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ISOLASI DAN SELEKSI PSEUDOMONAD FLUORESCENS PADA RISOSFER PENYAMBUNGAN TOMAT Nurcahyanti, Suhartiningsih Dwi; Arwiyanto, Triwidodo; Indradewa, Didik; Widada, Jaka
Berkala Ilmiah Pertanian Vol 1, No 1: AGUSTUS 2013
Publisher : Berkala Ilmiah Pertanian

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Abstract

[ENGLISH] Fluorescen pseudomonad had been isolated from the rhizosphere of grafting tomato with resisten rootstock (H 7996 and EG 203 from Asian Vegetable Research Development Center). Tomato varieties Permata and Fortuna were used as scion in grafting. Fluorescen pseudomonad was isolated on King’S B medium and used phosphate buffer 0,1 M + 0,1 % pepton. About 230 isolates of P. fluorescens were isolated from tomato rhizosphere at 14 HST and about 454 isolates at 28 HST. All isolates were tested for their capability to suppress the growth Ralstonia solanacearum in vitro. All isolates inhibited the growth of R. solanacearum with an inhibition zone of 1 mm to 7 mm or more. The mechanism growth of inhibition was bacteriostatic. About Ten isolates of P. fluorescens which had large inhibition zone, were not inhibit each other and inhibition against R. solanacearum due to nutrient competition. Keywords : tomato; grafting; Fluorescens pseudomonad [INDONESIAN] Pseudomonad fluorescens diisolasi dari risosfer tomat hasil penyambungan dengan batang bawah tahan yaitu tomat H 7996 dan terung EG 203 dari Asian vegetebles Research Development Center (Taiwan). Sebagai batang atas digunakan varietas Permata dan Fortuna. Isolasi dilakukan pada media King’s B dan menggunakan buffer phospat 0,1 M + pepton 0,1 %. Sejumlah 230 isolat P. fluorescens berhasil diisolasi dari risosfer pada 14 HST dan 454 isolat pada 28 HST. Semua isolat diuji kemampuannya dalam menghambat pertumbuhan Ralstonia solanacearum secara in vitro. Semua isolat P. fluorescens mampu menghambat R. solanacearum dengan zona hambatan antara 1 mm sampai dengan lebih dari 7 mm. Semua isolat mempunyai mekanisme penghambatan bakteriostatik. Sebanyak sepuluh isolat P. fluorescens yang mempunyai daya hambat besar, tidak saling menghambat satu dengan yang lain dan penghambatan terhadap R solanacearum yang terjadi karena adanya kompetisi nutrisi. Kata kunci: Tomat; Penyambungan; Pseudomonad fluorescens  How to citate: Nurcahyanti SD, T Arwiyanto, D Indradewa, J Widada. 2013. Isolasi dan seleksi pseudomonad fluorescens pada risosfer penyambungan tomat. Berkala Ilmiah Pertanian 1(1): 15-18
Sebaran Penyakit Hawar Daun Bakteri di Beberapa Sentra Produksi Bawang Merah di Indonesia Asrul, Asrul; Arwiyanto, Triwidodo; Hadisutrisno, Bambang; Widada, Jaka
Biota Biota Volume 18 Nomor 1 Tahun 2013
Publisher : PBI Yogyakarta

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Abstract

Penelitian ini bertujuan mengetahui daerah sebaran penyakit hawar daun bakteri di beberapa sentra pertanaman bawang merah di Indonesia dan kultivar bawang merah yang dapat diinfeksi, serta mengidentifikasi patogen penyebabnya. Penentuan lokasi pengamatan dan pengambilan sampel dilakukan secara stratified purpossive random sampling. Survei dilakukan dengan cara wawancara dan pengamatan di lapangan (observasi) terhadap kultivar bawang dan gejala penyakit yang terinfeksi oleh bakteri patogen. Sampel diidentifikasi melalui pengamatan morfologi koloni, uji postulat Koch, uji reaksi hipersensitif dan pengujian sifat-sifat biokimia dan fisiologi. Hasil penelitian menunjukkan bahwa penyakit hawar daun bakteri telah tersebar secara merata di seluruh daerah pertanaman bawang merah di Indonesia, yang meliputi Kabupaten Cirebon, Tegal, Nganjuk, Bantul, dan Sigi, dengan tingkat serangan mencapai 62,5–100%. Penyakit ini menginfeksi bawang merah kultivar Bima curut, Bauji, Biru-sawah, dan Palasa. Gejala hawar daun bakteri yang dijumpai berupa water soaking, terjadi lekukan daun, pengerutan daun,  klorosis, nekrosis, mati pucuk, pertumbuhan kerdil, dan kematian. Isolat bakteri yang ditemukan mempunyai bentuk koloni bulat, cembung, berlendir, dan berwarna kuning. Ciri morfologi koloni, gejala dan karakteristik isolat bakteri mirip dengan sifat-sifat bakteri Xanthomonas axonopodis pv. allii penyebab penyakit hawar daun pada bawang bombay.Kata kunci: Sebaran, bawang merah hawar daun bakteri, Xanthomonas axonopodis pv. allii
Succession of Actinomycetes During Composting Proccess of Dairy-Farm Waste Investigated by Culture-Dependent and Independent Approaches Faatih1, Mukhlissul; Widada, Jaka; N, Ngadiman
Indonesian Journal of Biotechnology Vol 13, No 2 (2008)
Publisher : Universitas Gadjah Mada

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Abstract

Mesophilic, thermophilic, and maturation phases were recognized in composting proccess. Temperaturechanges influence the microbial communities in compost within composting proccess. Actinomycetes account for alarger part of compost microbial population. The aim of this research was to study succession of actinomycetescommunity during composting of dairy-farm waste investigated by culture-dependent and independentapproaches.In culture-independent method, the succession of actinomycetes community was analyzed by nestedpolymerasechain reaction of ribosomal intergenic spacer (nested-PCR RISA) using spesific primer F243 and primerR23S followed by a second PCR using primers F968 and R23S. In culture-dependent method actinomycetes fromcompost were isolated on selective media, starch-nitrate medium and humic-acid + vitamins medium. DNA ofactinomycetes was extracted and amplified by repetitive sequence-based PCR (rep-PCR) using primer BOXA1R. Thebanding patterns were used to generate dendrograms by UPGMA clustering with NTSYS program. Microcosmcontaining sterile rice-straw and water which is inoculated with each actinomycetes isolates was used for examiningthe ability of each isolate in rice-straw degradation.The experiment results showed that succession of both bacteria and actinomycetes was occured withincomposting proccess of dairy-farm waste. Analysed by culture-independent method revealed that the highestcommunity of compost’s bacteria was on mesophilic, thermophilic, and maturation phases, respectively. WhereasPCR-nested RISA resulted the highest population of actinomycetes was on thermophilic, maturation, and mesophilicphases, respectively. By culture-dependent method was obtained 29 actinomycetes isolates from mesophilic phase,23 isolates from thermophilic phase, and 19 isolates from maturation phase. Genetic diversity analysis of the obtainedisolates showed the presence of phylogenetic grouping on each phase of composting proccess. This result illustratedthe occurance of succession of actinomycetes community in compost. The ability of each isolates in rice-strawdegradation was different, and SnT9 isolate was found to be a promising rice-straw degrader.Keywords: succession, actinomycetes, composting, nested-PCR RISA, rep-PCR
Diversity of Actinomycetes at Several Forest Types in Wanagama I Yogyakarta and Their Potency as a Producer of Antifungal Compound Nurjasmi, Reni; Widada, Jaka; N, Ngadiman
Indonesian Journal of Biotechnology Vol 14, No 2 (2009)
Publisher : Universitas Gadjah Mada

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Abstract

Actinomycetes are bacterial groups that produce many secondary metabolites, which different biological activities, such as antifungi, antibacteria, antivirus, antitumor, etc. Actinomycetes are widely distributed in soil and their diversity is influenced by type of forest. The aim of this study is to investigate diversity of actinomycetes in several forest types of Wanagama I forest in Yogyakarta and their potency as a producer of antifungal compound. Soil samples under the forest of Tectona grandis, Swietenia macrophylla King, Bamboosa vulgaris, Melaleuca leucadendron, and Gliricidia maculata were used as sources of soil bacteria. Bacteria and actinomycetes communities were analyzed through culture-independent approach by RISA and nested-PCR RISA using actinomycetes spesific primer (F243), respectively. Through culture-dependent approach, isolated actinomycetes diversity were analyzed by identification of morphology (colony and cell), genetic (BOX element by rep-PCR), and secondary metabolites (thin layer chromatography). In addition, isolates were assayed for their antifungal activity against Saccharomyces cerevisae, Candida albicans, Fusarium oxysporum and Aspergillus flavus. The presence of Polyketide Synthase-I (PKS-I) and NonRibosomal Peptide Synthetase (NRPS) genes were amplified by PCR to study their correlation with antifungal activity of the actinomycete isolates. The results showed that types of forest influence diversity of rhizobacteria especially actinomycetes. According to culture-independent approach, relatively, com-</div><div>munity of rhizobacteria from the highest were soil under the forest of B. vulgaris, G. maculata, T. grandis, S.macrophylla King, and M. leucadendron, respectively. Meanwhile, community of actinomycetes from the highest were soil under the forest of G. maculata, B. vulgaris, M. leucadendron, S. macrophylla King, and T. grandis, respec- tively. Fourty-three morphologically different isolates were found by using culture-dependent approach consisting of 17 isolates were found in soil under the forest of M. leucadedron, each of 9 isolates in G. maculata and T. grandis, 6 isolates in S. macrophylla King. and 2 isolates in B. vulgaris. More diversity of secondary metabolites were observed in soil actinomycetes under the forest of M. leucadendron. Of the 43 isolates, 100% were active against S.cerevisae, 37.20% against C. albicans, 95.30% against F. oxysporum, and 83.70% against A. flavus. Antifungal activity of actinomycete isolates did not always have correlation with the presence of PKS-I and NRPS.
Cloning and Sequencing cDNA Encoding for Rhoptry-2 Toxoplasma Gondii Tachyzoite Local Isolate M, Murwantoko; Widada, Jaka; Nuraini, Yani Lestari
Indonesian Journal of Biotechnology Vol 13, No 1 (2008)
Publisher : Universitas Gadjah Mada

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Abstract

Rhoptry protein belongs to an excretory and secretory antigens (ESAs) that play an important role during active penetration of parasite into the cell target. This protein an able Toxoplasma gondii to actively penetrate targeted cell, meanwhile ESAs protein stimulates intracellular vacuole modification. It is, therefore, after the parasite successfully enter the cell target then Granule (GRA) proteins are responsible for the formation of parasitophorus vacuole, which is protect the fusion with other intracellular compartments such as lysosomal vacuole. Consequently, this parasite is being able to survive and multiply at the cell target. The current study was aimed to clone and sequens cDNA encoding for ROP-2 of local isolated T. gondii tachizoite through DNA recombinant technique. Total ribonucleic acid (RNA) was isolated from tachyzoites of local isolated T. gondii that were grown up in Balb/c mice. Messenger RNA was isolated from total RNA using PolyAtract mRNA Isolation System. Messenger RNA was used as a template for synthesis cDNA using Riboclone cDNA Synthesis System AMV-RT. EcoRI adaptor from Riboclone EcoRI Adaptor Ligation System was added to Complementary DNA and than ligated to pUC19. Recombinant plasmid was transformed into E. coli (XL1-Blue). The transformed E. coli XL-1 Blue were plated on LB agar containing X-Gal, IPTG and ampicillin. Recombinant clones (white colony) were picked up and grown up in the LB medium at 37oC overnight. Expression of recombinant protein was analysed by immunoblotting in order to identify cDNA recombinant wich is express ESA of T. gondii local isolate. Recombinant plasmid were isolated using alkalilysis method and were elektroforated in 1% agarose gel. The isolated DNA recombinant plasmid was cut using Eco RI and then sequenced through Big Dye Terminator Mix AB1 377A Sequencer using M13 Forward and M13 Reverse primers. The conclusion of this results showed that the recombinant clone was coding for excretory and secretory protein which has molecular weight of 54 kDa. The DNA alignments of sequence from the cloned gene showed 97% homology with gene encoding for ROP-2 of T. gondii RH isolate., string),(99, en_US, subject, Toxoplasma gondii, tachizoite, ESA, complementary DNA, ROP2
Mineral Phosphate Solubilizing Bacteria Isolated from Various Plant Rhizosphere under Different Aluminum Content Damarjaya, Dolly Iriani; Widada, Jaka; Senoo, Keishi; Nishiyama, Masaya; Otsuka, Shigeto
Indonesian Journal of Biotechnology Vol 10, No 2 (2005)
Publisher : Universitas Gadjah Mada

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Abstract

The objectives of this study was to isolate and characterize the mineral phosphate solubilizing bacteriafrom rhizosphere and evaluate their potential as plant growth promoting bacteria in Al-toxic soils. The halozone formation method was used to isolate PSB using the media containing insoluble phosphates (Ca-P or Al-P)as a source of phosphate. Eight of acid and Al-tolerant PSB isolates that were able to solubilize Ca-P wereobtained from rhizosphere of clover, wheat, corn, and sunflower grown in Al-toxic soil. Identification of theisolates based on the 16S rRNA gene sequence analysis demonstrated that the isolates were strains of Burkholderia(5 strains), Pseudomonas (1 strain), Ralstonia (1 strain), and unidentified bacterium (1 strains). All PSB isolatesshowed the capability to dissolve Ca-P, and only 1 strain (Ralstonia strain) was able to dissolve Al-P in agar platemedium. The P-solubilization by these isolates was correlated with pH of medium. Inoculation of the bacterialstrains on clover on Al-toxic medium showed that all isolates increased the plant dry weight compared withuninoculated treatment. Our results showed that those PSB isolates have potential to be developed as a biofertilizerto increase the efficiency of P-inorganic fertilizer used in Al-toxic soils.
Legume Nodulating Bacterium, Achromobacter xylosoxidans Found in Tropical Shrub Agroecosystem, Sumatera, Indonesia Wedhastri, Sri; Fardhani, Dinar Mindrati; Kabirun, Siti; Widada, Jaka; Widianto, Donny; Evizal, Rusdi; Prijambada, Irfan Dwidya
Indonesian Journal of Biotechnology Vol 18, No 2 (2013)
Publisher : Universitas Gadjah Mada

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Abstract

Legume nodulating bacteria (LNB), known also as rhizobia, are soil bacteria, which are able to form rootnodules and fi x nitrogen in the leguminous plants. The LNB availability in the soil depends on the type ofagroecosystem, where plant grows. In this study, we isolated LNB from the shrub agroecosystem in Sumatera,Indonesia, and obtained four selected bacterial strains. Among them, the isolate UGM48a formed root nodulein Macroptilium atropurpureum and showed highest number of nitrogenase activity. UGM48a also contains nifHand nodA genes. An analysis of the PCR-amplifi ed 16S rDNA and BLASTn analysis showed that UGM48adisplayed 96% similarity with Achromobacter xylosoxidans. In addition, UGM48a were successfully nodulatedGlycine max (L.) merr var. wilis. This is the fi rst report detecting A. xylosoxidans as nodule-forming species forGlycine max possesing the positive copy of nodA gene.Keywords : Legume Nodulating Bacteria, shrub agroecosystem, Achromobacter xylosoxidans, nodA, Glycine max
The Diversity of Legume-Nodulating Bacteria from Several Agroecosystems in Sumberjaya, Lampung Wedhastri, Sri; Yuliana Prahastiwi, Yuliana; Widada, Jaka; Widianto, Donny; Kabirun, Siti
Indonesian Journal of Biotechnology Vol 18, No 1 (2013)
Publisher : Universitas Gadjah Mada

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Abstract

Bacteria that capable of forming root nodules on legumes are known as Rhizobia. They have also known as Legume- Nodulating Bacteria (LNB). They can fi x nitrogen from the atmosphere. Diversity of Legume-Nodulating Bacteria is affected by biotic factors (such as their genetic factors, plants, and competition with the other soil microbes) and abiotic factors (such as land use, soil’s temperature, pH, chemistry and soil’s properties). The aim of this experiment is to know the diversity of eleven Legume- Nodulating Bacteria based on their phenotypic and genotypic characters. The eleven LNB used in this experiments were isolated from several agroecosystems in Sumberjaya, Lampung. The analysis of these LNB diversity were carried out by characterizing both phenotypic and genotypic properties. The diversity analysis showed that the eleven LNB isolates had high diversity, based on nodule formation, and classifi ed into two groups of cross inoculation group.Key words: Rhizobia, phenotypic diversity, genotypic diversity
Antifungal Production of a Strain of Actinomycetes spp Isolated from the Rhizosphere of Cajuput Plant: Selection and Detection of Exhibiting Activity Against Tested Fungi A, Alimuddin; Widada, Jaka; Asmara, Widya; M, Mustofa
Indonesian Journal of Biotechnology Vol 16, No 1 (2011)
Publisher : Universitas Gadjah Mada

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Abstract

Actinomycetes are bacteria known to constitute a large part of the rhizosphere microbiota. Their isolation is an important step for screening of new bioactive compounds. Culturable actinomycetes populations from cajuput plant rhizosphere soils in Wanagama I Forest UGM Yogyakarta were collected to study about their antifungal activity. Among 17 of a total 43 isolates that showed activity were screened for producing antifungi substances. Screening for antifungal activity of isolates were performed with dual culture bioassay in vitro. One isolate that was designated as Streptomyces sp.GMR-22 was the strongest against all tested fungi and appeared promising for a sources of antifungal. Culture’s supernatant and mycelia were extracted with chloroform, ethyl acetate and methanol, respectively. Antifungal activity of crude extracts was tested by diffusion method against tested fungi. The result indicates that isolates of actinomycetes from cajuput plant rhizosphere could be an interesting sources of antifungal bioactive substances.
Combination Methods for Screening Marine Actinomycetes Producing Potential Compounds as Anticancer Farida, Yuyun; Widada, Jaka; Meiyanto, Edy
Indonesian Journal of Biotechnology Vol 12, No 2 (2007)
Publisher : Universitas Gadjah Mada

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Abstract

Marine actinomycetes is a robust source of secondary metabolites including anticancer compounds . The objective of this research was to select marine actinomycetes producing potential compounds as anticancer used combination methods that consist of amplification PKS I (polyketide synthases type I) and NRPS (non ribosomal peptide synthetases) genes, analysis the diversity of secondary metabolites and genetic. Selected isolates were used for cytotoxicity assay. PKS I and NRPS genes were amplified using sets of degenerate primers. K1F and M6R were used for amplify ketosynthase and methyl-malonyl-CoA transferase modules of PKS I gene which targeted sequences 1200-1400 bp. A3F and A7R were used for amplify adenilation domains of NRPS gene which targeted sequences 700-800 bp. The diversity of secondary metabolites was analized by TLC and densitometry of ethyl acetate extracts. Genetic diversity was analized by repetitive DNA fingerprinting using BOXA1R primers. The cytotoxicity of secondary metabolites on T47D and MCF7 breast cell lines cancer was measured by MTT assay method. Fifty two marine actinomycetes isolates were screened using combination methods. Ten isolates were detected encoding both PKS I and NRPS genes, whereas 11 isolates were detected encoding the NRPS gene. The screening by analysis of secondary metabolites and genetic diversity methods were obtained 6 selected isolates for cytotoxicity assay, which consist of 3 isolates encoding both PKS I and NRPS genes and 3 isolates encoding NRPS gene.Isolate 1 had high cytotoxicity with the IC50 on T47D cell was 19 μg/ml and the IC50 on MCF7 cell was 7 g/ml. This findings suggests that combination methods were effective and efficient way to select marine actinomycetes producing potential compounds as anticancer.