Prospects of Indigenous Bacillus subtilis Strain LS9.1 as a Potential Biocontrol Agent against Aedes aegypti Larvae

Biocontrol is one way that can be developed to overcome public health and agricultural problems. Biocontrol agents could be a microorganism, for example, from the Bacillus group. This study aims to determine the larvicidal toxicity of the Bacillus subtilis strain LS9.1 against Aedes aegypti larvae. The mode of action of the isolate caused larval death, which was detected with parasporal inclusions and secondary metabolites. Larvicidal toxicity with the bioassay method. Parasporal inclusion proteins were detected with the SDS-PAGE method. The Cry toxin coding gene with the PCR method and cell ultrastructure with transmission and scanning electron microscopy (TEM and SEM). The action of secondary metabolites was detected with hemolytic activity test. The isolate high toxicity to A. aegypti larvae. Parasporal inclusion proteins with molecular weights ranging from 72-7 kDa and the gene encoding a protein toxin in size 300 bp. The TEM and SEM results did not Cry-IV toxin. The hemolytic activity test showed a positive reaction correlated with biosurfactant production. Based on this study, the death of larvae is probably caused by secondary metabolites produced by Bacillus subtilis strain LS9.1. Thus, this isolate has the prospect of being an environmentally friendly biocontrol agent to disease vectors and insect pests

INDIGENOUS BACILLUS SPECIES ISOLATED FROM AEDES AEGYPTI LARVAE: ISOLATION, LARVICIDAL TOXICITY SCREENING, PHENOTYPIC CHARACTERIZATION, AND MOLECULAR IDENTIFICATION

Vector-borne diseases transmitted by mosquitoes are considered a significant public health problem worldwide. Aedes aegypti is one of the mosquito species responsible for transmitting these diseases. One environmentally friendly method of vector control is the use of microbial agents such as Bacillus species. This study aimed to explore investigate indigenous entomopathogenic bacteria of Bacillus species isolated from A. aegypti larvae. Larvae samples were collected from breeding sites of A. aegypti. All isolates underwent screening and affirmation confirmation tests to assess their larvicidal toxicity against A. aegypti larvae. Phenotypic characterizations and molecular identifications were conducted to determine the species of the Bacillus isolates based on similarity index and percent identity (%ID). Phylogenetic trees were used to compare the isolates with other Bacillus species. The results revealed 120 isolates of Bacillus species from A. aegypti larvae samples. Among them, three isolates (LS3.3, LS9.1, and LSD4.2) exhibited the highest larvicidal toxicity in the confirmation test, resulting in larval mortality rates of 100%, 96.7%, and 100%, respectively, after 48 hours of exposure. Molecular identifications, showed that LSD4.2 had a 99.16% ID with Bacillus velezensis, LS3.3 had a 98.22% ID with Bacillus mojavensis, and LS9.1 had a 99.93% ID with Bacillus subtilis. These three bacteria from the Bacillus genus have been reported to offer significant benefits to humans

Toksisitas Residual Bacillus Sp. Isolat Lsd4.2, Ls3.3, Dan Ls9.1 Di Tempat Perindukan Nyamuk Aedes Aegypti

Tujuan penelitian ini adalah: (1) mengetahui jenis isolat Bacillus sp. yang mempengaruhi toksisitas residual di tempat perindukan larva nyamuk A. aegypti., (2) mengetahui konsentrasi isolat Bacillus sp. yang mempengaruhi toksisitas residual di tempat perindukan larva nyamuk A. aegypti., (3) mengetahui interaksi antara jenis dengan konsentrasi isolat Bacillus sp. dalam mempengaruhi toksisitas residual di tempat perindukan larva nyamuk A. aegypti. Penelitian ini dilaksanakan secara eksperimental menggunakan rancangan faktorial 3x3, repeated measurement dengan pengulangan 3 kali. Variabel pertama, yaitu tiga jenis isolat lokal Bacillus sp. LSD4.2, LS3.3, dan LS9.1. Variabel kedua, yaitu variasi konsentrasi isolat yang digunakan 8%, 10%, dan 12%. Data dianalisis dengan ANAVA (Analisis Varian) dua arah dan uji Kruskal Wallis. Hasil penelitian menunjukkan: (1) jenis isolat Bacillus sp. mempengaruhi toksisitas residual di tempat perindukan larva nyamuk A. aegypti. Isolat LS9.1 paling optimal membunuh larva A. aegypti. Isolat ini efektif membunuh larva di Minggu I pada pengamatan 24 dan 48 jam, (2) berbagai konsentrasi isolat Bacillus sp. mempengaruhi toksisitas residual di tempat perindukan larva nyamuk A. aegypti. Konsentrasi yang paling optimal membunuh larva A. aegypti adalah 8% untuk pengamatan 24 jam dan 10% untuk pengamatan 48 jam selama pemaparan Minggu I dan Minggu II, (3) terdapat interaksi antara jenis dengan konsentrasi isolat Bacillus sp. yang dapat mempengaruhi toksisitas residual di tempat perindukan larva nyamuk A. aegypti. Jenis isolat LS9.1 dengan konsentrasi 8% dan 10% mempunyai daya bunuh larva yang lebih baik dibandingkan jenis isolat LSD4.2 dan LS3.3 dengan konsentrasi 8%, 10%, dan 12% pada Minggu I dan Minggu II selama pengamatan 24 jam dan 48 jam

Analysis of Lead (Pb) in Leaf of Tabebuia aurea from Polluted Air

One of the three major heavy metals which can be harmful to plants, animals, and humans is lead (Pb). Smoke from gasoline fueled motor vehicles become the sources of these pollutants. In plants, including Tabebuia aurea can be found in the roads that are often passed by vehicles, so lead exposure is unavoidable.The purpose of this research was 1) to analyze the lead levels in T. aurea leaves, 2) to analyze the chlorophyll levels in T. aurea leaves, 3) to analyze the growth (leaf area). The Pb level in plant leaves was calculated using AAS (Atomic Absorption Spectrophotometer), chlorophyll level using spectrophotometer, growth was measured with leaf meter. Data were analyzed by descriptive and Anova. Based on the research and analysis that have been carried out can be concluded that 1) Pb metal levels 0.09 – 0.187 mg/L, 2) leaf chlorophyll levels ranges from 2.719 – 7.594 mg/L, and 3) Leaf area ranges from 186.720 – 199.288 cm2.Analysis with Anova ahows that the sampling location affected the Pb and chlorophyll content in the leaves,while the location did not affect the surface area of T. aurea leaves. The results of this research indicate that T. aurea can be used as a plant to absorb Pb pollutants in the air

The Potential of Tabebuya as Phytoremediator of Lead (Pb) in Atmosphere

Lead (Pb) is one of the heavy metals that causes pollution in ecosystems, heavy metal Pb can cause disturbances to humans, including plants and animals. In Indonesia, especially in Surabaya, Tabebuya grows on main roads as a shade tree as well as an ornamental plant; This plant can be exposed to Pb metal, among others, from motor vehicle exhaust. This study aimed to determine the levels of Pb in Tabebuya and to study chlorophyll in the leaves. Pb levels in plant leaves were measured using an atomic absorption spectrophotometer (AAS), and chlorophyll levels using a spectrophotometer. Data in the form of Pb and chlorophyll levels at various locations were analyzed by analysis of variance (Anova). The results showed that the location of plant species affected the content of Pb metal and there is no effect of chlorophyll content in the leaves. The results also showed that the absorption of lead (Pb) content by the tabebuia 0.25-0.44 ppm and content of chlorophyll 12.89-14, 94. Lead (Pb) content do not affect the amount of leave chlorophyll produced and the plant had capabilities in absorbing lead like pollutants (Pb)

Potential biocontrol agent of indigenous Bacillus sp. EG6.4: Molecular identification, larvicidal toxicity, and mechanism of actions

Salamun, Susetyo RD, Nafidiastri FA, Zain RA, Sari RP, Geraldi A, Fatimah, Ni’matuzahroh. 2022. Potential biocontrol agent of indigenous Bacillus sp. EG6.4: Molecular identification, larvicidal toxicity, and mechanism of actions Biodiversitas 23: 5431-5438. This research was carried out for molecular identification, as well as the determination and mechanism of action of larvicidal toxicity of Bacillus sp. EG6.4 isolated from breeding sites of Aedes aegypti from Gresik, East Java, Indonesia. Bacillus sp. EG6.4 was a Gram-positive endospore-forming bacteria. Molecular species identification using 16S rRNA gene sequencing showed that isolate had 97.89% similarity with Bacillus mojavensis. The isolate showed larvicidal toxicity against A. aegypti larvae. The lethal concentration 50% (LC50) values ??at 24 and 48hours exposure were 8.99±1.01 ×107 cells/mL and 8.43±1.01 ×107 cells/mL, respectively, while lethal time 50% (LT50) value was 11.9±1.1 hours. Production of chitinolytic enzymes or biosurfactants, chitinolytic and hemolytic assays were conducted to determine the larvicidal mechanism. As a result, Bacillus sp. EG6.4 showed hemolytic, but not chitinolytic activity, indicating its potency to produce biosurfactants. Transmission Electron Microscopy (TEM) result showed that isolate had oval-shaped endospores located subterminal with massive-shape parasporal inclusions. The detection of srfA-D gene showed that isolate produced surfactin biosynthesis thioesterase. Thus, Bacillus sp. EG6.4 produced biosurfactant that potentially to be developed as a biocontrol agent for disease vectors and plant pathogens

Biosurfactant production of entomopathogenic Bacillus subtilis BK7.1, as potential biocontrol bacteria, isolated from Baluran National Park, East Java, Indonesia

Salamun, Susetyo RD, Ni’matuzahroh, Fatimah, Geraldi A, Supriyanto A, Nurhariyati T, Nafidiastri FA, Nisa’ N, Endarto. 2023. Biosurfactant production of entomopathogenic Bacillus subtilis BK7.1, as potential biocontrol bacteria, isolated from Baluran National Park, East Java, Indonesia. Biodiversitas 24: 1785-1792. Biosurfactants as biocontrol agents have received much attention for pest control and disease vectors. The research aimed to identify the species and genetic relationship, hemolytic activity, detect coding genes, and trial production of biosurfactants on various substrates of entomopathogenic Bacillus sp. BK7.1 isolated from natural soil in Baluran National Park, East Java, Indonesia. Biosurfactant screening was carried out by testing hemolytic activity, surface tension, and emulsification activities, detecting coding genes of biosurfactant biosynthesis, and testing biosurfactant production in various substrates. The results of the molecular identification by amplifying the 16S rRNA gene using the Polymerase Chain Reaction (PCR) method for Bacillus sp. BK7.1 has a genetic similarity of 98.68% with B. subtilis subsp. inaquosorum strain BGSC 3A28. Screening showed positive hemolytic activity results, reduced surface tension, increased emulsification activities, and the production of biosurfactant in glucose, glycerol, and molasses substrates. The PCR results showed that Bacillus sp. BK7.1 had srfAA and srfAD genes encoding surfactin biosynthesis, giving it the potential to produce bioinsecticide compounds. Based on these studies, the indigenous entomopathogenic B. subtilis BK7.1 can be developed as environmentally friendly microbial bioinsecticides for pest control and disease vectors

Isolation and screening of potential proteolytic and amylolytic microbes from Wonorejo Mangrove forest soil, Surabaya, Indonesia

The aims of this research were to isolate and to identify the microbes that have potential amylolytic and proteolytic activities. Microbial isolation was performed on mangrove soil sample from Wonorejo, Surabaya, by plating samples on Nutrient Agar (NA), Potato Dextrose Agar (PDA), Nitrogen free bromothymol blue (Nfb), Carboxymethylcellulose (CMC), and Pikovskaya agar media. Microbial isolates obtained were grown on Bushnell Haas agar enriched with starch or milk to test production of amylase and protease enzymes. The results revealed three kinds of bacteria and six kinds of moulds that were purified and characterised. The two kinds of bacteria had NA1 and CMC1 isolate codes and the two kinds of moulds were PDA20131 and PIKOV1513, which could grow on PDA and Pikovskaya media, and were used to test their amylolytic and proteolytic activities. Results of proteolytic tests revealed that there were two microbial isolates that could produce proteases, i.e. PDA20131 and a Gram-negative rod bacteria (NA1). The isolates that produced amylases were PIKOV1513 and a Gram-negative rod bacteria capable of growing on the CMC media (CMC1)

Screening and identifying of cellulolytic bacteria from Alas Purwo National Park

This research aimed to screen and identifies cellulolytic bacteria from Alas Purwo National Park. Soil samples were taken from five points in each location by indirect sampling method. Bacteria were isolated with CMC (Carboxymethylcellulose) agar. The result had successfully obtained 16 isolates which produced cellulase. The cellulase activity index assay of bacteria used CMC agar modified with adds 1 % YME (Yeast Malt Extract) and then was incubated during one day at room temperature. Bacterial colonies were soaked with Congo red 1 % and rinsed with NaOH 10 % to visualize the halo zone formed. Three isolates with highest cellulase activity index were identified by observing the colony morphology, cell morphology, and the biochemical characters. The results of this research showed that the three highest potential isolates producing cellulase were BPA-B, BSA-D, and BPA-A with cellulase activity indexes respectively 10.67, 9.86, and 8.16. BPA-B, BSA-D, and BPA-A each have the similarity with genera Pimelobacter, Micrococcus, and Cellulomonas