Tempe fermentation involved complex microbial communities which are only revealed partially through culture dependent methods. Culture-independent methods would be potential to unravel this complex microbial fermentation. Appropriate DNA extraction is an essential tool to obtain reliable data from culture independent method. In this study, we employed two commercial DNA extraction methods to find the best one for microbial community characterization employing amplified ribosomal intergenic spacer analysis (ARISA). Our result showed that PowerFood Microbial DNA Isolation Kit-MOBIO (PFMDIK) is an excellent method for microbial DNA extraction from tempe. It gave high quantity and quality of DNA suitable for PCR amplification of 16S-23S rRNA intergenic spacer to yield a diverse and reproducible ARISA profile.

Xanthomonas oryzae pv. oryzae (Xoo), a causal agent of bacterial leaf blight (BLB), is one of the most important pathogens of rice. The effectiveness of ten Streptomyces spp. isolates in suppressing Xoo disease was assessed in planta and in vitro. In planta experiments were carried out in a greenhouse and arranged in a randomized completely block design (RCBD) with three replications. Twenty treatments were tested which included plants inoculated with both Streptomyces spp. and Xoo, and plants inoculated with only Streptomyces spp. Plants inoculated with Xoo and sprayed with a chemical bactericide, and plants inoculated with only Xoo served as positive controls, whereas plants not inoculated with either Streptomyces spp. or Xoo were used as negative controls. The results showed that the effect of endophytic Streptomyces spp. on BLB disease expressed as area under disease progress curve (AUDPC) was not significantly different to that on control plants (P > 0.05). However, plants inoculated with endophytic Streptomyces spp. were significantly taller and produced higher tiller number than control plants (P < 0.05). Streptomyces spp. isolate AB131-1 gave the highest plant height. In vitro studies on biocontrol mechanisms of selected Streptomyces spp. isolates showed that isolate LBR02 gave the highest inhibition activity on Xoo growth, followed by AB131-1 and AB131-2. Two isolates (AB131-1 and LBR02) were able to produce chitinase, phosphatase, and siderophore which included biocontrol characteristics. Morphological and colonization studies under SEM and light microscopy confirmed that the three isolates were endophytic Streptomyces spp. from different species. These studies found that the paddy plant which was inoculated with endophytic Streptomyces spp. AB131-1 and infected by Xoo could increase the height of plant and number of tillers.

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.

The objective of this work is to clone flanking DNA derived from Tn-5 mutagenesis of wild type strain Xanthomonas axonopodis pv. glycines as first step to clone and to identify the gene involved in pathogenicity mechanism. We have localized the flanking DNA fragment from a nonpathogenic mutant of Xag M715. Southern hybridization analysis using 2.8 kb EcoRI from pYR103 as a probe showed that the fragment is located within 2.0 kb PstI fragment. A 0.7 kb flanking DNA was amplified using inverse PCR technique, and inserted into pGEM-T Easy vector generating a 3.7 kb recombinant plasmid (pAA01). Southern hybridization analysis of the wild type (YR32) with pAA01 as a probe indicated a hybridization signal located at approximately 3.0 kb PstI fragment. DNA sequence analysis revealed that the DNA fragment has a 64% identity to a vir gene of Bacillus anthracis.

The possibility of horizontal gene transfer of plant genomic DNA and bacteria in the soil, particularly as this relates to the possible transfer of genes encoding antibiotic resistance, has been seen as hazard associated with genetically engineered plants. It is hypothesized that introduction of bacterial genes into the plant genome leads to a higher probability of gene transfer from plants to bacteria due to the presence of homologous sequences. Bollgard (BG) cotton was constructed through the introduction of cry1A(c) gene, encodes for insecticidal activity againts Lepidopteran pests, together with genes for spectinomycin/streptomycin resistant (aad) and kanamycin resistant (nptII), into the genome of a conventional cotton variety, Delta Pine (DP). The aim of this study were to evaluate the ability of naturally competent Acinetobacter calcoaceticus strain ADP1 to take up and integrate transgenic plant DNA based on homologous recombination under optimized laboratory condition, and to compare phyllosphere microbial population resistant to antibiotic on leaves of transgenic and nontransgenic plant. The results showed that transformation of ADP1 cells with Bollgard DNA was not detected on nitrocellulose membrane nor in sterile soil. Total phyllosphere bacterial population on leaves collected from one month after planting were 1.3 x 108 and 1.6 x 108 cfu/g leave fresh weight for BG and DP, respectively. Samples collected after three month contained 5.9 x 107 and 7.1 x 107 cfu/g leave fresh weight for BG and DP, respectively. This study also showed that there was no significant difference of phyllosphere bacterial population resistant to streptomycin and kanamycin on leaves of BG or DP samples collected from one or three month after planting.

A rapid polymerase chain reaction (PCR)-based procedure was developed for detection of Xanthomonas axonopodis pv. glycines, the causal agent of bacterial pustule disease on soybean. A set of primers was designed from partial sequence of the pathogenicity gene of X. axonopodis pv. glycines strain YR32. Specific PCR product of 490 base pairs was produced from strains of X. axonopodis pv. glycines originally from Indonesia as well as from Taiwan. No other pathovars and bacterial species among those tested showed amplification product under optimized PCR conditions. Shaking infected soybean leaves in phosphate buffer saline during six hours was proved to be an essential in order to increase cell number of the bacterial. The procedure was applicable and reliable for detecting of pathogens in infected plant materials. The procedure was proved to be more effective than that of conventional detection and could be of great help for monitoring of pustule bacterial disease in the soybean fields. Key words: Xanthomonas axonopodis pv. glycines, bacterial pustule disease, rapid detection, PCR, specific primer

Tempe is an Indonesian fermented food prepared by fermenting dehulled cooked soybeans with Rhizopus oligosporus. Many types of bacteria are also involved during tempe fermentation, and one of these is Klebsiella spp.  Some isolates of K.  pneumoniae produces vitamin B12 in tempe but it has also been classified as an opportunistic pathogen. For this reason Klebsiella spp. in tempe is important to be studied. The aim of this study was to investigate the genetic diversity of Klebsiella spp. from tempe employing ERIC-PCR method. Sixty-one isolates of Klebsiella have been isolated from sixteen tempe producers  in Bogor, Jakarta, Malang, Tengerang, Bandung and Cianjur. 63F and 1387R primers were used to amplify 16S rDNA sequences, and 1R and 1F primers were used for ERIC analysis. The results of this research showed that sixty-one strains of Klebsiella were clustered into 17 groups. Based on ERIC-PCR analysis, isolates of Klebsiella could be grouped into different profiles which some of these groups consisted of isolates with identical ERIC-PCR profiles. Several identical ERIC-PCR profiles were found in tempe from the same producer. There was no correlation observed between genetic similarity  among isolates with the origin of tempe.

Pengembangan Perangkat Lunak Bantu untuk Penentuan Promotor pada Sekuen DNA Escherichia coli

Pengembangan Aplikasi T-RFLP untuk Pencarian Nama Bakteri

Bacteriocin is proteinaceous compound that has bactericidal action against other microorganisms. Bacteriocin-producing lactic acid bacteria (LAB) is generally considered safe for human consumption and can be applied in food preservation. One source of indigenous LAB is from Indonesian fermented fish products, bekasam. This study aimed to obtain LAB isolates from bekasam that have high potential as  producer of bacteriocin. The steps were screening of bacteriocin compound and protein precipitation using ammonium sulfate with a concentration of 0-10% to 70-80%. Screening of bacteriocin compounds of 25 isolates LAB from bekasam showed that there were 11 isolates (44%) that have the potential as  producer of bacteriocin, in which the cell-free supernatant to pH 5 and or pH 6 produce inhibitory zones on the indicator bacteria Escherichia coli, Salmonella typhimurium ATCC 14028, Bacillus cereus, Staphylococcus aureus and Listeria monocytogenes. Then, the precipitation of proteins from the cell-free supernantant was done for the selected four isolates that have the potential as  producer of bacteriocin. The supernatant and  the precipitate from yield of protein precipitation in the selected four isolates showed that inhibition zone against the indicator bacteria E. coli, S. typhimurium ATCC 14 028, and L. monocytogenes with inhibition zone around 3.0 to 10.0 mm. Inhibition zones in the supernatant and the precipitate were indication that  active compound is organic acid and bacteriocin, respectively. The highest inhibition zone of the supernatant and the precipitate of the BP(3) and SK(5) isolates against L. monocytogenes and S. typhimurium, respectively.  The highest inhibition zone of the supernatant of the BP(20) and BI(3) isolates against S. typhimurium and  S. typhimurium and E. coli, respectively. While the highest inhibition zone of precipitate of the BP(20) and BI(3) isolates were same, that is against E. coli. Each with ammonium sulfate concentrations were different.Key words: Bacteriocin, lactic acid bacteria, bekasam