Aims: Our previous study demonstrated that Klebsiella IIEMP-3 associated with tempeh was genetically different from those of medical isolates. In addition to the whole genome sequence of Klebsiella IIEMP-3, the draft genome sequence of another isolate, i.e. IWJB-6 was employed for comparison. In this study, the details of the virulence genes and unique gene in both Klebsiella isolates were compared employing in silico and in vitro analysis. Methodology and results: Whole genome of Klebsiella IIEMP-3 and IWJB-6 were annotated to investigate the virulence factor. Klebsiella IIEMP-3 and IWJB-6 were obtained from tempeh producers in Bogor, West Java - Indonesia. Genome sequences were analyzed employing BLAST Ring Image Generator (BRIG) software. The results showed that all of the samples, including isolates IIEMP-3 and IWJB-6 did not harbor rmpA, i.e. DNA sequence for K. pneumoniae virulence factor. Conclusion, significance and impact of study: Klebsiella could be found in almost all tempeh samples from Indonesia and could be harmless for human due to the absence of rmpA and other virulence-associated genes. The significance of this study showed that IIEMP-3 and IWJB-6 isolates were more closely related to K. variicola. However, K. variicola At22 harbored sdsA gene which is lacking in those two tempeh isolates. Combined with PCR analysis for specific gene/s; our study suggested that isolates from Indonesian tempeh were closely related to K. variicola, and proposed to be designated as K. variicola subsp. tempehensis.

Aims: Tempeh is a soy-based traditional food fermented by Rhizopus oligosporus. Although this mold is the main microorganism responsible for tempeh fermentation, various unknown bacteria presence in tempeh could enhance tempeh’s nutritional value. This study is aimed to examine the identity of bacteria in tempeh bacterial community by combining metagenomics analysis and culturable technique. Methodology and results: Samples were obtained from a tempeh producer which consists of raw soybeans, fresh water used to soak the beans, soaking water after the beans were soaked for 18 h, dehulled-soybean before inoculation, starter culture, and fresh tempeh. All samples were plated onto Enterobacteriaceae and Lactic Acid Bacteria agar media, and the total DNA was extracted for metagenomics analysis based on 16S rRNA gene cloning and High-Throughput Sequencing (HTS). Metagenomic analysis indicated that Firmicutes and Proteobacteria were the predominant and subdominant bacteria, respectively, while the culturable technique showed Proteobacteria were the predominant bacteria. Firmicutes species detected in tempeh were similar to the ones in the soaking water, which were populated by Lactobacillus. However, another predominant bacteria from tempeh, Enterococcus, was similar to minor population of Enterococcus detected in dehulled-soybean before inoculation. Based on the cloned 16S rRNA genes, we observed L. agilis, L. fermentum, and E. cecorum as the predominant bacteria in tempeh. The starter culture, which was dominated by Clostridium, did not alter bacterial community in tempeh, since its proportion was only 2.7% in tempeh clean reads. Conclusion, significance and impact of study: The dominant bacteria in tempeh was Lactobacillus from Firmicutes. The bacterial community in tempeh was not affected by the starter culture used, but mainly because of the soybean soaking process.

The primary objective of the current research was to detect genetic variations within the Indonesian isolates of Cochliobolus heterostrophus collected from ecologically different places of the country at molecular level using PCR-RFLP analyses. The primer pair of NS3 and NS6 produced amplification fragment in all of the isolates tested. A single fragment of estimated 907 bp was observed in the PCR product pattern. RFLP analysis of the PCR product employing three restriction enzymes, HaeIII, HhaI, and RsaI, respectively, did not reveal intraspecific variations within the fungus. Similarly, nucleotide sequences of portion of small subunit of the ribosomal DNA gene of two of the isolates collected showed no appreciable differences, indicating the absence of genetic diversities among the isolates tested. A phylogenetic tree was constructed and the Indonesian C. heterostrophus, represented by SM-1 isolate, was found to be phylogenetically located near C. sativus, a closely related species.
Ascomycota* ; Base Sequence ; DNA, Ribosomal ; Fungi ; Genetic Variation ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length

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

Aims: Biocontrol of fungal plant pathogens using beneficial microorganisms is a safer alternative over synthetic fungicides. PHP12 is a bacterial strain isolated from healthy oil palm rhizosphere and is closely related to the recently described Burkholderia stagnalis, a member of the Burkholderia cepacia complex. This study aimed to characterize the antifungal activity spectrum of PHP12 and identify the antifungal compounds produced by the strain. Methodology and results: The antifungal activity of PHP12 was characterized by growing fungal strains in the presence and absence of PHP12 and measuring the radius of the antifungal zone. PHP12 inhibited the growth of fungal pathogens including Ganoderma boninense, Curvularia oryzae, Phellinus noxius and Colletotrichum capsici. However, PHP12 did not inhibit the growth of Trichoderma asperellum, a known fungal biocontrol agent. The antifungal compounds of PHP12 were precipitated using ammonium sulfate and further purified with HPLC followed by identification using Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC/ESI-MS). The LC/ESI-MS analysis showed the presence of an oligopeptide with a molecular weight of 1210.63 Da. The peptide consists of heavily modified amino acids that are linked by a hexose residue. Conclusion, significance and impact of study Although characteristics of the antifungal compounds are similar to other antifungal peptides from Burkholderia such as occidiofungin, there have been no reports of antifungal peptides from B. stagnalis with the corresponding molecular weight or fragmentation profile. The novelty of the compound, as well as its antifungal spectrum, makes PHP12 an interesting strain to be investigated further as a biocontrol agent.
Fungicides, Industrial ; Burkholderia cepacia complex--pathogenicity

Aims: Lytic polysaccharide monooxygenase (LPMO) is an enzyme capable of cleaving glycoside bonds of recalcitrant polysaccharides through an oxidative mechanism. LPMO activity, in synergy with hydrolytic enzymes, increases the production of monomer sugars from the biodegradation of lignocellulose. This study was aimed at evaluating actinomycete S2 strain LPMO activity based on the release of xylose as one of reducing sugar and hydrogen peroxide (H2O2) in the course of lignocellulosic biodegradation. Methodology and results: The oxidation activity of LPMO from actinomycete S2 strain was measured by using the substrate of Avicel supplemented with ascorbic acid and copper ions (Cu2+) to identify its effect on the release of xylose as one of reducing sugar. The optimum incubation time for the LPMO production was also conducted. Further, H2O2 quantitative analysis was performed as by-product of LPMO activity and 16S rRNA gene sequence of actinomycete S2 strain were subsequently determined. We found that supplementation of 1 mM ascorbic acid and 0.2 mM Cu2+ increased xylose as one of reducing sugar production by up to 5-fold from 255.03 to 1290 μg/mL after an optimal incubation period of 6 days. Based on H2O2 production, the LPMO activity of actinomycete S2 strain was 0.019 ± 0.001 U/mL. There is likelihood that LPMO activity derived from actinomycete S2 strain has a synergistic effect with the activity of other lignocellulose-degrading enzymes. This actinomycete showed 99% similarity to the 16S rRNA gene sequence of Streptomyces avermitilis strain EAAG80. Conclusion, significance, and impact of study LPMO enzyme activity from actinomycete S2 strain as determined by the production of reducing sugar and H2O2 was greatly increased by supplementation with ascorbic acid as an electron donor and Cu2+ ions. To the best of our knowledge, this is the first elucidation of LPMO activity from an indigenous Indonesian actinomycete.
Mixed Function Oxygenases--metabolism ; Lignin--metabolism