Aims: Phosphate and nitrogen are major macronutrients needed by plants. Phosphates in the soil are present in the organic and inorganic form. The amounts of phosphate and nitrogen in marginal soil can be increased by plant growth promoting rhizobacteria (PGPR). The aim of this study was to isolate and characterize phosphate solubilizing bacteria which has ability to fix nitrogen from the soil around limestone mining area. Methodology and results: There were 22 isolates that could solubilize phosphate and fix nitrogen. There were 9 isolates that could not cause hypersensitivity symptoms (necrotic) on tobacco leaf. Quantitative assay of phosphate solubilizing was done by colorimetric method. Quantitative assay of phosphate showed that isolate GPC1.7 had the highest phosphate solubilizing activity on Pikovskaya broth (450 mg/L) on the 6th and 7th day of incubation whereas isolate GPA2.2 had the highest nitrogen fixing activity (0.162 ppm/h), measured with Acetylene Reduction Assay whereas nitrogenase activity of GPC1.7 was unidentified. Isolate GPA2.1 and GPA2.2 were Gram negative bacteria whereas isolate GPC1.7 was Gram positive bacteria. Identification based on 16S rRNA gene showed that GPA2.1 was closely related to Pseudomonas psychrotolerans, GPA2.2 was closely related to Stenotrophomonas maltophilia, GPC1.7 was closely related to Bacillus megaterium and B. aryabhattai. Conclusion, significance and impact of study: Phosphate solubilizing bacteria and nitrogen fixing bacteria isolate could be further used for revegetation process of the ex-limestone mining area.

Aims: Paddy field is one of the sources of greenhouse gasses such as methane (CH4) and nitrous oxide (N2O), which causes global warming and other negative effects in agricultural sector. An alternative to optimize paddy productivity and reduce emissions of CH4 and N2O is by using methanotrophic bacteria and Ochrobactrum anthropi BL2. Methodology and results: This study consisted of two parts, i.e. positive control and experimental treatments. Positive control consisted of 250 kg/ha NPK inorganic fertilizer NPK (15:15:15) (100% of the recommended normal dose) without any methanotrophic bacteria. Meanwhile the experimental treatment consisted of 50 kg/ha inorganic fertilizers NPK (20% of the recommended normal dose) with methanotrophic bacteria (Methylocystis rosea BGM 1, M. parvus BGM 3, Methylococcus capculatus BGM 9, Methylobacter sp. SKM 14) and N2O reducing bacteria (Ochrobactrum anthropi BL2). Using weight indicator of 1000 grams, all the bacteria are capable of increasing paddy productivity by 42.07%, compared to conventional method which can only increase the productivity by 2.51% (Cepy and Wangiyana, 2011). The increasing productivity and growth of paddy plants were due to the nitrogen fixation activity of M. rosea BGM 1, M. capculatus BGM 9, and Methylobacter sp. SKM 14. In the experimental treatment using bacteria, the emission of CH4 and N2O was reduced with the highest CH4 and N2O sinks of 24018.8 mol CH4/day/ha and 68.48 mol N2O/day/ha, respectively. However, the positive control treatment with 100% of the recommended fertilizer dose showed the highest CH4 and N2O emissions which were up to 74346.45 mol CH4/day/ha and 26.21 mol N2O/day/ha, respectively. Conclusion, significance and impact study: All the methanotropic bacteria and O. anthropi BL2 are significantly increase paddy production, compared to positive control treatment. The addition of bacteria in paddy fields results in CH4 and N2O sinks.
Ochrobactrum anthropi

Aims: Potassium is an essential macronutrient for the growth and development of plants. Most of potassium in the soil presented in mineral forms or non-exchangeable forms which are not available for plants. The microbial activity facilitated to release of mineral forms or non-exchangeable potassium to the exchangeable or water-soluble. This study was aimed to isolate, select, and characterize of the selected potassium solubilizing bacteria from limestone mining area in Palimanan, Cirebon Quarry. Methodology and results: Isolation and selection of bacteria was done based on potassium dissolving index in Aleksandrov media containing feldspar, non-exchangeable potassium. Thirty seven isolates of potassium solubilizing bacteria were obtained in this study. Three isolates showed higher dissolution index, namely KQC.4B.1, KQC.5A.4, and KQC.5C.5. All of isolates were Gram negative bacteria, short-rod formed, and able to dissolve potassium concentration on 10th and 20th days. The three isolates showed 99.9% physiologically similar with Burkholderia cepacia. Futhermore by using 16S rRNA gene identification, isolate KQC.5C.5 closely related with B. cepacia with 99% identity. The application of isolate KQC.5C.5 on soil showed that the isolate was able to release the solution K formed after 10th day incubation. Conclusion, significance and impact of study: Potassium solubilizing bacteria (B. cepacia) could use as a biological fertilizer for providing potassium which is available to plants grown on reclamation area of limestone quarry.
Potassium ; Burkholderia cepacia

Aims: Some of methanotrophic bacteria and nitrous oxide (N2O) reducing bacteria have been proven able to support the plant growth and increase productivity of paddy. However, the effect of application of the methanotrophics and N2O reducing bacteria as a biofertilizer to indigenous nitrogen-fixing bacteria and total bacterial community are still not well known yet. The aim of the study was to analyze the diversity of nitrogen-fixing bacteria and total bacterial communty in lowland paddy soils. Methodology and results: Soil samples were taken from lowland paddy fields in Pelabuhan Ratu, Sukabumi, West Java, Indonesia. There were two treatments applied to the paddy field i.e biofertilizer-treated field (biofertilizer with 50 kg/ha NPK) and control (250 kg/ha NPK fertilizer). There were nine different nifH bands which were successfully sequenced and most of them were identified as unculturable bacteria and three of them were closely related to Sphingomonas sp., Magnetospirillum sp. and Ideonella dechloratans respectively. In addition, there were 20 different 16S rDNA bands which were successfully sequenced. Phylogenetic analysis of the sequence showed that there were 5 phyla of bacteria, i.e. Proteobacteria (Alphaproteobacteria and Gammaproteobacteria), Chlorofexi, Gemmatimonadetes, Clostridia, and Bacteroidetes respectively. Alphaproteobacteria was the most dominant group in lowland paddy field. Microbial diversities in the biofertilizer-treated field were lower than that of 100% fertilizer-treated field either based on nifH and 16S rDNA genes. Conclusion, significance and impact study: Biofertilizer treatment has lower microbial diversity than control, either based on nifH and 16S rDNA genes.

Objective: To identify bioactive compound in pigeon pea leaves (Cajanus cajan) that inhibits Salmonella thypi (S. thypi). Methods: The leaf sample was powdered and macerated with methanol and fractioned by liquid–liquid extraction using ethyl acetate. The fraction was chromatographed and the isolates were identified for major component with liquid chromatography-mass spec-trometry and the antibacterial activity was tested against S. thypi by Kirby–Bauer method. Results: Subfraction 1 from the ethyl acetate fraction formed a yellowish solid with m/z 272, identified as naringenin. The naringenin-rich fraction shows fairly well inhibitory toward S. thypi in comparison with chloramphenicol. Conclusions: Naringenin shows antibacterial activity and can be developed to treat typhoid.

Aims: The objectives of this study were to screen chitinolytic bacteria isolated from soil of Taman Nasional Bukit Duabelas, Jambi, Indonesia. Isolates were selected based on chitinolytic index and antagonism activity of Colletotrichum capsici. Chitinase enzyme from selected isolates was investigated for growth inhibition of C. capsici. Methodology and results: Two chitinolytic bacteria were selected based on their ability to degrade colloidal chitin and inhibit of the growth of C. capsici. Those isolates were KAHN 15.12 and SAHA 12.12, identified as Serratia marcescens and Bacillus thuringiensis respectively based on 16S rRNA gene. The chitinase maximum specific activity of isolate KAHN 15.12 was 52.03 U/mg after 36 h of incubation and SAHA 12.12 was 45.67 U/mg after 24 h of incubation. The enzyme was precipitated by ammonium sulfate 40% and 60% respectively for KAHN 15.12 and SAHA 12.12. The precipitated chitinases were active over a broad range of pH (5 to 10) and temperature (20 to 80 °C). Enzymes were stable in optimum temperature for 180 min. The precipitated of chitinase KAHN 15.12 and SAHA 12.12 had five and two protein bands respectively on SDS-PAGE gel. Chitinases exhibited an antifungal activity against C. capsici at concentration of 60 ppm. Conclusion, significance and impact of study: Isolates KAHN 15.12 and SAHA 12.12 were successfully selected by their ability to degrade colloidal chitin and inhibit the growth of C. capsici. The isolates had a broad range of pH and temperature, moreover relatively stable at the optimum temperature. Chitinase was effective as biological control for anthracnose caused by C. capsici in chilli.
Chitinase

Aims: Cricula trifenestrata is one of natural insects which has not been domesticated yet, thus called as the wild silkworm. C. trifenestrata is known as a silk producer which has high economic and market value. However, the fungi attack on C. trifenestrata cocoon decreased quality and quantity of silk yarn. Chitinolytic bacteria have a high potential as biological control against pathogenic fungi. This research aimed to isolate, select, characterize, and identify chitinolytic bacteria as pathogenic fungi growth inhibitors on C. trifenestrata cocoon. Methodology and results: Chitinolytic bacteria was isolated from the uninfected and infected cocoon while fungi was isolated from the uninfected cocoon. Inhibition test was conducted by Fokkema method and chitinase activity was measured by Spindler method. A total of 36 chitinolytic bacteria and 10 suspected pathogenic fungi isolates have been isolated. Fungal pathogenicity test showed that isolate CSAJ.2 was suspected as fungal pathogen. In vitro inhibition test indicated that chitinolytic bacteria isolate BSEP.3 could inhibit the growth of pathogenic fungi CSAJ.2 with percentage of inhibition 50%. Isolate BSEP.3 showed highest chitinase activity (5.11 U/mL) at the 15th h. It able to inhibit the growth of pathogenic fungi with percentage of inhibition of 47.5% and 46.25%, respectively. Conclusion, significance and impact of study: Identification of bacteria targeted on 16S rRNA gene showed that isolate BSEP.3 had 98% identity with Bacillus amyloliquefaciens B5 while identification of fungi using ITS region of the rDNA showed that isolate CSAJ.2 had 100% identity with Trichoderma virens TV242. Chitinase crude extract was effective to be used as a biological control agent of T. virens CSAJ.2.
Chitinase ; Biological Control Agents

Aims: The use of rhizobacteri as biofertilizer may help plants in obtaining nutrients from soil. A consortium inoculant (co-inoculant) consisting of nitrogen fixing and phosphate solubilizing bacteria is formulated to maintain its ability as booster of plant growth. This is easy to be stored and applied on plants. The aims of the study were to formulate rhizobacterial co-inoculant and its application on chili plants at greenhouse experiment. Methodology and results: Isolates of Burkholderia cepacia KD 2.10, Serratia marcescens KAHN 15.12, and Bacillus thuringiensis SAHA 12.12 which have the ability in fixing nitrogen and solubilizing phosphate were used in this study. The three isolates did not show antagonistic activity and hypersensitivity reaction on chili plant. Biofertilizer as carrier material with talc-based powder was mixed with three isolates. This 109 CFU/g cell population of rhizobacterial consortium could be maintained up to six months of storage. Based on result of completely randomized design (CRD) using two factorials and four replicates, application of rhizobacterial co-inoculant significantly affected plant height, number of leaves, flowering age, dry weight of upper plant and root, and root length of chili plant. Conclusion, significance and impact of study Rhizobacterial co-inoculant was effective as biofertilizer to improve the growth of chili plants and it reduced the use of chemical fertilizer.

Objective To identify bioactive compound in pigeon pea leaves (Cajanus cajan) that inhibits Salmonella thypi (S. thypi). Methods The leaf sample was powdered and macerated with methanol and fractioned by liquid–liquid extraction using ethyl acetate. The fraction was chromatographed and the isolates were identified for major component with liquid chromatography-mass spectrometry and the antibacterial activity was tested against S. thypi by Kirby–Bauer method. Results Subfraction 1 from the ethyl acetate fraction formed a yellowish solid with m/z 272, identified as naringenin. The naringenin-rich fraction shows fairly well inhibitory toward S. thypi in comparison with chloramphenicol. Conclusions Naringenin shows antibacterial activity and can be developed to treat typhoid.

Aims: Oryctes rhinoceros beetle is one of the most damaging pests of oil palm and cause high oil palm mortality. The empty fruit bunch mulch and rotten old trunk of oil palm in the field provide the organic matter for the breeding sites and increases the number of O. rhinoceros larvae. Bacillus thuringiensis as bioinsecticide can synthesize crystal proteins toxic to the larvae. The present study was aimed to find effective B. thuringiensis isolates as biopesticide against O. rhinoceros larvae. Methodology and results: Screening process was carried out through heating of soil sample suspension at 80 °C to eliminate the non-spore formers and plated onto T3 medium. Colony morphology was observed, followed by Gram and endospore staining. The crystal protein was observed by Coomassie Brilliant Blue (CBB) staining. Bioassay test was conducted by force-feed method followed by food contamination method. The results showed isolates SBB33 and SBB35 were able to infect and caused high mortality to the O. rhinoceros larvae. Isolates SBB33 and SBB35 showed the highest mortality against 1st instar larvae (94.44% and 75% respectively) and 3rd instar larvae (64.8% and 60% respectively) compared to control treatments. The 16S rRNA gene sequencing showed SBB33 has high similarity with B. thuringiensis strain 3S2-3, while SBB35 has high similarity with B. thuringiensis strain GCU_BTi10. Protein separation of the spore-crystal mixture by SDS-PAGE showed the prominence of 66 kDa protein band that was predicted to be Cry toxins which is specific to coleopterans insect. Conclusion, significance and impact of study Bacillus thuringiensis isolates SBB33 and SBB35 have high potential as biopesticides against O. rhinoceros larvae and could be used to control major pests in oil palm plantation.
Bacillus thuringiensis--isolation & ; purification ; Coleoptera