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Abubakar H, Astuti RI, Listyowati S, Batubara I, Wahyudi AT. 2022. An orange pigment from the marine bacterium Paracoccus haeundaensis SAB E11 as a prospective source of natural antioxidants. Biodiversitas 23: 4730-4737. Bacterial pigment extracts are the source of many natural antioxidant substances. The present study aimed to assess the antioxidant activity of an orange pigment derived from a marine bacterium, identified as Paracoccus haeundaensis SAB E11, analyzed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azino-bis(ethylbenzothiazoline-6-sulfonic acid) (ABTS). Methanol-pigmented crude extract of P. haeundaensis SAB E11 exhibited the best DPPH scavenging activity with an IC50 value of 54.7 mg/mL, while the n-hexane crude pigment extract of the bacterium reduced ABTS optimally with an IC50 value of 24.46 mg/mL. The analysis of antioxidant compounds in P. haeundaensis SAB E11 was identified by combining liquid chromatography with tandem mass spectrometry (LCMS/MS). The five antioxidant compounds from methanol and n-hexane crude pigment extract of P. haeundaensis SAB E11 have been detected. Polyphenols were found to be the major compound, such as 3,5,3’,4’-tetrahydroxystilbene, scopoletin, and liquiritigenin. The other compounds were dihydroactinidiolide (terpenes) and ricinoleic acid (a fatty acid compound). The orange-colored extract of marine bacterium P. haeundaensis SAB E11 could be promising pharmacological capabilities as an antioxidant.

Abstract. Suryanti E, Rusamana I, Wahyudi AT, Akhdiya A, Sukamdjaja D. 2022. Constructing and expressing acyl-homoserine lactone lactonase (aiiA) gene for enhancing Solanum tuberosum resistance against soft rot disease. Biodiversitas 23: 1780-1787. Expression of the genes associated with pectinase production in soft rot disease of Dickeya spp. are regulated by quorum sensing mechanism using acyl homoserine lactone(AHL). Degradation of AHL using AHL lactonase can be developed to reduce pectinase production in Dickeyaspp.The aiiA gene encodes AHL lactonase from Bacillus spp. This study aims to construct binary vector of inserted aiiA gene (pCAB10) from Bacillus cereus B10 and express the aiiA gene in potato Medians cultivar genome. The aiiAgene was cloned into binary vector and transformed into Agrobacterium tumefaciens LB4404. The Agrobacterium-mediated genetic transformation was conducted with co-cultivation technique.The transgenic plant was confirmed through PCR andselected through kanamycin and soft rot disease resistance to Dickeya dadantii A3. The result showed that aiiA gene was successfully integrated top CAB10 and introduced to A. tumefaciens LB4404. The percentage of pCAB10 transformation was 57.6%, while there generation efficiency was 11.9% with 50 mg/L acetosyringone addition. Four transgenic potato Medians cultivar exhibited significantly higher resistance against soft rot disease than non-transgenic plants. Mahl1 clone had the highest resistance to D. dadantii with soft rot disease incidence as 7.1% and percentage of soft rot disease suppression as 92.85%.

Mesrian DK, Purwaningtyas WE, Astuti RI, Hasan AEZ, Wahyudi AT. 2021. Methanol pigment extracts derived from two marine actinomycetes exhibit antibacterial and antioxidant activities. Biodiversitas 22: 4440-4447. In recent years, natural pigments produced by marine microorganisms, especially actinomycetes, have become an important source of medically valuable drugs and other active compounds. Based on that crucial fact, this study aimed to determine the toxicity, antimicrobial, and antioxidant activities of methanolic pigment extract derived from marine actinomycetes HV11.P3 and SCA54.P2. Both extracts exhibited high toxicity activity based on Brine Shrimp Lethality Test (BSLT). The results showed the lethal concentration 50% (LC50) values of actinomycetes HV11.P3 and SCA54.P2 pigment crude extracts were 94.60 µg/mL and 131.22 mg/mL, respectively. The antimicrobial activity of those extracts was also tested against several microbes and showed the best results for inhibiting against Bacillus subtilis and Escherichia coli. Furthermore, we assessed the antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals and the results showed the inhibitory concentration 50% (IC50) values ??of actinomycetes HV11.P3 and SCA54.P2 pigment crude extracts were 231.08 µg/mL and 369.3 µg/mL, respectively. By using GC-MS analysis, we identified chemical compounds of actinomycete pigment crude extracts, revealing that the most dominant compounds were 1H-Purine-2,6-dione, 3,7-dihydro-1,3,7-trimethyl- (CAS) (44.65%) for HV11.P3 and 5-Methoxypyrrolidin-2-one (30.23%) for SCA54.P2. The 16S rRNA gene sequence analysis showed that actinomycete HV11.P3 and actinomycete SCA54.P2 had the closest similarity to Micromonospora chalcea strain 1464-217L and Micromonospora tulbaghiae strain TVU1, respectively. This study revealed that two pigment crude extracts derived from actinimycetes HV11.P3 and SVA54.P2 had antibacterial, antioxidant, and toxicity activities. The actinomycete SCA54.P2 pigment crude extract had the best activity as an antibacterial against E. coli ATCC 8739, while actnomycete HV11.P3 pigment crude extract had the best activity as an antioxidant.

Wahyudi AT, Fitriansyah NG, Amri MF, Priyanto JA, Nawangsih AA. 2021. Screening of chitinase-producing rhizosphere actinomycetes and their genetic diversity. Biodiversitas 22: 4186-4192. Soil microbial community, especially rhizosphere actinomycetes, plays an important role in protecting plants from phytopathogenic fungi by producing various agroactive compounds, including mycolytic enzymes particularly chitinase. The objective of this study was to investigate chitinase activity and analyze the diversity of family 19 chitinase genes of actinomycetes isolated from maize and soybean rhizosphere. Of fivety actinimycetes, Seventeen isolates exhibited chitinolytic activities and formed a hydrolytic zone around the colony with chitinolytic index ranging from 0.49±0.01 to 2.15±0.69, qualitatively tested in 0.3% chitin agar medium. Six selected isolates (ARJ 36, ARJ 81, ARJ 15, ARK 17, ARK 143, ARK 103) showed chitinase activities ranging from 0.157±0.04 to 0.440±0.09 U/mL, based on chitinolytic index. The production of chitinase was confirmed by the presence of family 19 chitinase encoding genes from Streptomyces sp. Some conserved regions and essential amino acid residues were also detected. This study suggests that chitinolytic actinomycetes isolated from maize and soybean rhizosphere can be studied further as biological control candidates for controlling phytopathogenic fungi.

Wijayanti M, Wahyudi AT, Yuhana M, Engelhaupt M, Meryandini A. 2019. Impact of Bukit Dua Belas rainforest transformation to oil palm plantation on phylogenetic of soil bacterial communities in Sarolangun, Jambi, Sumatra, Indonesia. Biodiversitas 20: 811-818. Land use change from forest to oil palm plantation at Sumatra could decrease biodiversity, including bacterial diversity. The case of Bukit Dua Belas transformation from forest to oil palm plantation was gotten for measuring shift community of soil bacterial in both areas. The diversity of bacterial communities from rainforest and oil palm plantation topsoil in Sumatra was studied using pyrosequencing of 16S rRNA gene and common biodiversity indices. Phylogenetic approach was used to reveal the community shift of bacterial phyla and genera in both areas. Ecological approach was carried out by measuring soil pH, TC (total carbon), TN (total nitrogen), AP (available phosphorous), bacterial diversity with Shannon and Simpson indices, and bacterial richness with Chao1-ACE index and OTUs. Bacterial diversity and richness on lowland forest topsoil and oil palm plantation soil were not different, as soil pH, TC, and TN as substrate factors were not different significantly. The majority of sequences related to Acidobacteria (56.33%), Proteobacteria (27.43%), Actinobacteria (7.11%), and Cyanobacteria (5.55%) were from forest; whereas those related to Acidobacteria (50.11%), Proteobacteria (31.63%), Actinobacteria (7.58%), Chloroflexi (2.60%), and Gemmatimonadetes (2.71%) invented from oil palm plantation. Acidobacteria was the most dominant phyla in both habitats, because soil pH in both areas was acidic (3.77 - 4.80 pHH2O). The genera of alpha-proteobacteria dominated in genera phylotype of bacterial 16S rRNA phylogenetic revealed in both forest and oil palm plantation topsoil. The most genera in phylogenetic tree are Burkholderia from Beta-proteobacteria. The bacterial community shift occurred in forest transformation, even though the oil palm plantation showed more bacterial phyla and genera than the lowland rainforest.

Lingga R, Budiarti S, Rusmana I, Wahyu AT. 2020. Isolation, characterization and efficacy of lytic bacteriophages against pathogenic Escherichia coli from hospital liquid waste. Biodiversitas 21: 3234-3241. Escherichia coli is known as a pathogenic contaminant bacteria in hospital wastewater hazardous to humans and the environment. Concerns about the emergence of chlorine- and antibiotic-resistant bacteria increase the urgency to find an alternative strategy to control pathogenic bacteria in hospital wastewater. One of the alternatives is using lytic bacteriophage. This study aimed to isolate, characterize, and examine the efficacy of lytic bacteriophage against pathogenic Escherichia coli from hospital wastewater. It isolated, characterized (plaque morphology, host range, virion electron micrograph, and sensitivity to temperature, pH, and chlorine treatments), and tested the efficacy of lytic bacteriophages in controlling pathogenic E. coli isolated from hospital wastewater. Five phages were successfully obtained, all of which had clear plaques (lytic phage character). Based on host range assay, most of the phages could lyse all tested E. coli strains but not for other species. Electron micrograph photography revealed that the phages belonged to Myoviridae. The phages showed stability in high temperature, broad-ranged pH, and high concentrations of chlorine treatments. Assay on phages efficacy suggested that the phages are capable of significantly reducing the E. coli population both in sterilized and non-sterilized wastewater. The combination of phage treatment and chlorine was more effective than single phage treatment. The efficacy test revealed that phage application in wastewater had the best result seen from cocktail treatment and a combination of phage treatment and chlorine. These results suggested that the phage can be a potential candidate for disinfection purposes.

Wiraswati SM, Nawangsih AA, Rusmana I, Wahyudi AT. 2020. Rice phyllosphere bacteria producing antifungal compounds as biological control agents of blast disease. Biodiversitas 21: 1273-1278. In vitro analysis of rice phyllosphere bacteria can be developed as biocontrol agents of blast disease using their antifungal activity. To prove the efficacy of bacterial isolates in reducing the severity of blast disease, a greenhouse experiment was conducted. Furthermore, antifungal compounds produced by potential bacterial isolate were also identified using Thin Layer Chromatography (TLC) and Liquid Chromatography-Mass Spectrometry/ Mass Spectrometry (LC-MS/MS) analysis. The application of seven rice phyllosphere bacterial isolates significantly reduced the severity of blast disease in rice (var: Ciherang). Surprisingly, the isolate STGG 14 is the most effective with 70.83% of blast disease reduction. Isolate STGG 14 has been identified as Bacillus subtilis subsp. subtilis which is known as various bioactive compounds producer. It was confirmed through identification of antifungal compounds produced by isolate STGG 14. The bioautography test shows that crude bioactive compounds from isolate STGG 14 could inhibit P. oryzae race 173. On the further analysis using LC-MS/MS, four bioactive compounds isolated from isolate STGG 14 were identified as moracin C, psoralen, 4,6-dimethyl-3 (4’-hydroxyphenyl) coumarin and xanthotoxin. All identified compounds except moracin C, belong to the coumarin group. According to the previous studies, among all compounds, psoralen and xanthotoxin displayed antifungal activity against several fungal pathogens.

Maharsiwi W, Astuti RI, Meryandini A, Wahyudi AT. 2020. Screening and characterization of sponge-associated bacteria from Seribu Island, Indonesia producing cellulase and laccase enzymes. Biodiversitas 21: 975-981. Exploration of new enzymes from an extreme environment is important to improve industrial efficiency. This study aimed to get sponge-associated bacteria from Seribu Island with the capability to produce cellulase and laccase. These enzyme activities were indicated by the clear zones on CMC medium for cellulase and the reddish-brown zone on Guaiacol medium for laccase. About 100 of sponge-associated bacteria have been isolated from 5 marine sponges used SWC and NA modified media. As screened, one isolate (AGN89) could produce both enzymes and 11 isolates could produce cellulase. Quantitative analysis was performed using the DNS method and obtained the activities of 4 best cellulolytic isolates ranged from 0.04-0.06 UmL-1 and 0.70-1.18 UmL-1 in enzyme and specific activities, respectively. Gene-based determination for the isolate producing laccase resulted in a ±1100 bp amplicon fragment which identified as multicopper oxidase family protein. Based on the 16S-rRNA gene, AGN89 and these 4 cellulolytic isolates were identified as Pseudomonas luteola strain NBRC 103146, Bacillus aerius strain 24k, Pseudomonas aeruginosa strain DSM 50071, Mycobacterium maritypicum strain DSM 20578, and Brachybacterium conglomeratum strain J 1015. This result suggests that the sponge-associated bacteria from Seribu Island could become new enzymes producer for further applications in industry.

Wiraswati SM, Wahyudi AT, Rusmana I, Nawangsih AA. 2018. TRFLP analysis for revealing the diversity of rice phyllosphere bacteria. Biodiversitas 19: 1743-1749. Phyllosphere environment of rice plant is usually inhabited by diverse bacteria which mostly contribute beneficial effects to the plant fitness. TRFLP method is a rapid and straightforward method to determine the bacterial diversity of many environments, including rice phyllosphere environment. This study aimed to analyze rice phyllosphere bacterial diversity of healthy rice plant cultivar Ciherang obtained from Sukabumi, Jasinga, and Situgede. The bacterial genomes were amplified and digested with two restriction enzymes, i.e., MspI and BstUI. The bacterial diversity (H’ index) and evenness (E index) were calculated from the peak value. From TRFs analysis, Betaproteobacteria and Pseudomonadales were dominantly found in nearly all samples with different relative abundance. In addition, Alphaproteobacteria and Gammaproteobacteria were also dominant in the several samples. The unique bacteria groups were inhabited in the sample from specific regions with certain growth phase. This finding informs us that the geographical factors might be more influent than the growth phase factor. Furthermore, the bacterial diversity and evenness of the metagenomic approach are higher than cultivation-dependent approach.

Raharja NI, Widanarni, Wahyudi AT. 2019. Marine sponge-associated bacteria as biocontrol agents of vibriosis on whiteleg shrimp caused by Vibrio parahaemolyticus. Biodiversitas 20: 3164-3169. Sponge-associated bacteria are commonly known as an excellent source of microbial's bioactive compounds attributed to biological properties, including antibacterial activity. In our previous study, 12 isolates from sponge Aaptos sp. and Hyrtios have been screened to have anti-Vibrio spp. activities in vitro. The objective of this study was to evaluate these bacterial isolates for controlling Vibrio parahaemolyticus infection in vivo, identify the most potential isolate, and analyze chemical composition of bacterial crude extract. As tested by pathogenicity assay on post-larvae of whiteleg shrimp stadia PL 10, all 12 isolates were not pathogenic bacteria as indicated by the survival rate of shrimps ranging from 80 ± 8.1 - 93 ± 4.7%. Conversely, more than 50% died after inoculated with pathogenic V. parahaemolyticus. Interestingly, all isolates could increase the survival rate of shrimps infected with V. parahaemolyticus (105 cells/mL) reach of 80 ± 0.8 - 91 ± 0.4%, nearly 40% higher than the positive control, as evaluated by challenge test. The best anti-Vibrio activity showed by D2.Z13 isolate which could improve the survival rate up to 91.11 ± 0.4%. 16S-rRNA-based identification showed that this isolate was closely related to Pseudomonas aeruginosa 1-4-b-9 strain. The extract derived from this isolate contained some major compounds such as 4,8-dihydroxy-2-2 (1’-hydroxyheptyl) and 1,2,4-oxadiazol which have been reported as antibacterial compounds. Our study indicates that these bacterial isolates could be developed as biocontrol agents of vibriosis in whiteleg shrimp.

Fatmawati U, Meryandini A, Nawangsih AA, Wahyudi AT. 2019. Screening and characterization of actinomycetes isolated from soybean rhizosphere for promoting plant growth. Biodiversitas 20: 2970-2977. Actinomycetes which colonized plant rhizosphere has a vital role in improving plant growth by producing Indole-3-Acetic Acid (IAA). The aim of this study was to evaluate the potency of actinomycetes isolated from soybean rhizosphere as promoter agents for plant growth in vitro. Fifty actinomycetes isolates were successfully isolated from soybean rhizosphere. Based on the colorimetric methods, 35 isolates can produce IAA in various concentration, in the range of 0.46-30.6 mg/L. Seed germination assay using Ragdoll methods revealed that 26 isolates significantly promoted germination parameters, including the hypocotyl and the radicular length, the number of the lateral roots, and dry weight of the plant. Also, 14 from 26 isolates showed phosphate solubilizing activity in different phosphate-solubilizing index ranging from 1.25-2.62. Eight isolates were able to grow in N-free medium, indicating that these isolates have the ability in fixing nitrogen. Out of 23 from 26 isolates were detected to produce siderophore. All the tested isolates show chitinase production except ASR 55. Based on the observed parameters, it showed that there are four potential isolates (ASR 46, ASR 58, ASR 75 and ASR 76) as promising plant-growth promoters, phosphate solubilizer, nitrogen fixer, siderophore and chitinase producer. Based on the result of 16S rRNA sequence analysis, four potential isolates were identified as Streptomyces spp. in different taxa of strains and species.

Wahyudi AT, Priyanto JA, Fijrina HN, Mariastuti HD, Nawangsih AA. 2019. Streptomyces spp. from rhizosphere soil of maize with potential as plant growth promoter. Biodiversitas 20: 2547-2553. Actinomycete is one group of rhizobacteria that plays an important role as a plant growth promoter. This study was aimed to evaluate the potential of Actinomycetes isolated from maize rhizosphere in promoting plant growth in vitro including their ability to produce IAA, promote maize sprout growth, solubilize phosphate, and grow in N-free medium. Thirty isolates have been isolated from maize rhizosphere using a spread plate method. All 30 isolates were probably not pathogenic to plants as tested by hypersensitivity reaction test on tobacco leaves. Based on the colorimetric assay, 30 isolates (100%) were able to produce IAA with concentrations ranging from 1.05 to 26.89 ppm. The highest concentration of IAA (26.89 ppm) produced by ARJ 21 and the lowest IAA concentration (1.05 ppm) produced by ARJ 12. By using the Ragdoll method, it showed that 9 isolates (30%) were able to promote maize sprout growth significantly on five growth parameters including primary root length, stem length, number of lateral roots, wet weight and dry weight. Twenty-one isolates (70%) were capable of solubilizing phosphate in Pikovskaya medium containing tricalcium phosphate. Also, 30 isolates (100%) were able to grow on N-free medium, suggesting their ability to fix nitrogen. Based on 16S-rRNA, five potential isolates with plant growth-promoting properties were highly similar to Streptomyces spp. Based on their potential characters, these Actinomycetes isolates have the potential to be further developed as a biofertilizer agent for sustainable maize farming.