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Potential Pseudomonas Isolated from Soybean Rhizosphere as Biocontrol against Soilborne Phytopathogenic Fungi SUSILOWATI, ARI; WAHYUDI, ARIS TRI; LESTARI, YULIN; SUWANTO, ANTONIUS; WIYONO, SURYO
HAYATI Journal of Biosciences Vol 18, No 2 (2011): June 2011
Publisher : Bogor Agricultural University, Indonesia

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Abstract

Plants are liable to be attacked by soilborne fungal pathogens which are responsible to reduce plant growth and losses in yield. In Indonesia, indigenous soybeans’ rhizobacteria such as antifungal producing Pseudomonas sp. have not many been reported yet. Therefore, the potential of the Pseudomonas sp. as biocontrol agent should be deeply explored. The aim of this study was to screen the indigenous soybeans’ rhizobacteria Pseudomonas sp. that possessing biocontrol characters against soilborne mainly i.e. Sclerotium rolfsii, Fusarium oxysporum, and Rhizoctonia solani, in vitro and in planta. Eleven isolates identified Pseudomonas sp. CRB numbered by CRB-3, CRB-16, CRB-17, CRB-31, CRB-44, CRB-75, CRB-80, CRB-86, CRB-102, CRB-109, and CRB-112 were affirmed to be candidates of biocontrol agents toward the soilborne fungal pathogens. Pseudomonas sp. CRB inhibited growth of the pathogenic fungi approximately 11.1-60.0% in vitro. Among of them, 7 isolates were also produced siderophore, 2 isolates produced chitinase, and 4 isolates produced hydrogen cyanide. Seed coating with the Pseudomonas sp. CRB accomplished disease suppression in planta about 14.3-100% in sterile soil condition and 5.2-52.6% in non sterile soil condition. Consistency in high performance more than 30% of disease suppression in non sterile soil condition suggested that 5 isolates i.e. CRB-16, CRB-44, CRB-86, CRB-102, and CRB-109 isolates have great promising to be developed as biocontrol agents of soilborne pathogenic fungi.
Diversity of Antifungal Compounds-Producing Bacillus spp. Isolated from Rhizosphere of Soybean Plant Based on ARDRA and 16S rRNA WAHYUDI, ARIS TRI; PRASOJO, BRAMANTYO JATI; MUBARIK, NISA RACHMANIA
HAYATI Journal of Biosciences Vol 17, No 3 (2010): September 2010
Publisher : Bogor Agricultural University, Indonesia

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Abstract

Plant growth promoting rhizobacteria (PGPR) play an important role in improvement of seed germination, root development, and water utilization by plants. These rhizobacteria can stimulate plant growth directly by producing growth hormones or indirectly by producing antifungal compounds/antibiotics to suppress phytopathogenic fungi. The objective of this research was to analyze the diversity of 22 antifungal-producing rhizobacteria of Bacillus sp. isolated from rhizosphere of soybean plant based on Amplified rDNA Restriction Analysis (ARDRA) and 16S rRNA Sequence. Restriction enzymes in ARDRA analysis, HinfI, HaeIII, and RsaI were used to digest 22 16S rDNA amplified from Bacillus sp. genomes. Based on this analysis, genetic diversity of 22 Bacillus sp. producing antifungal compounds were classified into eight different groups. Moreover, six selected isolates randomly from each ARDRA group that have strong activity to suppress fungal growth were analyzed for their 16S rDNA sequences compared with reference strains. The distributions of these isolates were genetically diverse on several species of Bacillus sp. such as B. subtilis, B. cereus, and B. fusiformis. ARDRA is a reliable technique to analyze genetic diversity of Bacillus sp. community in the rhizosphere.
Screening and Characterization of Protease Inhibitors from Marine Bacteria Associated with Sponge Jaspis sp. WAHYUDI, ARIS TRI; QATRUNNADA, .; MUBARIK, NISA RACHMANIA
HAYATI Journal of Biosciences Vol 17, No 4 (2010): December 2010
Publisher : Bogor Agricultural University, Indonesia

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Abstract

Three isolates among 138 sponge-associated bacteria were isolated from Waigeo Island, Raja Ampat West Papua Province, Indonesia, have been shown protease inhibitory activity against subtilisin (serine protease), thermolysin (metalloprotease), and crude extract from pathogenic bacteria (Eschericia coli enteropathogenic/EPEC K.1.1, Staphylococcus aureus, and Pseudomonas aeruginosa). Those three isolates were designated as sponge associated bacteria SAB S-12, SAB S-21, and SAB S-17. A simple casein and Sea Water Complete (SWC) double layer agar method was used to screen the bacteria against pathogenic bacteria producing protease, i.e. EPEC K.1.1, S. aureus, and P. aeruginosa. Among them, SAB S-12 isolate showed no inhibitory zone indicated. The isolate had the highest inhibitory activity against subtilisin and crude extract enzyme of pathogenic bacteria, the inhibitory activity was 91.6 and 98.9%, respectively. In addition, the SAB S-21 isolate had the highest inhibitory activity against thermolysin, it was 70.4%. The optimum pH and temperature for protease inhibition of the three isolates was at pH 7.0-8.0 and 40-50 oC respectively. Based on 16S rRNA gene sequence, the closest related with SAB S-12, SAB-17, and SAB-21 isolates was Providencia sp. (92% identity), Paracoccus sp. (86% identity), and Bacillus sp. (% identity), respectively.
Involvement of a Gene Encoding Putative Acetate Kinase in Magnetosome Synthesis in Magnetospirillum magneticum AMB-1 WAHYUDI, ARIS TRI
HAYATI Journal of Biosciences Vol 13, No 1 (2006): March 2006
Publisher : Bogor Agricultural University, Indonesia

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Abstract

A nonmagnetic mutant of Magnetospirillum magneticum AMB-1, designated NMA40, was constructed by mini-Tn5 transposon mutagenesis to identify genes involved in magnetosome synthesis. Transposon delivery was carried out through conjugation between M. magneticum AMB-1 as a recipient and Escherichia coli S17-1 (λ pir) carrying pUTmini-Tn5Km1 as a donor strain. NAM40 did not respond to the magnetic fields and completely lacked of magnetosome in the cell. DNA sequence/gen interrupted by transposon (called flanking DNA) was isolated by inverse PCR and cloned into pGEM-T Easy. Alignment of the DNA sequence of the flanking DNA allowed the isolation of an open reading frame (ORF2) within an operon consisting of three genes. The amino acid sequence deduced from ORF2 showed homology with acetate kinase from Sinorhizobium meliloti (50% identity and 67% similarity), which function for acetate metabolism. Further analysis revealed that upstream of ORF2 is ORF1, had homology with phosphotransacetylase of S. meliloti (67% identity, 77% similarity), and ORF3 located downstream of ORF2, had homology with hypothetical protein of Thermotoga maritima (30% identity, 60% similarity). ORF2 was subsequently isolated, cloned, and overexpressed in Escherichia coli BL21 (DE3) pLysS as an ORF2-Histag fusion polypeptide. Key words: Magnetospirillum magneticum AMB-1, magnetosome synthesis, transposon mutagenesis, cloning, overexpression
Kandungan IAA, serapan hara, pertumbuhan dan produksi jagung dan kacang tanah sebagai respon terhadap aplikasi pupuk hayati Wibowo, Sigit Tri; Hamim, Hamim; Wahyudi, Aris Tri
Jurnal Ilmu Pertanian Indonesia Vol 14, No 3 (2009): Jurnal Ilmu Pertanian Indonesia
Publisher : Institut Pertanian Bogor

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Abstract

The aim of this research was to study IAA content, nutrient uptake, growth and productivity of maize and peanut in response to application of biofertilizer. The research was conducted in a green house of Cikabayan IPB Farm, Bogor Agriculture University, Darmaga, Bogor, West Java. A completely randomized design was applied in single factor experiment for maize and peanut with 3 replications. The treatments consisted of 4 factors: I. Without fertilizer, II. 100% biofertilizer (dosage 100g/pot), III. 100% inorganic fertilizer, and IV. Combination of biofertilizer and inorganic fertilizer with 50% dosage. Biofertilizer was applied using compost enriched by Pseudomonas sp., Bacillus sp., Azotobacter sp., Azospirillum sp., Rhizobium sp, and P-solubilising bacteria. The dosage of inorganic fertilizer was 0.5 gfpot of Urea; 0.5 g/pot of SP-36; 0.375 g/pot of KCI for maize, and 0.125 g/pot of Urea; 0.5 g/pot of SP-36; 0.375 g/pot of KCI for peanut. Application of biofertilizerenhanced auxin content of maize by 73-159°/o, but not in peanut. The treatment also increased the uptake of N,P, and K of both plants by 2 to 35 times as compared to control plant. The production increased by 270% onmaize and 66% on peanut due to application of biofertilizer. The result showed that application of compost enriched by microbial activator was able to supplement inorganic fertilizer for growth and production of maize and peanut.Keywords: Biofertilizer, nutrient uptake, hormone IAA, morphology responses.
Skrining Bakteri yang Berasosiasi dengan Spons Jaspis sp. Sebagai Penghasil Senyawa Antimikroba Abubakar, Hermawaty; Wahyudi, Aris Tri; Yuhana, Munti
ILMU KELAUTAN: Indonesian Journal of Marine Sciences Vol 16, No 1 (2011): Jurnal Ilmu Kelautan
Publisher : Marine Science Department Diponegoro University

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Abstract

Organisme bentik laut seperti spons, seringkali hidup berasosiasi dengan bakteri yang menghasilkan senyawa antimikroba.  Penelitian  ini  bertujuan  untuk  mengetahui  kemapuan  antagonis  isolat-isolat  bakteri  yang berasosiasi dengan spons Jaspis sp. terhadap beberapa bakteri patogen, dengan metode skrining secara kualitatif. Sebanyak 32 (45,71%) dan 20 (29,41%) isolat yang berasal dari bagian mesohyl dan permukaan Jaspis sp. menunjukkan kemampuan antimikroba, karena mampu menghambat pertumbuhan Staphylococcus aureus, Vibrio harveyii, Escherichia coli, Pseudomonas aerogenosa, EPEC K-11, Candida albicans, and C. tropicalis. Uji fenotipik dilakukan pada beberapa isolat dengan aktivitas antimikroba terbaik, yaitu SAB E-8, SAB E-33, SAB E-35, SAB E-38, SAB E-40 dan SAB S-43. Hasil pewarnaan Gram menujukkan isolat  SAB E-8, SAB E35, and SAB E-40 adalah Gram negatif, sedangkan isolat SAB E-33, SAB E-38, and SAB S-43 adalah gram positif yang dilanjutkan dengan identifikasi parsial (pengecatan gram dan uji katalase) untuk kelompok Bacillus.Kata kunci: Bakteri, Asosiasi, Jaspis sp., antimikroba Living benthic marine organisms such as sponges are frequently assosiated with as bacteria that may be produce antimicrobial compounds. This study aims to determine antagonistic of bacterial isolates that associated sponge Jaspis sp., with a qualitative screening method. Screening of bacteria from marine sponge Jaspis sp. which have bility to produce antibacterial subtances was investigated. There are 32 (45,71%) and 20 (29,41%) isolates from mesohyl and surface sponge respectively. Those isolated bacterial showed the antibacterial activity against Staphylococcus aureus, Vibrio harveyii, Escherichia coli, Pseudomonas aerogenosa, EPEC K-11, Candida albicans, and C. tropicalis. However, use of a few additional simple phenotypic tests for those isolate can be used to differentiate among isolates. The simple phenotypic test divided two ways based on staining gram. Gram negative bacteria were desingned SAB E-8, SAB E-35, and SAB E-40 and gram positive bacteria were desingned SAB E-33, SAB E-38, and SAB S-43. Parsial identification that directed to Bacillus was used for positive gram bacteria, involve gram staining, endospora staining and katalase test. Key words: Bacteria, Assosiation, Jaspis sp, antimicrobe
Role of Bacteria in Tempe Bitter Taste Formation: Microbiological and Molecular Biological Analysis Based on 16S rRNA Gene BARUS, TATI; SUWANTO, ANTONIUS; WAHYUDI, ARIS TRI; WIJAYA, HANNY
Microbiology Indonesia Vol 2, No 1 (2008): April 2008
Publisher : Indonesian Society for microbiology

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Abstract

Tempe is traditional Indonesian food. It has a variety of tastes, sometimes with a hint of bitterness, which may differ in intensity. The cause of bitterness in tempe has never been reported previously. In this study, the aim is to identify whether bacteria play a role in the formation of bitter tastes in tempe. Sensory tests were carried out in order to determine the scoresof bitter-taste-intensity in tempe. The sensory test on EMP, WJB, CLR, DRG, and MLB tempe shows that EMP tempe has the highest score (2.3) and WJB has the lowest (1.3). It is revealed that the processing method has no impact on the formation of the bitter taste in tempe. Plating analysis, showed that EMP soaking water contained a higher number of Enterobacteria groupbacteria, approximately 103-104 CFU ml-1 and spore-forming bacteria groups, 102 CFU ml-1, compared to WJB. Similarly, other bacteria groups in fresh EMP tempe was 102 CFU g-1 higher than those in fresh WJB tempe. Based on sequencing the16S rRNA gene, the dominant bacteria on PCA media in EMP tempe are Acetobacter indonesiensis, Klebsiella pneumoniae, Bacillus subtilis, and Flavobacterium sp. On the other hand those in WJB tempe were Klebsiella sp., Brevundimonas sp., Bacillus sp., Pseudomonas putida, and Acinetobacter sp. Bacillus, a group of proteolytic bacteria was found 105 CFU m-1 higher in the soaking water of EMP compared to WJB. Nevertheless, the types and numbers of fungi were not significantly different betweentempe types. Accordingly, it is concluded that the difference in the number and the types of bacteria involved in the tempe production process leads to the difference in the bitter taste intensity in both EMP and WJB tempe.
Rapid and Simple Amplification of Genomic DNA Sequences Flanking Transposon WAHYUDI, ARIS TRI
Microbiology Indonesia Vol 1, No 1 (2007): April 2007
Publisher : Indonesian Society for microbiology

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Abstract

A rapid and simple method to amplify genomic DNA sequences flanking mini-Tn5 transposon insertion was developed. This technique can be used to determine the location and orientation of the transposon insertion within genomic DNA of the bacteria. Based on the mini-Tn5Km1 transposon sequence, PCR primers can be designed to specifically amplify the DNA sequences flanking mini-Tn5 transposon by inverse polymerase chain reaction (inverse PCR) directly, upstream and downstream of the transposon insertion. The method involves: (i) digestion with a restriction enzyme that does not cut mini-Tn5Km1 sequence; (ii) self-ligation under conditionsfavoring the production of monomeric circles; and (iii) inverse PCR reaction using primers designed from mini-Tn5Km1 sequence to amplify the DNA sequences flanking mini-Tn5Km1 transposon insertion. Feasibility and reliability of this method were demonstrated with mini-Tn5Km1 mutants of the microaerobic magnetic bacterium Magnetospirillum magneticum AMB-1 which are defective in magnetosomes synthesis. The inverse PCR products amplified from these mutant genomes showed the correct fragments as determined through Southern hybridization and DNA sequence analysis.
Genetic Diversity of Antifungi-Producing Rhizobacteria of Pseudomonas sp. Isolated from Rhizosphere of Soybean Plant SUSILOWATI1, SUSILOWATI1; WAHYUDI, ARIS TRI; LESTARI, YULIN; WIYONO, SURYO; SUWANTO, ANTONIUS
Microbiology Indonesia Vol 4, No 1 (2010): April 2010
Publisher : Indonesian Society for microbiology

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Abstract

Antifungi-producing rhizobacteria have been recognized playing an important role in plant disease suppression. In our laboratory, 13 indigenous soybeans´ rhizobacteria Pseudomonas sp. that showed strong growth inhibition of root pathogenic fungi, Rhizoctonia solani, Fusarium oxysporum and Sclerotium rolfsii, have been isolated from rhizosphere of soybean plant. For further understanding, the genetic diversity of the antifungi-producing Pseudomonas sp. was investigated using Amplified 16S rDNA Restriction Analysis (ARDRA) and 16S rRNA gene sequences analysis. 16S rDNA were amplified by PCR technique and digested with restriction endonuclease HaeIII, RsaI and AluI. Sequences of 16S rRNA gene were analyzed using the BLAST program for similarity searches on sequence databases. ARDRA based dendrogram analysis was carried out by neighbor-joining of TREECON 1.3b software package. ARDRA indicated the variability of Pseudomonas sp. based on the digestion sites. Dendrogram clustering analysis based on the restriction enzymes profile of the amplified rDNA distinguished Pseudomonas sp. into 7 ribotype groups. The sequences of 16S rRNA gene confirmed that the isolates belonging to Pseudomonas sp. and the phylogenetic tree formed 4 clusters. There was a quite overlap among ARDRA groups and 16S rRNA sequence clusters. This finding suggested that antifungal producing Pseudomonas sp. were present in the rhizosphere of soybean plant and the level of genetic diversity exist within these species. Sequence analysis of the 16S rRNA gene of the Pseudomonas sp. with an identical ARDRA pattern confirmed that members of an ARDRA group were closely related to each other.
Evidence for a Link Between Pathogenicity and the Role of ImpBacterial Transport Effector Proteins in Soybean Infection by Xanthomonas axonopodis pv. glycines SUWANTO, ANTONIUS; WAHYUDI, ARIS TRI; TJAHJONO, BUDI
Microbiology Indonesia Vol 1, No 2 (2007): August 2007
Publisher : Indonesian Society for microbiology

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Abstract

Xanthomonas axonopodis pv. glycines (Xag) is the causal agent of bacterial pustule disease of soybeans. A nonpathogenic mutant of Xag (M715) was constructed employing transposon mutagenesis which showed similar epiphytic survival in planta to its wild type strain (YR32). The objective of this work was to identify and to analyze genes involved in pathogenicity in Xag YR32. Inverse Polymerase Chain Reaction (IPCR) was used to isolate the DNA flanking transposon insertion. A 1.3 kb flanking DNA fragment was sequenced and analyzed employing BLAST program to study homology, the position of transposon insertion and to predict the structure and function of the gene. One of the Open Reading Frames (ORFs) shared homology with inner membrane proteins (imps) of Xanthomonas axonopodis pv. citri (GenBank accession No. NC 003919). Northern blot analysis revealed that an imps gene was monocistronic and the size of imps mRNA in YR32 was slightly longer than in M715. Reverse Transcriptase- PCR analysis demonstrated that the imps transcript in M715 was much less abundant than in the wild type YR32. Transposon (mini-Tn5-Kmr-Tpr) was determined to be inserted close to the end of C-terminal region of imps gene and might be sufficient to destabilize the imps transcript in M715 and so influence effectors transportation from Xag to plant cell.