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Bio-based fungicides with nanotechnology delivery systems employing bacterial products, such as antifungal compounds, could be an alternative substitution for synthetic fungicides. Antifungal activity of extract from B. subtilis strain CR.9 and its nanoemulsion formula using medium-chain triglycerides (MCT) and Tween-80 was evaluated in vitro and in planta effectiveness against Fusarium oxysporum f. sp. glycines associated with soybean plants. For this purpose, the antifungal compounds from bacterial culture were recovered using ethyl acetate as an organic solvent. The fungitoxic activity of antifungal compounds strains CR.9 demonstrated fungal growth inhibition at 394.12 µg/mL (IC50), 250 µg/mL (MICEE), and 100 µg/mL (MICnanoemulsion), respectively. The EtOAc extract (EE)-nanoemulsion formula developed with a ratio of 2:1 (MCT: Tween-80) showed homogenous size and stability for 25 days without creaming, sedimentation, or separate layers. Moreover, seedlings treatment and soil amendment of EE-nanoemulsion performed high efficacy in suppressing wilt and in agreement with commercial fungicide, Dithane M−45. Overall, the antifungal properties of strain CR.9 may have potency as a bio-based fungicide in plant diseases biocontrol strategy.

Actinomycetes are known to be a source of natural products and drugs. Gordonia terrae, an actinomycete pigment producer, shows potential in producing pigment with antimicrobial activity. This study aims to determine the antimicrobial activity of the active pigment fraction produced by the actinomycete G. terrae, assess the effects of the active pigment fraction on microbial cells, and identify the types of compounds present in the fraction. The pigment extract exhibited antimicrobial activity against Gram-positive bacteria and fungi. Specifically, it showed activity against Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25293, and Candida albicans ATCC 10231. Furthermore, the study evaluated the antimicrobial activities of the active fractions, revealed the active fraction had better antimicrobial activity than the crude extract. Scanning electron microscopy (SEM) confirmed that the active fraction of the pigment causes damage to B. subtilis ATCC 6633 cells, inhibits the formation of filaments in C. albicans ATCC 10231, and alters the normal shape of the cells. LC-MS/MS results showed that the active fraction contained several compounds known for their antimicrobial activity. Among the dominant compounds identified were cholestyramine, aminopregnane, and sphinganine. Thus, this study demonstrated that the orange pigment extract derived from G. terrae JSN1.9 exhibits promising antimicrobial activity.

Traditional nata de coco fermentation often results in inconsistent nata thickness. From the producer's perspective, thin nata sheets are detrimental because most fermentation media will be wasted. The main cause of this condition may be that the microbial population in the starter culture is different in each batch. It is necessary to observe the cultured microbial community on various qualities of available thick and thin nata to design a better nata de coco starter culture. This study showed thick nata had more Komagataeibacter intermedius bacteria (pellicle forming) than thin nata. In traditional nata fermentation, K. intermedius always coexists with other microbes from the bacteria and yeast groups. Random Amplified Polymorphic DNA (RAPD) analysis indicated that the genetic diversity of bacteria was higher than that of the yeast group. Keywords: dendrogram of relationship, fermented food, microbial genetic diversity, nata de coco

Sponge-associated bacteria can produce bioactive compounds similar to the host. Here, the investigation of antioxidant properties of the yellow-red pigment produced by sponge-associated bacterium AGS112 was conducted using in vitro and in vivo analysis. The 16S rRNA gene sequence showed the sponge-associated bacterium AGS112 has the closest similarity with Bacillus haikouensis C-89 (99%). The crude pigment extract produced by Bacillus haikouensis AGS112 contained both phenolic and flavonoid. The peaks occurred at 412 nm and 664 nm, which indicated as the presence of carotenoids and flavonoids, respectively. The active fraction extract obtained through bio-autography TLC, and had more potent antioxidant activity against DPPH (2,2-diphenyl-1-picrylhydrazyl) compared to the crude pigment extract with an IC50 value of 68.62±0.59 µg/ml and 198.88±1.66 µg/ml, respectively. Moreover, the active fraction extract at the concentration of 35 µg/ml could better enhance the viability of Schizosaccharomyces pombe and achieve the highest expression of sod1 and ctt1 genes. Metabolite profiling using LC-MS analysis confirmed the active fraction extract contains eudesmin and artelastin that might be contributed as antioxidants. These results suggest that the active fraction extract of the yellow-red pigment produced by B. haikouensis AGS112 had potency to be used as candidate for natural antioxidants.

Soybean Fusarium wilt and root rot disease caused by a necrotrophic ascomycete pathogen, F. oxysporum, triggered severe damage to the plant tissues and organs and impacted heavy losses. Biocontrol agents, Bacillus subtilis, were commonly used to produce a broad spectrum of antifungal substances and were gradually used in biocontrol studies for plant disease management. Investigation and determination of the inhibiting mechanism of antifungal substance produced by B. subtilis on F. oxysporum should be done to protect the soybean plant. This study revealed that basal nutrient broth (NB) gives the best antifungal activity. The stationary phase of the bacterial growth curve was obtained on two days of cultivation and showed the maximum antifungal activity against F. oxysporum. Ethyl acetate (EA) extraction of bacterial supernatant generated crude EA extract, which showed half inhibition (IC50) at 306.42 µg/ml obtained from the dose-response regression curve. Post-treatment mycelia of F. oxysporum with bacterial extract were demonstrated as hyphal deformation followed by malondialdehyde (MDA) accumulation. Furthermore, cellular leakage on fungal cells that may be triggered by antifungal compounds from strain W3.15 occurred. Last, the related antifungal compounds were predicted to be epicatechin and benzophenone from the LC-MS/MS analysis of crude EA extract. Accordingly, the biocontrol agent B. subtilis strain W3.15 promises a strong potency for biofungicide development.

Abstract Objective Amplicon sequencing targeting 16S ribosomal RNA (rRNA) has been widely used to profile the microbial community from fermented food samples. However, polymerase chain reaction (PCR) steps on amplicon sequencing analysis and intragenomic heterogeneity within 16S rRNA are believed to contribute to bias in estimating microbial community composition. As potential paraprobiotics sources, a comprehensive profiling study of tempeh microbial ecology could contribute to tempeh product development. This study employed a shotgun metagenomic approach, where metagenome fragments from tempeh samples were sequenced directly for taxonomic and functional profiling analysis. Results Taxonomic profiling showed that Proteobacteria, Firmicutes, and Bacteroidetes were the dominant phyla from the shotgun metagenomic analysis in all tempeh samples. In terms of composition, this shotgun metagenomic study revealed that Proteobacteria was the most abundant phylum. Functional profiling showed that iron complex outer-membrane recepter protein (KEGG ID: K02014) was the most transcribed gene based on this metagenomic analysis. The metagenome-assembled genomes (MAGs) results from the binning pipeline could reveal almost complete whole genome sequence of Lactobacillus fermentum, Enterococcus cecorum, Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii.

Background and Aim: Melioidosis is a potentially fatal disease affecting humans and a wide range of animal species; it is often underdiagnosed and underreported in veterinary medicine in Indonesia. This study aimed to characterize morphological and molecular features of Burkholderia pseudomallei, the causative agent of melioidosis which caused the death of a Bornean orangutan. Materials and Methods: Pulmonary abscess samples were cultured on several types of media, including Ashdown agar, Ashdown broth, and MacConkey agar. Type three secretion system orf 2 real-time polymerase chain reaction (PCR) and latex agglutination tests were performed to identify the bacteria. Morphological characteristics were compared to all previously published morphotypes. Subsequently, the bacteria were characterized by multilocus sequence typing (MLST) and Yersinia-like flagellum/Burkholderia thailandensis-like flagellum and chemotaxis PCR. The results of the genotyping were afterward compared to all genotypes from Southeast Asia. Results: Multiple morphotypes of B. pseudomallei were perceived during the growth on Ashdown agar. Furthermore, it was identified by MLST that the Type I and Type II morphotypes observed in this study were clones of a single ST, ST54, which is predominantly found in humans and the environment in Malaysia and Thailand, although a very limited number of reports was published in association with animals. Moreover, the E-BURST analysis showed that the ST is grouped together with isolates from Southeast Asian countries, including Malaysia, Thailand, Singapore, and Cambodia. ST54 was predicted to be the founding genotype of several STs from those regions. Conclusion: B. pseudomallei ST54 that caused the death of a Bornean orangutan has a distant genetic relationship with other STs which were previously reported in Indonesia, implying a vast genetic diversity in Indonesia that has not been discovered yet.

Actinomycetes are considered to be the biggest producer of bioactive compounds which are expected to have antifungal activity for controlling many fungi such as Rhizoctonia solani. The objective of this study was to obtain potential soybean rhizosphere actinomycetes as a biocontrol agent for R. solani which cause damping-off disease both in vitro and in vivo, including their ability to produce siderophore, chitinase, and HCN. Out of 26 isolates, 18 (56%) showed diverse antifungal activities against R. solani with percentages of inhibition radial growth (PIRG) from 18.9 to 64.8%, as evaluated by a dual culture method. Ten isolates with the strongest antifungal activity were numbered for further characterization. All the tested isolates were not antagonistic towards Bradyrhizobium japonicum. These isolates were able to suppress damping-off disease caused by R. solani in the greenhouse experiment. Isolate ASR53 showed the highest disease suppression, 68% and 91% in sterile and non-sterile soil, respectively. Based on 16S rRNA sequence analysis this isolate belonged to Streptomyces violaceorubidus LMG 20319 (similarity 98.8%) according to GenBank data base available at www.ncbi.nlm.gov.nih. Furthermore, isolate ASR53 had significantly longer roots and shoots, as well as greater fresh and dry weights of seedlings than the control. Crude extract derived from ASR53 isolates contained 10 dominant compounds that were biologically active against fungal pathogens. Thus, this study suggests that the application of potential actinomycetes of the soybean rhizosphere can act as a promising biocontrol agent against damping-off disease caused by R. solani.

Actinomycete is one of the beneficial bacteria groups inhabiting rhizosphere soil. They can promote plant growth through various mechanisms. In the previous study we have isolated rhizosphere actinomycetes from maize rhizosphere with direct plant growth promotion characters. The aims of the present study were to analyze the ability of maize rhizosphere actinomycetes to solubilize phosphate, determine alkaline phosphatase activity, and study their genetic diversity based on phoD gene. Thirteen rhizosphere actinomycete isolates were able to solubilize phosphate at concertation range 55.84±2.27 mg/L to 144.48±5.71 mg/L. The activity of extracellular alkaline phosphatase was exhibited by all maize rhizosphere actinomycetes isolates in various level ranging from 0.08 mU/mL to 0.51 mU/mL. The phoD gene, one of the three homologous genes which encode alkaline phosphatases, was successfully detected in all isolates and identified as alkaline phosphatase D of Streptomyces spp. The partial phoD sequences of the isolates were located within metallophosphatase domain of alkaline phosphatase D. Alignment analysis showed that the deduced amino acid sequences of PhoD were mostly conserved in the isolates and Streptomyces spp. Essential residues involved in the active core arrangement of PhoD which binds metal ion cofactors were conserved. Constructed phylogenetic tree showed that the isolates were divided into two groups within PhoD cluster. PhoD of the isolates and Streptomyces spp. had closer relationship to purple acid phosphatase compared to other homologous PhoA and PhoX which form separate cluster. Generated three-dimensional structure model of partial PhoD had high similarity to alkaline phosphatase D of Bacillus subtilis (2YEQ) and showed overlapping structure based on super-positioning analysis.

Marine bacteria associated with sponges can be used as a source of new enzymes, especially cellulase which are potentially used for various industries. The aim of this study was to screen and identify the cellulolytic bacteria associated with sponges. A total of 38 isolates have been successfully isolated from sponges Aaptos sp., Euryspongia sp., and Haliclona sp. by a spread plate method. Amongst 38 isolates, 14 (37%) showed cellulolytic activities in carboxy methyl cellulose (CMC) medium with a cellulolytic index ranging from 0.31 to 1.63. The three highest cellulolytic index was exhibited by bacterial isolates coded Y.5.10, Y.5.11, and Y.40.6. Isolates Y.5.10, Y.5.11, and Y.40.6 showed cellulolytic activity of 0.0053 U/mL; 0.0083 U/mL; and 0.0124 U/mL, respectively, as were tested by spectrophotometry. The highest specific enzymatic activity was showed by isolate Y.40.6 with a value of 0.3391 U/mg. Based on 16S rRNA gene, isolate Y.5.10, Y.5.11, and Y.40.6 were highly similar to (similarity ≥ 99%) Bacillus sp., B. subtilis, and B. cereus, respectively. Keywords: cellulase, isolation, sponge-associated bacteria, 16S rRNA

Rice blast is the main disease of rice plants in Indonesia and several countries worldwide. Controlling this disease using chemical fungicides has harmful effects on the environment. Therefore, we need biocontrol agents which are more environmentally friendly such as rice phyllosphere bacteria. This study aimed to explore bacteria producing bioactive compounds from the rice phyllosphere environment to control blast disease. A total of 88 isolates were successfully isolated from rice leaves in Sukabumi, Situgede, and Jasinga (West Java, Indonesia). From them, we obtained 22 bacteria isolates with antifungal activity against Pyricularia oryzae in vitro assay. In addition, seven non-pathogenic bacteria were obtained from further screening in hypersensitivity, hemolysis and pathogenicity assays, namely STGG 3, STGG 7, STGG 8, STGG 14, SKBV 1, STGV 8, and SKBG 78. To show their antifungal activity, we tested crude extracts of these seven isolates and the results revealed that all the crude extracts can inhibit the growth of P. oryzae. Based on a genetic approach, isolates STGG 3, STGG 7, and STGG 14 were found to have both nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) genes, while isolate SKBV 1 only had the NRPS gene. The NRPS and PKS genes from potential isolates were similar to NRPS and PKS genes of Bacillus sp. in different strains. Furthermore, molecular identification based on the 16S rRNA gene revealed that the seven potential isolates belong to three genera, i.e. Bacillus (STGG 3, STGG 7, STGG 8, STGG 14, SKBV 1), Enterobacter (STGV 8) and Brachybacterium (SKBG 78). We suggest that the seven isolates found in this study have potency and could be recommended as biocontrol agents of blast disease caused by P. oryzae.

Xylanase is an important hydrolytic enzymes with many application in several industries, but to obtain enzyme derived products is not easy. Thus, the optimization of efficient xylanases production is a great interest for biotechnological application. This study aims to determine the type of substrate, medium composition, and optimum conditions of xylanase production by S. costaricanus 45I-3. Determination of substrate type was done by growing the tested bacteria on birchwood xylan, beechwood xylan, oat spelled xylan, corn cobs xylan, and tobacco xylan substrate, meanwhile the determination of medium composition and enzyme production were done by measuring xylanase activity at various substrate concentration and replacing the carbon, nitrogen, phosphate and surfactants source. The results showed that the highest enzymatic index (EI) produced from corn cob xylan substrate at 3.60 meanwhile the second highest was beechwood xylan substrate at 2.87 EI, however this substrate is purer, thus this substrate was selected and used as xylan sources for further optimization measurement. The best xylanase activity (2.29 U/mL) obtained on eighth day after inoculation on rotary incubator at 120 rpm in 28 ºC. Arabinose as the source of carbon generate the highest activity at 3.161 U/mL meanwhile the most preferred source of phosphate is Na2HPO4 (2.37 U/mL). Both source of nitrogen i.e. nitrogen ammonium sulphate (NH4)2SO4 and yeast extract were able to produce xylanase at 2.57 and 2.36 U/mL. The addition of surfactant in production medium showed addition of SDS surfactant (0.146 U/mL) and Tween 80 (0.438 U/mL) showed a negative response by decreasing the activity. The conclusion showed that the xylanase activity was increased after optimization at various C, N, and P sources, and the use of nitrogen source (NH4)2SO4), become a more economical alternative to replacing a nitrogen source yeast extract so it can lower the production costs of xylanase enzyme.

Paddy field is one of the anthropogenic sources that produce greenhouse gases emission. This study aimed to investigate the impact of methanotrophic and N2O-reducing bacterial inoculation on CH4 and N2O emissions, paddy growth and bacterial community structure in paddy field. Two treatments of 100% synthetic fertilizer (250 kg urea/ha) without biofertilizer and 50% synthetic fertilizer (125 kg urea/ha) with biofertilizer consisted of methanotrophic and N2O-reducing bacteria were applied in the paddy field. Inoculation of methanotrophic and N2O-reducing bacteria was able to reduce CH4 and N2O emission up to 4.19 mg CH4/m2/day and 351.29 µg N2O/m2/day, respectively. Those bacterial applications were also able to increase paddy growth and yield productivity. According to DGGE profile, inoculation of the biofertilizer seemed to have a transient impact on bacterial communities in paddy soil at 36 days after transplanting (DAT) which showed the lowest similarity with all samples (a similarity index of 0.68). DGGE bands successfully excised have closest relative to uncultured bacteria which comprised 5 phyla, i.e. Proteobacteria (Alphaproteobacteria and Deltaproteobacteria), Nitrospirae, Actinobacteria, Firmicutes, and Acidobacteria. In this study, Alphaproteobacteria was the most dominant phylum. We provide basic information for developing the biofertilizer which supports sustainable agriculture.

Actinomycetes are the one of the components of the rhizospheric microbial population and useful for producing secondary metabolites such as lytic enzymes, antibiotics, and antifungal. The aim of the study was to isolate the actinomycetes from maize rhizosphere collected from Kupang, East Nusa Tenggara. The screening was focused on the actinomycetes that showed the ability to produce antibacterial, antifungal, and extracellular enzymes such as amylase, cellulase, and protease. The actinomycetes were isolated using Humic-Acid Vitamin B (HV) agar media. The antagonistic assay was tested against Escherichia coli, Staphylococcus aureus, Sclerotium rolfsii and Fusarium oxysporum. Isolate JKP-8 was an isolate that showed the highest activity in inhibiting the growth of E. coli and S. aureus bacteria. Isolate JKP-5 showed the highest activity in inhibiting the growth of F.oxysporum. There were no actinomycetes isolates that showed an ability to inhibit the growth of S. rolfsii fungus based on dual culture assay. JKP-3 and JKP-4 isolates exhibited the highest ability to hydrolyze amylum, while JKP-5 and JKP-8 isolates exhibited the highest ability to hydrolyze CMC. The results of the amplification of 16S rRNA gene in selected potential isolates JKP 5 and JKP 8 indicated that both isolates belong to the genus Streptomyces.

ABSTRACT The aims of this study were to obtain sponge-associated bacteria as biocontrol to inhibit vibriosis in vitro and in vivo, to identify the bacterial isolates based on 16S-rRNA gene, and to detect the presence of nonribosomal peptide synthetase (NRPS), and polyketide synthase (PKS) genes to prove its ability of bioactive compounds synthesis. Aaptos sp. and Hyrtios sp. sponges were collected from Pramuka Island, Jakarta. The isolation using sea water complete (SWC) and zobel marine agar (ZMA) medium obtained 174 isolates. A total 69 isolates were screened successfully based on their antibacterial activity. 47 isolates showed negative haemolysis through hemolytic assays. The pathogenicity test used twelve selected isolates that have a broad spectrum of antibacterial activity and haemolysis negative. The result of pathogenicity test showed that 12 isolates were not pathogenic to the shrimp post larvae with no significantly different (P>0.05) between treatment and negative control. Results of challenge test with Vibrio harveyi have a significant difference survival (70±5.0–90±0.0%) (PPseudomonas, Staphylococcus, and Alcaligenes. Based on amplification of NRPS and PKS genes, four bacterial isolates have been detected to have only NRPS gene, one isolate has only PKS, and one isolate has both genes. The results indicate that the potency of six sponge-associated bacteria as bioactive compounds producers. Keywords: NRPS, PKS, anti-vibriosis, Pacific white shrimp ABSTRAK Penelitian ini bertujuan untuk memperoleh isolat bakteri asosiasi spons yang mempunyai kemampuan dalam menghambat vibriosis secara in vitro, in vivo dan mendeteksi gen 16S-rRNA, nonribosomal peptide synthase (NRPS) serta polyketide synthase (PKS) untuk memastikan kemampuan mensintesis senyawa bioaktif. Spons Aaptos sp. dan Hyrtios sp. berhasil dikoleksi dari perairan Pulau Pramuka, Kep. Seribu Jakarta. Isolasi bakteri dengan menggunakan media sea water complete (SWC) dan zobel marine agar (ZMA) diperoleh 174 isolat. Sebanyak 69 isolat terdeteksi memiliki aktivitas antibakteri. Uji hemolisis menunjukkan 47 isolat adalah hemolisis negatif. Uji patogenisitas menggunakan 12 isolat terpilih yang memiliki spektrum luas dan hemolisis negatif. Hasil uji patogenisitas tehadap 12 isolat menunjukkan bahwa semua isolat tidak bersifat patogen terhadap pascalarva udang vaname. Hal ini dibuktikan dengan sintasan pascalarva udang vaname yang tidak berbeda nyata (P>0,05) dengan kontrol negatif. Hasil uji tantang terhadap Vibrio harveyi diketahui sintasan pascalarva udang vaname (70±5,0–90±0,0%) memiliki perbedaan yang signifikan jika dibandingkan dengan kontrol positif (38,3±2,9%). Berdasarkan analisis sekuen gen 16S-rRNA, menunjukkan bahwa isolat-isolat tersebut memiliki kemiripan dengan genus Pseudomonas, Staphylococcus, dan Alcaligenes. Deteksi gen NRPS dan PKS menggunakan PCR diperoleh empat isolat bakteri memiliki hanya gen NRPS, satu isolat memiliki hanya gen PKS, dan satu isolat memiliki kedua gen NRPS-PKS. Hal ini membuktikan bahwa keenam isolat memiliki potensi sebagai penghasil senyawa bioaktif. Kata kunci: NRPS, PKS, antivibriosis, udang vaname

This study aimed to isolate and explore the actinomycetes of CIFOR forest origin as an antimicrobial and antifungal agent, to produce an extracellular xylanase, and to identify isolates based on 16S rRNA gene sequences. Actinomycetes were isolated using Humic-acid Vitamin-B agar (HV) media. Actinomycetes colonies that grow on the medium HV was subsequently purified by growing them on yeast malt agar (YMA) media), then an antagonistic test of selected bacteria against Bacillus sp., Escherichia coli, Fusarium oxysporum, and Sclerotium sp was performed. Xylanase activity test was detected by observing a clear zone, followed by identification. Total of 35 isolates of actinomycetes isolated based on their colony morphology characteristics and diverse types of spore chains showed Streptomyces spp. of isolates CFR-06, CFR-15, CFR-17, CFR-18, and CFR-19 were able to inhibit the growth of Bacillus sp.. The highest inhibition zone has a diameter of 10.1 mm (isolate CFR-17). Isolates CFR-01 and CFR-15 were able to inhibit the growth of E. coli with the highest inhibition zone diameter of 5.1 mm (isolate CFR-15). Isolates CFR-29 and CFR-12 were able to inhibit the growth of F. oxysporum while isolate CFR-35 were able to inhibit the growth of Sclerotium sp.. Xylanase activity test showed that isolates CFR-12, CFR-20, CFR-22, CFR-24, CFR-25, CFR-30, CFR-33, CFR-34 have an ability to produce extracellular xylanase enzyme. Actinomycetes isolate (Xyl_22) as a potential xylanase enzyme producer was closely related with Streptomyces drozdowicii by the maximum similarity of 99%.How to CiteSipriyadi, S., Lestari, Y., Wahyudi, A., Meryandini, A., & Suhartono, M. T. (2016). Exploration Potential CIFOR Forest actinomycetes origin as Antimicrobial, Anti Fungus and Producing Enzymes Extracellular Xylanase. Biosaintifika: Journal of Biology & Biology Education, 8(1), 94-102.

Burkholderia pseudomallei, the Gram-negative bacterium which causes melioidosis, is a threat to human and a wide range of animal species. There is an increased concern of melioidosis in Indonesian primate facilities, especially following case reports of fatal melioidosis in captive macaques and orangutans. Our preliminary serosurveillance of immunoglobulin G (IgG) to B. pseudomallei lipopolysaccharide showed that a significant number of captive and wild macaques in the western part of Java, Indonesia, have been exposed to B. pseudomallei. To better characterize the humoral immune response in those animals, a panel of assays were conducted on the same blood plasma specimens that were taken from 182 cynomolgus macaques (M. fascicularis) and 88 pig-tailed macaques (M. nemestrina) reared in captive enclosures and wild habitats in the western part of Java, Indonesia. The enzyme-linked immunosorbent assays (ELISAs) in this study were conducted to detect IgG against B. pseudomallei proteins; alkyl hydroperoxide reductase subunit C (AhpC), hemolysin-coregulated protein (Hcp1), and putative outer membrane porin protein (OmpH). The performances of those immunoassays were compared to ELISA against B. pseudomallei LPS, which has been conducted previously. Seropositivity to at least one assay was 76.4% (139/182) and 13.6% (12/88) in cynomolgus macaques and pig-tailed macaques, respectively. Analysis of demographic factors showed that species and primate facility were significant factors. Cynomolgus macaques had higher probability of exposure to B. pseudomallei. Moreover, macaques in Jonggol facility also had higher probability, compared to macaques in other facilities. There were no statistical associations between seropositivity with other demographic factors such as sex, age group, and habitat type. There were strong positive correlations between the absorbance results of AhpC, HcpI, and OmpH assays, but not with LPS assay. Our analysis suggested that Hcp1 assay would complement LPS assay in melioidosis serosurveillance in macaques.

The diversity of bacterial communities in aquatic sediment of rainforest and oil palm plantation at Sumatra was studied using pyrosequencing of 16S rRNA gene and common biodiversity indices. Phylogenetic approach was used for revealing the community shift of bacterial phyla and genera in both areas. Ecological approach used soil pH, total Carbon (TC), total Nitrogen (TN), available Phosphorus (AP) measurement and bacterial diversity with Shanon and Simpson indices, and bacterial richness with Chao1-ACE indices and OTUs. Bacterial diversity and richness in aquatic sediment of forest area was higher than that ones in oil palm plantation area, although their pH, TC, and TN in both areas were not different significantly. The majority of sequences related to Proteobacteria (34.85%), Acidobacteria (32.67%), Nitrospirae (6.86%), Chloroflexi (4.31%), and Actinobacteria (4.02%) were from forest; whereas those related to Acidobacteria (46.10%), Proteobacteria (25.86%), Nitrospirae (9.20%), Chloroflexi (4.99%), and Actinobacteria (2.34%) invented from oil palm plantation. The genera of Alphaproteobacteria and Betaproteobacteria dominated in genera phylotype of bacterial 16S rRNA phylogenetic revealed both aquatic sediment of forest and oil palm plantation. The most genera in the phylogenetic tree from aquatic sediment of both areas was Burkholderia. The bacterial community shift in aquatic sediment of forest transformation indicated higher bacterial diversity index, richness index, some of phyla and genera in aquatic sediment from forest than from oil palm plantation.

Melioidosis is an emerging zoonotic disease caused by the saprophytic Burkholderia pseudomallei, which infects human and a wide range of animal species. Melioidosis may lead to septicemia and pneumonia in human patients, which can be fatal if the patient is not treated accordingly. The disease is spread in tropical areas and is highly endemic to Southeast Asia and Northern Australia. However, melioidosis is poorly reported in Indonesia, especially in the veterinary field. This research provides serological evidence of antibodies to B. pseudomallei in both captive and wild nonhuman primates. Plasma samples were taken from a total of 390 monkeys in captivities and wild habitats in West Java and Bali, Indonesia. Enzyme-linked immunosorbent assay (ELISA) showed that the seroprevalence was 42.21% for Macaca fascicularis and 43.59% for Macaca nemestrina. Furthermore, the seroprevalence was 53.41% for captive macaques and 17.83% for wild macaques. The findings showed that exposure to B. pseudomallei happened in both captive and wild macaques. Based on this serosurveillance results, further studies such as comprehensive culture and clinical study are required to discover the clinical burden of the disease in nonhuman primates.

Streptomyces costaricanus 45I-3 is a bacterial strain belongs to actinomycetes group isolated from peat soil. The bacterium is known to produce extracellular xylanase. The aims of this study were to analyze DNA sequence and sub-clone gene involved in the synthesis of extracellular xylanase. Complete DNA sequence predicted to encode xylanase genes was isolated from bacterial genome using Inverse Polymerase Chain Reaction (I-PCR). Total DNA sequence of 1664 bp in size obtained from I-PCR consisted of two open reading frames (ORF) in opposite direction. ORF1 was 1029 bp and ORF2 (partial sequence) was 309 bp. Analysis sequence using BlastX indicated that ORF1 was homologous with xylanase bacterium enrichment culture clone Xyl8B8 (GenBank accession No. AFH35005.1), i.e. 95% in identity and 99% in similarity. In addition, ORF2 was homologous with glyoxalase bacterium enrichment culture clone Xyl8B8 (GenBank accession No. AFH35007.1), i.e. 95% in identity and 98% in similarity. Analysis of amino acid sequence revealed that ORF1 consisted of 2 domains, i.e. glyco-hydrolase 11 (GH11) and Carbohydrate Binding Type 2 (CBM2). Active site was found at 130th amino acid on GH11 domain. Visualization of 3-dimension structure showed that 1029 bp fragment is of 19 areas.

Endophytic actinomycetes associated with medicinal plants is very important as source of various bioactive compounds. The fact that more than 99% of microbes that exist in nature may have the potency but still unexplored. Published data regarding diversity of endophytic actinomycetes from T. crispa is mainly based on a culturable approach. This paper describes the first reported data regarding metagenomic analysis on the diversity of endophytic actinomycetes from T.crispa based on 16S rRNA gene using PCR-DGGE. There were some similarities amongst endophytic actinomycetes found in stems, roots, and leaves with soil actinomycetes community in the rhizosphere of T. crispa. There were a total of 21 bands found from the DGGE analysis which were interpreted using Phoretix 1D software. Diversity of actinomycetes in the stems, leaves, roots were represented by 17, 16, and 14 bands, respectively. Whereas only 10 bands represented diversity of actinomycetes in the soil rhizosphere. The 12 dominant and or different bands with 180 bp in size were molecularly sequenced. The A4 and A9 bands have 95% and 86% similarities with Williamsia and Streptomyces, respectively. These similarities were less than 97% thus may indicate novel actinomycetes. The other 10 sequenced bands have closed similarity ranging from 97-100% and they were closely related to the genus Streptomyces, Microbacterium, Amycolatopsis, Actinomadura, Actinoplanes, Actinokineospora, Kibdelosporangium, Williamsia and Kocuria. These findings indicate that diversity of actinomycetes can be found associated with T. crispa.

Agricultural land in Indonesia was largely dominated by untapped potential land, almost of that potential land is dry land. Maize (Zea mays L.) is one of the staple crops that are known could be cultivated on dry land. Rizosphere has been known to containmicroorganisms that can improve the plant growth. In the previously study, we have screenedin vitro of rhizobacteria (6 isolates of Pseudomonas sp CRB and 7 isolates of Bacillus sp CR) as growth promoter of maize and drought tolerant. A total of 6 rhizobacterial formula were further applied in vivo in greenhouse to promote the growth of maize. In planta test on maize showed that formula CRB 19 and F3 (CR 83 + CRB 10) were the best formula that could enhance plant growth parameters under drought stress conditions. After that abundance and activities of microbe were analyzed by enumeration of total fungus, bacteria and actinomycetes, dehidrogease test, soil respiration test dan C-microbe test and those tests showed that formula CRB 19 and F3 (CR 83 + CRB 10) were the best formula.These formula could be recommended as inoculants of maize planted in dry land agriculture. Keywords : drought tolerant,formulation, growth promoter, maize, rhizobacteria

An endophytic bacterium isolate G062 was characterized base on its molecular genetic potents, morphology, physiology, and biochemistry reactions. Analysis of 16S rDNA sequences of G062 showed the highest similarity to Paracoccus halophilus (98%). Detection of the phlD and prnC genes occurrence indicated that the bacterium had this antibiotic-like genes of Diacethylphloroglucinol (DAPG) and pyrrolnitrin. The cells are rod shaped (0.59-0.89 × 1.85-3.3 μm), aerobic, Gram negative, non motile, non spore forming, positive catalase, positive oxydase, could reduce NO3 to N2, nitrogen fixing, producing siderophore and plant growth hormones-like compounds (IAA, Gibberellin, and zeatin), and solubilizing phosphate. The G062 isolate could grow on media containing 2.5% NaCl. Range of the temperature and pH growth were 15-40 and 5.0-9.5 °C, respectively. The bacterium did not cause red blood cells lysis. There was no hypersensitive response when it was injected into tobacco leaves, and it was not pathogenic against potato plantlets. Moreover, the bacterium promoted the growth of the potato plant and had high colonization ability. These results suggested that the bacterium had beneficial and good traits as biological agent candidate to promote potato plant growth.

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.

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.

Xanthomonas oryzae pv oryzae the Causal Agent of Bacterial Leaf Blight of rice: Isolation, Characterization, and Study of Transposon Mutagenesis. X. oryzae pv. oryzae (Xoo) causes bacterial leaf blight (BLB) of rice (Oryza sativa L.), a major disease that constrains production of the staple crop in many countries of the world. Identification of X. oryzae pv. oryzae (Xoo) was conducted based on the disease symptoms, pathogenicity, morphological, physiological, and genetic characteristics of bacterial cultures isolated from the infected plants. Fifty bacterial isolates predicted as Xoo have been successfully isolated. They are aerobic, rod shaped, and Gram negative bacteria. The isolates were evaluated for their hypersensitivity in tobacco and pathogenicity in rice plant. Fifty isolates induced hypersensitive reaction in tobacco and showed pathogenicity symptom in rice in different length. Based on physiological test, hypersensitivity and pathogenicity reactions, three bacterial isolates strongly predicted as Xoo, i.e. STG21, STG42, and STG46, were non indole formation, non pigment fluorescent, hydrolyzed casein, catalase activity positive, but negative oxidase. Partial sequencing of 16S rRNA genes of STG21 and STG42 showed 80% and 82% homology with X. oryzae, respectively, while STG46 showed 84% homology with X. campestris. Mini-Tn5 transposon mutagenesis of STG21 generated one of the mutants (M5) lossed it’s ability to induce hypersensitive reaction in tobacco plant and deficient in pathogenicity on rice. The lesion length of rice leaf caused by the mutant M5 decreased up to 80%.

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 °C 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. (100% identity), respectively.

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.

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 g/pot of Urea; 0.5 g/pot of SP-36; 0.375 g/pot of KCl for maize, and 0.125 g/pot of Urea; 0.5 g/pot of SP-36; 0.375 g/pot of KCl for peanut. Application of biofertilizer enhanced auxin content of maize by 73-159%, 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% on maize 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

Magnetospirillum magneticum AMB-1 synthesizes intracellular magnetic particles, magnetite (Fe3O4), enveloped by membrane called magnetosome under micro-aerobic conditions. Initial study of random transposon-based mutagenesis generated 62 nonmagnetic mutants of AMB-1 in a mini-Tn5 library. In order to identify a gene involved in bacterial magnetic particle (BMP) synthesis in the magnetic bacterium M. magneticum AMB-1, a nonmagnetic mutant from the library designated as NMA38-4, was analyzed. h e amino acid sequence deduced from the gene directly interrupted by transposon, ORF4 (1482 bp), showed homology to ATP binding cassette (ABC) transporter of Mesorhizobium loti with 62 % identity and 74 % similarity. It was strongly indicated by the occurrence of putative consensus sequence of ATP-binding motifs (ATP-binding protein). h e ORF4 was subsequently cloned in pET-15b and the recombinant ORF4-Histag fusion protein was heterologously expressed in Escherichia coli BL21 (DE3) pLysS. A 55 kDa protein corresponding to the ORF4-Histag fusion protein was obtained after purifi cation using Ni-NTA column. h is is the fi rst report describing a gene cluster containing gene encoding protein belonging to ABC transporter organized in an operon which is involved in BMP synthesis.

Utilization of soil bacteria as biocontrol agent is becoming popular due to its valuable and effective mechanisms to suppress plant pathogenic microbes. We have previously isolated Bacillus sp, designated as Bacillus sp CR64, which exhibited effective plant growth promoting and antifungal activities. In this study, CR64 was examined in inhibiting the growth of Rhizoctonia solani, the causing agent of root rot disease. Partial sequence analysis of 16S rRNA gene revealed that this isolate similar with Bacillus cereus (94%). Furthermore, a gene designated zmaR was detected by means of specific amplification of DNA fragment approximately 950 bp. This fragment was then cloned onto pCRII-TOPO (3.9 kb) and sequenced using DNA sequencer ABI PRISM 310. Sequence analysis revealed that it had highest homology with the ZmaR protein (89% identity; 90% similarity) of B. thuringiensis serovar kurstaki (AAF82729.2). Alignment analysis with other ZmaR sequences from other antibiotic-producing Bacilli exhibited an almost fully conserved region within ZmaR sequences.Key words : PGPR, Bacillus sp CR64, Zwitermicin A, Cloning, Antifungal.

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.

Composition and abundance of culturable bacteria of four soil samples (Ktr50II, D50II, G50II, and A50II) were analyzed. The soil samples were collected from maize rhizosphere that planted in dryland Lombok Island. Each soil sample give different growth performance of maize in greenhouse experiment. This study was to investigate the relation of maize growth performance with culturable bacterial community of their rhizosphere and the effect of culture media on number of bacterial isolates recovery. The rhizosphere bacteria were cultured and isolated on commercial media (SEA) and non commercial modification media (NAln, NAln-SE and NAln- RE). Thirty four strains rhizosphere bacteria were obtained from four maize rhizophere soil samples. D50II is the soil sample that caused the better growth performance to the maize, contrary to Ktr50II. D50II has significantly highest number of culturable bacterial types, while significantly lowest on Ktr50II. In D50II, at least 17 bacterial isolates contributed to better growth performance in maize and have relative abundance of dominant isolate not more than 35.34%. In comparing the rhizosphere bacteria recovered using different culture media, bacteria cultivated on SEA have different growth characteristic compared with bacteria cultivated on NAln, NAln SE and NAln-RE. Six bacterial isolates showed antagonistic ability when grew on SEA but not in all of three media. Compared with commercial media, non commercial modification media can increase total isolates recovery about 70.6%.

Rhizobacteria have been known for their capability as plant growth promoter through some mechanisms, directly and indirectly. The purpose of this research to screen rhizobacteria of Bacillus spp. and Pseudomonas spp. for their drought tolerance as plant growth promoter of maize (Zea mays). Screening of rhizobacteria as growth promoter of 47 isolates of Bacillus CR and 34 isolates of Pseudomonas CRB resulted 24 and 9 isolates were able to stimulate the growth of maize sprouts, respectively. Further screening of those growth promoter of the rhizobacterial isolates to drought tolerance resulted 7 isolates of Bacillus CR and 6 isolates of Pseudomonas CRB that were able to grow on medium with osmotic pressure -1 and -2 MPa, respectively. Potential rhizobacterial isolates of growth promoter and drought tolerance were tested for antagonist mechanisms which aims to determine ability to live together in one carrier medium if to be made formulation. Both non antagonist rhizobacterial isolates were evaluated for their potential in producing exopolysaccharide (EPS) revealing that CRB 19 and CR 90 exhibited the highest activity of EPS production up to 0.346 mg mL-1 on medium with -2.0 MPa and 0.107 mg mL-1 on medium with -0.73 MPa, respectively. Based on 16S rRNA sequence analysis, it revealed CRB 19 and CR 90 had highest similarities to Pseudomonas aeruginosa strain B2 and Brevibacillus brevis B33, respectively. Those growth promoter and drought tolerant of Bacillus CR and Pseudomonas CRB had potency to be developed as inoculants in dry land agriculture.

Plant-growth promoting rhizobacteria (PGPR) are rhizosphere associated soil-borne bacteria that can enhance plant growth and inhibit the development of root pathogens. Many soil bacteria have been used as PGPR, and one of them is Bacillus sp. The implementation of PGPR is constrained by genotype fluctuation that makes it inactive on the rhizosphere. Our previous study had characterized and revealed that 11 Bacillus sp. isolated from the soybean plant rhizosphere were PGPR. To asses and compare the genetic diversity of these isolates, Amplified Ribosomal DNA Restriction Analysis (ARDRA) and DNA sequence analysis of 16S rRNA were conducted. The construction of Neighbor-joining trees and bootstrap analysis of 100 resamples of ARDRA and 16S rRNA gene sequences were performed using Treecon software for windows ver. 1.3b. ARDRA analysis was done by using four restriction enzymes (RsaI, HaeIII, CfrI and HinfI), resulting in four phylotypes, respectively phylotype I (Bacillus sp. Cr24, Cr33, Cr64 and Cr68), phylotype II (Bacillus sp. Cr 31 and Cr66), phylotype III (Bacillus sp. Cr44 and Cr71) and phylotype IV (Bacillus sp. Cr67, Cr28 and Cr69). Results of BLASTN from 16S rRNA gene sequences showed that these isolates are genetically diversed. The evolution relationship of Bacillus sp. could be shown by the 16S rRNA gene sequences analysis, while ARDRA based on the digestion sites showed their variability.

The 1-aminocyclopropane-1-carboxylate (ACC) deaminase (EC4.1.99.4) is an enzyme produced by some soil bacteria to degrade ACC (the immediate precursor of ethylene) to reduce ethylene biosynthesis in higher plants. Increased concentrations of ethylene in plant tissues, which are triggered by various biotic and abiotic stresses, inhibits plant growth and weakens the plant defense against the stressors. Various findings on the successful use of ACC deaminase producing bacteria for plant growth under unfavorable soil conditions are inspiring their use in tropical peat-soil-agriculture, which possesses bio-physical constraints. It has been proven that inoculation of plants with ACC deaminase producing bacteria decreased ethylene inhibition generated by unfavorable environmental conditions, such as nutrient shortage, flooding, drought, high salts, and the presence of heavy metals and organic pollutants. Understanding the mechanisms by which ACC deaminase-producing bacteria act to reduce plant stress and the fitness of bacterial traits with the properties and constraints of peat-soils becomes a key to utilize these bacteria in improving crop productivity. The bacteria may ameliorate plant stress as well as promote plant growth under seasonal bio-physical changes of peat-soils that are usually encountered in the field.

Acid-aluminium sensitive mutants of symbiotic bacterium Bradyrhizobium japonicum BJ11 (designated as AAS11) and KDR15 (designated as AAS15) were constructed by mini-Tn5 transposon mutagenesis to study genes involved in acid-aluminium tolerance (AAT) in B. japonicum. Transposon delivery was carried out through conjugation between B. japonicum strains as recipients and Escherichia coli S17-1 (ë pir) carrying pUTmini- Tn5Km1 as a donor strain. The result showed that frequency of transconjugation was in the range of 6.7 x 10-7 to 7.1 x 10-6 cell per recipients. AAS11 and AAS15 mutants did not grow on Ayanaba media (pH 4.5) containing 50 μM Aluminium. These mutants remained able to form root nodules of Siratro (Macroptilium arthropurpureum) plants revealing genes interrupted by transposon which were responsible for acid-Al tolerance did not correlate with the nodulation genes. Strains tolerant to acid-aluminium and their mutants with a wild type sensitive to acidaluminium were characterized by accumulating phosphate and aluminium absorption. Compared to the wild type acid-aluminium tolerant B. japonicum, there was approximately a three- to eight-times decrease in phosphate accumulation and a five- to seven-times increase in aluminium absorption by these mutants. These results suggest that aluminium and phosphate contents in the bacterial cells may be involved in mechanisms of acid-Al tolerance of B. japonicum grown in acid-aluminium stress conditions.

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.

Utilization of soil bacteria as biocontrol agent is becoming popular due to its valuable and effective mechanisms to suppress plant pathogenic microbes. We have previously isolated Bacillus sp, designated as Bacillus sp CR64, which exhibited effective plant growth promoting and antifungal activities. In this study, CR64 was examined in inhibiting the growth of Rhizoctonia solani, the causing agent of root rot disease. Partial sequence analysis of 16S rRNA gene revealed that this isolate similar with Bacillus cereus (94%). Furthermore, a gene designated zmaR was detected by means of specific amplification of DNA fragment approximately 950 bp. This fragment was then cloned onto pCRII-TOPO (3.9 kb) and sequenced using DNA sequencer ABI PRISM 310. Sequence analysis revealed that it had highest homology with the ZmaR protein (89% identity; 90% similarity) of B. thuringiensis serovar kurstaki (AAF82729.2). Alignment analysis with other ZmaR sequences from other antibiotic-producing Bacilli exhibited an almost fully conserved region within ZmaR sequences. Key words : PGPR, Bacillus sp CR64, Zwitermicin A, Cloning, Antifungal.

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.

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 scores of 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 group bacteria, 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 between tempe 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.

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 conditions favoring 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.