.id/id/eprint/183687
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