Abstract
Background
Mosquitoes are ancient enemies of humans and act as vectors of many life-threatening diseases, including dengue, malaria, chikungunya, filariasis. In Bangladesh, more than a hundred thousand individuals suffered from dengue in 2019 and this number is increasing consistently at every year. For sustainable control of mosquitoes, we aimed to isolate and characterize mosquito controlling Bacillus species from mangrove forests and municipal areas in Bangladesh.
Results
Initially, 38 bacteria were isolated in laboratory via the heat-shock method and categorized by morphological studies, Gram staining, and catalase test. To conduct different in vitro and in vivo trials, 16S rRNA gene sequencing-based molecular prediction of 12 bacterial strains was conducted via amplification with 27 F and 1492R universal primer pairs. Antimicrobial assays, salt and pH tolerance tests, amylase, cellulase, protease, and antibiotic susceptibility test were performed on Bacillus sp. to identify their multiplication potential in diverse and challenging environments. However, larvicidal assay was conducted against Aedes (Aedes aegypti) and Culex (Culex quinquefasciatus) mosquitoes to select the best potential biopesticide among the 6 selected Bacillus species (Bacillus tropicus, Bacillus thuringiensis, Bacillus zanthoxyli, Bacillus altitudinis, Bacillus pseudoflexus, and Fictibacillus barbaricus), and a lethality test was conducted for four potential biopesticides, all of which showed no lethality to Artemia salina in aquatic environment. The whole genome sequence (WGS) data obtained with the “K-mar” finder revealed that the best biopesticide bacteria was Bacillus altitudinis.
Conclusions
To the best of our knowledge, this is the first report that Bacillus altitudinis has mosquito larvicidal activity and can be a commercial and ecofriendly mosquito controlling biopesticides after scaling up.
Keywords: Mosquitoes; 16S rRNA gene; Differential assays; Whole genome sequencing,; Bacillus altitudinis; and Biopesticide
Background
Mosquitoes transmit etiological agents that produce several life-threatening illnesses like malaria, filariasis, Japanese encephalitis, dengue, and chikungunya, as well as various arbovirus diseases, including West Nile virus, Saint Louis encephalitis virus, and Eastern Equine encephalitis virus [1]. There has been an alarming increase in the occurrences of chronic vector-borne diseases in tropical regions worldwide over the past few decades, and Bangladesh is no exception. This is mainly because mosquitoes have high reproductive capacity and a significant degree of genetic flexibility making them difficult to control. Since 2000, Bangladesh has faced nearly annual dengue outbreaks, with at least six of these outbreaks leading to more than 3000 dengue death cases [2]. In Bangladesh, more than 100,000 individuals were hospitalized in Dhaka in 2019 due to dengue infection, with 164 confirmed dengue-related deaths reported by the Directorate General of Health Services (DGHS) [3]. In addition, a significant Chikungunya virus (CHIKV) outbreak occurred in 2017 from April to September, putting over two million people at risk of viral infection [4]. According to the observations of Dhaka Medical College Hospital, Bangladesh, 690,399 cases were confirmed, and 291 most likely suffered during that period [5]. Therefore, it is crucial to take safer preventive measures against mosquitoes and the eco-friendly step ensures sustainable mosquito management.
The current methods for controlling mosquitoes include chemical control (DDT, malathion, parathion, fenthion, chlorpyrifos), genetic control (sterile insect technique or SIT), net use, and mosquito traps. The chemical insecticides used to control mosquitoes are not very selective and may negatively affect beneficial insects and other nontarget animals [6–8]. Over time, genetic variations may cause mosquitoes to become resistant to certain chemicals. Some chemical pesticides accumulate in soil, water, and sediment due to their extended environmental durability [9, 10]. Excessive exposure to chemical insecticides used for mosquito control can pose health risks to humans, such as respiratory irritation, allergic reactions, neurological effects, and increased cancer risk, particularly for individuals involved in the application or living in treated areas [11, 12]. The use of larvicides and insecticides potentially harms nontarget organisms, including humans, and the environmental damage caused by synthetic pesticides is becoming a great concern. Therefore, the WHO and the Pan American Health Organization (PAHO) jointly emphasize that the use of biological control involves using “biological agents” to interact with mosquito populations via parasitism, competition, or predation [13].
Compared with chemical pesticides, bacteria are less harmful to nontarget organisms and ecosystems and are, therefore, an environmentally friendly alternative [14]. Because they target mosquito larvae and do not harm other organisms, bacterial agents have a selective mechanism of action for example, Bacillus thuringiensis israelensis works by its Cry proteins, Bacillus sphaericus works by its paraspore protein crystal [15, 16]. Therefore, collateral damage to aquatic species and beneficial insects can be reduced. Mosquitoes do not develop resistance easily against bacterial biopesticides as they have different modes of action. Moreover, bacteria can persist in nature for a long time, and this strategy could keep mosquito larvae under control. Bacterial agents effectively target mosquito larvae at breeding sites and decrease adult mosquito populations, resulting in lower rates of infection with mosquito-borne diseases [17]. This study was conducted to develop a novel bacterial biopesticide against mosquito larvae and elucidate its antimicrobial and larvicidal properties.
Methods
Sample collection
A total of 17 environmental samples (soil, sediment, dead plants, and water) were collected in sterile plastic bags and bottles from the Sundarbans mangrove forest of Khulna (22°27’30.2”N89°23’49.9”E) as well as Wari (23°45.04’N 90°22.79’E) and Nakhalpara (23°45.5’N 90°23.5’E) of Dhaka city, Bangladesh (Table 1). The samples were transferred to the laboratory and stored at 4 °C.
Table 1.
Details about the collected samples, bacterial isolates, gram staining, and catalase test
| Sample location | Sample | Isolates | Gram Staining | Gram staining with KOH | Catalase test |
|---|---|---|---|---|---|
| Sundarbans mangrove forest, Khulna | Crab soil | (1WS) | - | - | ++ |
| (1WR) | + | + | -- | ||
| (1T3dot) | + | + | ++ | ||
| 3’deep soil | (2WR | + | + | ++ | |
| (2OR) | - | - | ++ | ||
| (2YR) | + | + | -- | ||
| Sea bed soil | (5WR) | + | + | ++ | |
| (5TW) | + | + | -- | ||
| Dead Bain plant | (6TW) | + | + | ++ | |
| (6RW) | + | + | ++ | ||
|
Rhizospheric Soil of Sundori plant |
(8WR) | + | + | ++ | |
| (8T) | - | - | -- | ||
| River bead water | (9WR) | + | + | -- | |
| (9YR) | - | - | -- | ||
| Sea bead soil | (10OR) | + | + | ++ | |
| (10YR) | + | + | -- | ||
| (10RW) | + | + | ++ | ||
| Dead Sundori plant | (11RWL) | + | + | ++ | |
| Sea bed soil | (12RW) | - | - | -- | |
| Water smear | (15YR) | + | + | -- | |
| (15R) | - | - | ++ | ||
| Surface soil | (17YR) | + | + | -- | |
| (17WR) | + | + | ++ | ||
| Rhizosphere soil of Golpata plant (Nypa fruticans) | (18IW) | - | - | -- | |
| (18RW) | + | + | ++ | ||
| (18YR) | + | + | -- | ||
| (18W1) | - | - | ++ | ||
| Wari, Dhaka |
Soil sample of drain |
(20RW) | + | + | ++ |
| (20W2L) | + | + | ++ | ||
| (20IWL) | + | + | -- | ||
| Drain water | (21RWL) | + | + | ++ | |
| (21RWM) | + | + | -- | ||
| Nakhal para, Dhaka | Soil of drain | (22RW) | + | + | ++ |
| (22IW) | - | - | ++ | ||
| (22T) | + | + | -- | ||
| (22WR) | + | + | ++ | ||
| Drain water | (23WR) | + | + | -- | |
| Soil of drain | (24 WR) | + | + | ++ |
WS white spread, WR white round, T3dot transparent 3 dot, OR orange round, TW transparent white, R rhizoid, IW irregular white, RW round white, W1 round white, IWL large irregular white, RWM round white medium, IW irregular white, IW irregular white
1,2,5,6,8,9,10,11,12,15,17,18,20,21,22,23,24 = sample collection code
+ = Gram positive and - = Gram negative
++ = catalase positive and -- = catalase negative
Isolation and cultivation of bacteria
A one-gram sample was placed into a sterile test tube containing 9 mL distilled water and mixed gently by vortexing to displace bacterial cells from the soil particles. In the case of the water sample, 1 ml water sample was taken. The samples were heated in the water bath (Memmert, Germany) at 80oC for 10 min. When the sample reached room temperature, a tenfold serial dilution (10−1 to 10−5) was performed. Nutrient agar (NA) media (HIMEDIA, India) was sterilized in autoclave (Taisite, USA) and poured into 100 mm sterile petri plates. After the media were cooled, 100 µl of the sample was placed on the plate containing NA media. The sample was spread onto the plate by using a sterile glass rod. The spread plates were incubated at 35 °C in an incubator (BINDER, Germany) for 24 h. To obtain pure culture, isolated bacteria were taken using a sterile inoculating loop and streaked into NA media. The subculturing technique was used to obtain pure bacterial cultures from each of the purified colonies, which were allowed to grow for 24 h, and stored at 4 °C.
Morphological and biochemical study of the Bacteria
The bacteria were characterized based on the shape, color, and average diameter of the colony. Gram staining and catalase tests were performed according to the protocol of the American Society of Microbiology. In the case of Gram staining, violet or purple color represents gram-positive, and pink or red color represents gram-negative when examined under a microscope (Olympus CX23LEDRFS1, China). In the catalase test, bubble formation indicates a positive reaction.
16S rRNA gene sequencing and prediction of bacterial species
DNA isolation was performed according to the protocol of the TIANamp bacterial DNA kit. A UV/Vis biophotometer (Eppendorf, Germany) was used to check the quality and quantity of the isolated bacterial genomic DNA. PCR amplification of 16S rRNA gene was performed via mini-PCR, m16-1541 universal primers; forward (27F): 5’-AGAGTTTGATCCTGGCTCAG-3’ and reverse (1492R): 5’-TACGGTTACCTTGTTACGACTT-3’ [18–20]. The PCR products were loaded on an agarose gel tank (Bluegel), after which the desirable PCR band was observed, that were selected by measuring the 1500 bp ladder marker. The amplified products were run on a Sanger machine using the dideoxy chain termination method at Genecreate Biotech, China. After the sequencing chromatogram was observed via Finch TV chromatogram viewer, the nucleotide BLAST was used to identify the bacteria most similar to each other by comparing the sequences found after 16S rRNA gene sequencing.
Antimicrobial test
The agar well diffusion method [21] was conducted to identify the Bacillus species’ antimicrobial activity and the bacteria, including Salmonella typhi, Staphylococcus aeruginosa, and Escherichia coli (American Type Culture Collection: ATCC type) were used in this assay. The procedure involved cotton swabbing the entire surface of the Mueller Hinton agar (HIMEDIA, India) plate (25 ml/100 mm) repeatedly six times, rotating the plate at a 30º angle each time. Wells were prepared by making a hole with a diameter of 5 mm by aseptic punching with a sterile tip, and a volume of 100 µL of the overnight cultured broth of turbidity 0.1 OD and 0.5 OD was introduced into the well. Then, the 5 µg disc of rifampicin and autoclaved distilled water were placed on each of the plates as positive and negative controls, respectively. After that, the plates were incubated for approximately 24–96 h at 37 °C. The diameters of the inhibition zones were observed and recorded.
Salt and pH tolerance test
Nutrient broth medium was prepared with different concentrations of NaCl (laboratory grade) of 1%, 3%, 5%, 7%, and 9%, and different pH values including 2, 4, 6, 8, and 10 were prepared with 1% HCl and 1 N NaOH. The bacteria were inoculated and incubated in a rotary incubator at 35 °C and 200 rpm for 24 h, and the bacterial growth was measured by observing the optical density (OD). Nutrient Broth containing no significant NaCl concentration (0%) and normal pH was inoculated with isolates and incubated for 24 h as a positive control. On the other hand, nutrient broth without inoculation of bacteria was incubated for 24 h, was used as a negative control. The OD was measured by using a spectrophotometer (C7100 Peak Instruments Inc., USA) at a wavelength of 600 nm to measure bacterial growth in each broth after 24 h of incubation.
Enzymatic assay
Amylase
For the amylase assay, the bacterial isolates were spot inoculated on starch agar media (HIMEDIA, India), and the plates were incubated at 30 °C for 24–96 h. After the incubation, the plates were flooded by using the iodine solution. A halo zone was observed around the colonies after 5–10 min that indicates the production of amylase enzyme by the isolate [19].
Cellulase
Cellulase enzyme assay was performed by culturing the sample bacteria on carboxymethyl cellulose (CMC) agar medium (carboxymethyl cellulose 26 g/l, agar 15 g/l). The plates were incubated at 37 °C for 5 days to allow for the secretion of cellulase enzyme by the bacteria. Following the incubation, the plates were flooded with a 1% (w/v) aqueous solution of Congo red for 15 min. After draining out the Congo red solution, the plates were further treated with 1 M NaCl for 15 min by flooding. The difference between the diameter of the halo and colony indicates cellulose breakdown by the bacterial cellulase, and the clear zone was measured in millimeters (mm) [22].
Protease
For the protease assay, the bacterial isolates were spot inoculated on skim milk agar (2% skim milk, 1% glucose, 1.8% agar, pH 9.0), and the incubation was performed at 30 °C for 24–96 h [23]. Protease production was also calculated similarly to the cellulase assay, in which we have measured the difference between the diameter of the halo and the colony, that creative halo zone indicates the protease enzyme producing isolate.
Antibiotic susceptibility assay
The test was performed according to the American Society of Microbiology’s Kirby-Bauer disc diffusion protocol [24]. The cotton swabs of isolates (0.1 OD) were streaked over the whole surface of Mueller Hinton agar (HIMEDIA, India) plates (25 ml/100 mm), which was repeated six times, with the plates rotated 30° each time. Afterward, the plate was allowed to dry for 5–10 min. The sterile forceps were used carefully to remove one disk from the cartridge and then placed on the streaked plate. The disc was gently pressed on the plate to ensure complete contact with the agar surface. Antibiotic discs containing ciprofloxacin (5 µg), tetracycline (30 µg), vancomycin (30 µg), ampicillin (25 µg), chloramphenicol (30 µg), and rifampicin (5 µg) were used every time the incubation was conducted at 35 °C for 24 h. Each zone of inhibition diameter revealed the intensity of susceptibility of the bacteria against the corresponding antibiotic. In our study, the results were explained based on standardized criteria provided by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). However, the bacteria are classified as susceptible, intermediate, or resistant according to the EUCAST of each antibiotic tested.
Mosquito larvicidal test
To collect the experimental mosquito (Aedes sp. and Culex sp.) larvae, a bucket with full of clear water was kept in a bush with open air for several days [25, 26]. After 5 days, mosquito’s larvae appeared on the water, and 10 mosquito larvae of both mosquitoes were collected for each bacterial sample in different petri dish, along with 15 ml of their habitat water. After that, 5 ml of corresponding bacterial isolate was inoculated into each petri dish containing larvae, where every ml of bacterial liquid culture was carried about 1 × 108 CFU. However, the bacterial culture was picked from the mother culture plate that were incubated overnight on the nutrient broth medium at 37 °C by using rotary shaker. The petri dish containing only 5 ml of media and 15 ml of their habitat water was used as the negative control. The larvicidal activity of the experimented bacteria was observed for 48 h, in which the data were collected after 24 h intervals in which the data were analyzed by Microsoft excel.
Lethality Test on Artemia salina
The lethality test was conducted on Artemia salina, an aquatic organism consumed as feed by fish. At first, the eggs of A. salina were hatched in saline water with continuous oxygen supplementation. Then, ten larvae of A. salina were collected in 100 ml of saline water in a sterile glass beaker to test against each bacterium. After that, 10 ml of each bacterial isolate was inoculated into a beaker containing larvae. Moreover, a beaker containing 10 ml of bacterial media and 100 ml of saline water was used as control, and the lethality of the given organism was observed after 24 h intervals [27, 28].
Whole-Genome sequencing (WGS) of the best isolates and analysis
After evaluation of the isolate’s mosquito larvicidal activity and others considering parameters of a potential biopesticide, the WGS data analysis of the best bacteria was conducted. Firstly, the DNA isolation was done according to the protocol of the TIANamp bacteria DNA kit, and the quality and quantity of the isolated bacterial genomic DNA were checked via a uv/vis biophotometer (Eppendorf, Germany) at 260/280 nm wavelength. The WGS of the strain was conducted using the Illumina NovaSeq PE150 platform for paired-end 150 × 2 library preparation (Novogene, China). The sequence read quality was analyzed by the FastQC Galaxy Version 0.74 and the common adapters as well as primers were removed using Trim Galore Galaxy Version 0.6.7. The trimmed read sequences were assembled by the de novo approach by the SPAdes Galaxy Version 3.15.5 and the errors were corrected again. After the genome was assembled, we downloaded the scaffold and contig files for further analysis. However, the genome assembled output quality was checked by using Quality Assessment Tools (QUAST) Galaxy Version 5.2.0 and the HTML reports were saved. Default parameters were used for all software. The genome circular view has been designed by using the genome circular view of the BV-BRC (Bacterial and Viral Bioinformatics Resource Centre) website (https://www.bv-brc.org/).
Species identification
In our study, we used KmerFinder 3.2 of the Center for Genomic Epidemiology (https://cge.food.dtu.dk/services/KmerFinder/) to identify species. We selected the bacterial organisms database and uploaded the FASTA file of scaffold that had been generated by SPAdes Galaxy Version 3.15.5. We have selected the highest-matched species based on some distinct parameters. Among these parameters, we have considered crucially the highest score and lowest E-value.
Construction of Codon-based phylogenetic tree
The comprehensive WGS data analysis provides a phylogenetic tree (codon tree) based on the reference and representative genomes which are provided by PATRIC. The PATRIC global protein families (PGFams) were chosen from the relevant genomes, and their placement occurred according to the PGFams. The closest reference and representative genomes were identified by Mash/MinHash [29], and the common proteins were aligned with MUSCLE [30].
Data analysis
Most of the data were analyzed and visualized via the Microsoft Excel-2019 and all the experiments were conducted triplicate manner except the WGS study. In this study, we have applied differential approach for the data interpretation that was clearly explained in the methodology.
Results
Twelve samples, including soil, water, and dead plant samples, were collected from the Sundarbans mangrove forest of Khulna, and five samples were collected from Wari and Nakhal para of Dhaka. Thirty eight bacterial isolates were taken from these seventeen samples (Table 1) because of their unique colony size, shape, and color (Fig. 1a).
Fig. 1 .
Morphological and Biochemical analysis of isolates, (a) Round white colony, (b) Gram-positive bacteria, and (c) Catalase-positive bacteria
Gram staining was conducted on 38 isolates, 22 of which were gram-positive and 16 of which were gram negative. In this study, gram positive bacteria were selected for further investigation (Fig. 1b). In the catalase test, 16 of the 38 isolates were catalase positive (Fig. 1c), and 22 were catalase negative. Thus, 16 isolates were both gram positive and catalase positive.
The bacteria including 1T3dot, 5WR, 6TW, 6RW, 8WR, 10 RW, 11RWL,17WR, 18YR, 24RW, 20 RW, 21RWL, 18RW, 20W2L, 22RW, 23WR were both gram positive and catalase positive and so the DNA of these 16 isolates were processed for PCR. Eppendorf bio-photometer (Germany) measured the quality and quantity of the isolated bacterial genomic DNA, and then these isolates PCR operation were done. Due to minute DNA content, PCR was not possible for 18YR, 24RW, 20RW, and 21RWL.
The gel electrophoresis of PCR products revealed that all the gene sequences of the 16S rRNA gene derived from 12 bacterial isolates (Fig. 2a, b) gave a unique band, and all presented a nearly 1500 bp band when compared with the ladder. The 16S rRNA gene sequencing was conducted on PCR amplified products following gel electrophoresis. We subsequently assessed the sequence (Fig. 2c) via the Basic Local Alignment Search Tool (BLAST) program within the National Center for Biotechnology Information (NCBI) database (accessed 17 April 2024).
Fig. 2.
Gel electrophoresis of PCR products and Chromatogram of 16S rRNA gene sequence, (a) Gel electrophoresis of 1T3dot, 5WR, 6TW, 6RW, 8WR, 10 RW, 11RWL, 17 WR (b) Gel electrophoresis of 18RW, 20W2L, 22RW, 23WR, and (c) Chromatogram of 16S rRNA gene sequence
The results of the NCBI BLAST analysis revealed that six out of the twelve bacterial isolates presented substantial sequence similarity to species within the Bacillus genus, such as Bacillus tropicus, Bacillus thuringiensis, Bacillus zanthoxyli, Bacillus pseudoflexus, and Bacillus stratosphericus. The findings suggests a close genetic relationship between these samples and known Bacillus species. Some isolates were found to be similar to Fictibacillus, Metabacillus and Halobacillus species. However, the sequence of bacteria 10OR showed similarity to another different genus Rossellomorea. This findings indicates that 10OR may possess genetic characteristics that are more similar to Rossellomorea species rather than Bacillus species. The similarity to multiple genera suggests genetic diversity within the bacterial samples and highlights the importance of further genetic analysis to elucidate their taxonomic classification and evolutionary relationships.
Among the 12 16S rRNA gene based predicted isolates, 6 isolates (10 RW, 22 RW, 18 RW, 6 RW, 11 RWL, 20 W2L) were selected for further tests according to our target genus. The antimicrobial activity test, salt tolerance test, pH tolerance test, enzyme assay, antibiotic susceptibility test, mosquito larvicidal test, and lethality test were subsequently performed on these isolates.
In case of antimicrobial test of six isolates, 6RW (B. tropicus), 11RWL (B. thuringiensis), 10WR (B. zanthoxyli), 18RW (B. altitudines), 20W2L (B. pseudoflexus), and 22RW (F. barbaricus), revealed that 6RW and 18RW created an inhibition zone after 24 h and 96 h of incubation. However, the isolates 10RW, 22RW, 11RWL, and 20W2L showed no antimicrobial activity and inhibition zone against the same pathogenic bacteria. Moreover, isolate 6RW showed antimicrobial properties against S. typhi and E. coli, and isolate 18RW showed antimicrobial activity against S. aureus and S. typhi (Fig. 3a and b). Surprisingly, 18RW inhibited the S. typhi after 96 h, whereas no zone was observed after 24 h. Therefore, the antimicrobial activity was observed in 2 among the 6 isolates that were tested for antimicrobial activity against S. typhi, S. aureus, and E. coli.
Fig. 3.
(a) Antimicrobial assay (b) Antimicrobial assay (graphical view)
The result of the salt tolerance test (Fig. 4a) revealed that most of the isolates could not tolerate high salt contents. However, the isolate 6RW (B. tropicus) presented the highest salt tolerance. The pH tolerance test (Fig. 4b) represented that the isolates could not grow on under highly acidic and/or basic conditions, but most of them grew better at pH 6–8.
Fig. 4.
(a) Salt tolerance test, and (b) pH tolerance test
In the enzyme assay, amylase activity was detected in 6RW (B. tropicus) and 10RW (B. zanthoxyli) (Fig. 5a). Therefore, these isolates use carbon as their energy source and are able to produce amylase enzyme. Protease activity was detected in 6RW (B. tropicus), 11RWL (B. thuringiensis), 10WR (B. zanthoxyli), 18RW (B. altitudinis), 20W2L (B. pseudoflexus), and 22RW (F. barbaricus) (Fig. 5b). These isolates produce protease and are able to use nitrogen as their nutrient source. Cellulase activity was observed only in 10RW (B. zanthoxyli), hence it consume the cellulose as nutrient (Fig. 5c). Compared with the other isolates, 10WR (B. zanthoxyli) was shown the highest enzyme activity because this have amylase, protease, and cellulase activities. As a result, it can survive by using multiple nutrient sources (Fig. 6). All isolates observed in this study took nitrogen as their energy source, few took carbon as their energy source, and only one isolate took cellulose as their energy source.
Fig. 5.
Enzyme assay of Bacillus isolates, (a) Amylase activity (b) Protease activity and (c) Cellulase activity
Fig. 6.
Enzymes activity of bacterial isolates in percentage (%)
In antibiotic susceptibility test, ciprofloxacin (5 µg), tetramycin (30 µg), vancomycin (30 µg), and chloramphenicol (30 µg) displayed effectiveness against the tested strains, while ampicillin (25 µg) and rifampicin (5 µg) encountered resistance in some instances (Fig. 7a and Fig. 7b).
Fig. 7.
(a) Antibiotic susceptibility test, (b) Antibiotic susceptibility test of six bacterial isolates
The number of larval deaths was recorded and the percentage of mortality was determined after 24 and 48 h of inoculation with the cultures of 6 RW, 11RWL, 10WR, 18RW, 20W2L, and 22RW. The mortality of 10 larvae exposed to 6RW (B. tropicus), 10WR (B. zanthoxyli), and 20W2L (B. pseudoflexus) were not shown mortality after 24 and 48 h respectively. The mortality rate of 22RW (F. barbaricus) was 2.0 ± 0.00 i.e. 20% after 48 h. Moreover, 11RWL (B. thuringiensis) have killed two larvae after 24 h and another one larva was observed dead after 48 h that totally exhibited 3.0 ± 0.00 i.e. 30% larval mortality. However, the isolate 18RW (B. altitudinis) caused the death of eight larvae of both mosquito genus after 48 h and the isolate revealed highest larval mortality that was 7.67 ± 0.57 i.e. 77% (Fig. 8a & Table 2). In the negative control, all the larvae were alive even after 48 h of observation (Table 2). In our study, we did not find out any variation of larvicidal activity between the two tasted mosquito species.
Fig. 8.
(a)Mosquito larvicidal activity test, (b) Lethality test on Artemia salina
Table 2.
Findings of mosquito larvicidal activity and lethality test of selected isolates
| Sample Name | Identified Organism (Bacteria) | No. of treated larvae in each treatment | Death after treatment by bacteria | Percentage of death after 48 h | Lethality test finding | |
|---|---|---|---|---|---|---|
| After 24 h M ± SE |
After 48 h M ± SE |
|||||
| 6RW | Bacillus tropicus | 10 | 0 | 0 | 0% | - |
| 11RWL | Bacillus thuringiensis | 10 | 2.66 ± 0.33 | 3.0 ± 0.00 | 30% | Non-lethal |
| 10RW | Bacillus zanthoxyli | 10 | 0 | 0 | 0% | - |
| 18RW | Bacillus altitudinis | 10 | 5.67 ± 0.66 | 7.67 ± 0.57 | 77% | Non-lethal |
| 20W2L | Bacillus pseudoflexus | 10 | 0 | 0 | 0% | - |
| 22RW | Fictibacillus barbaricus | 10 | 1.33 ± 0.33 | 2.0 ± 0.00 | 20% | Non-lethal |
| (-) Control | Only the media | 10 | 0 | 0 | 0% | - |
Here, M Mean value, SE Standard Error of mean; we have conducted this study three times
Here, M = Mean value; SE = Standard Error of mean; we have conducted this study three times.
The isolates 11RWL, 22RW, and 18RW were showed mosquitocidal activity and the lethality tests of these particular isolates were conducted on A. salina for 48 h. All the tested isoltes were non-lethal, as no larvae of Artemia were killed after inoculation with these isolates. All the larvae were alive in case of negative control. Therefore, (B) altitudinis (18RW) has larvicidal properties and appears to be non-lethal for other aquatic organisms (Fig. 8b). This studies prove the bacteria as a potential environmentally friendly biopesticide to control mosquitoes.
The whole genome sequencing was conducted via the Illumina platform in which the machine generated two paired-end reads of 9,755,756 after trimming the raw reads by Trime Galore. However, the total number of contigs 32, the largest contig 888,707, the whole genome sequence length 3,872,751, the GC percentage 41.07, and the contig L50 value 3 were found after assembling the trimmed raw reads through the de novo assembly approach. The genome does not contain any plasmid (Table 3). However, the genome showed 100% completeness whenever we did the task through the RAST toolkit.
Table 3.
Genomic feature of the best isolate (Bacillus altitudinis)
| Genome Feature | Value |
|---|---|
| Total Number of Contigs | 32 |
| Contig L50 | 3 |
| Genome Size (bp) | 3,872,751 |
| GC Percent | 41.07 |
| Protein Coding Genes | 4137 |
| Ribosomal RNA (rRNA) | 3 |
| Transfer RNA (tRNA) | 68 |
| Miscellaneous RNA | 0 |
| CRISPR Repeat | 5 |
| Number of Plasmids | 0 |
We performed both classical and comprehensive annotation by using the BV-BRC website. The results revealed that the bacteria was very close to the B. altitudinis strain HQ-51-Ba (Genome ID: 293387.52). The comprehensive genome analysis has shown 4137 protein-coding genes present in the genome and were no partial protein-coding genes. The WGS data results provided information about 3 ribosomal RNAs, 68 transfer RNAs, no Miscellaneous RNAs, and 5 CRISPR repeats. The circular view of the genome depicts the quality of both forward and reverse reads. It also reveals from outer to inner rings, the contigs, CDS on the forward strand, CDS on the reverse strand, RNA genes, CDS with homology to known antimicrobial resistance genes, CDS with homology to known virulence factors, GC content, and GC skew (Fig. 9a and b).
Fig. 9.
(a) Different color represent different feature of the circular view of the genome (b) Circular view of the genomeof Bacillus altitudinis, (c) Overview of codon-based phylogenetic tree
The codon-based phylogenetic tree indicated that B. pumilus strain NJ-M2 and B. altitudinis exhibited the maximum homology. Moreover, Bacillus sp. M2-6 and B. stratosphericus LAMA 585 are also close relatives of our desired isolate (Fig. 9c). The distance of the tree was 0.1 in which the B. psychrosaccharolyticus was the most distanced strain from B. altitudinis according to the codon based phylogenetic tree.
Discussion
Compared with the chemical based mosquito controlling pesticides, bacterial biopesticides can be a solution for mosquito control because they are highly selective in targeting specific pests, have low toxicity levels, and pose minimal risks to human health. In a previous study, Bacillus amyloliquefaciens was isolated from the mangrove forests of Andaman, Nicobar Islands, and Malabar Coast of India and was found to have significant mosquitocidal activity [31, 32]. Hence, in this study, mangroves were chosen for sample collection as these environments harbor a diverse array of microorganisms, including bacteria such as Bacillus species and there is the possibility of obtaining novel strains with increased adaptability. The heat shock method was done in a water bath as, Bacillus are spore-forming bacteria that are able to survive in adverse conditions such as high temperatures, but other non-spore-forming bacteria can die. Previously, the thermal heat shock method was used to isolate several species of sporulated Bacillus, with a total of 77 isolates [33].
In this study, 6RW (B. tropicus) and 18 RW (B. altitudinis) showed antimicrobial activity among the 6 isolates tested for antimicrobial activity. B. altitudinis showed a significant inhibition zone of 25.6 mm to 31.3 mm after 96 h but surprisingly was not revealed any antimicrobial activity after 24 h. Therefore, more time is needed to release the secondary metabolites, resulting in antagonism against pathogenic bacteria. In previous studies, Bacillus sp. were reported to show antimicrobial activity against Yersinia enterocolitica, Micrococcus flavus, S. aureus, E. coli, Pseudomonas aeruginosa and Micrococcus luteus [34].
In the salt tolerance test, the 6RW (B. pacificus) was the most salt tolerant as it was capable of growing at 1–9% salt concentrations. As B. pacificus was isolated from the soil of high saline-containing mangrove forest that could be the reason of its high salt tolerance. In previous studies, it was found that Bacillus aerius survives in the salinity up to 6% [35]. Previously, 39 Bacillus strains were isolated from soil, and observed five isolates were grown at only 2% NaCl [36]. Similarly, in this study, the six isolates tested for salt tolerance showed maximum growth at 1-3% NaCl. In pH tolerance test, it was observed most of the isolates did not survive in acidic environments but survived in basic environments.
In the enzyme assay, protease activity was found in 6RW (B. tropicus), 11RWL (B. thuringiensis), 10WR (B. zanthoxyli), 18RW (B. altitudinis), 20W2L (B. pseudoflexus), and 22 RW (F. barbaricus). Previously, protease activity was reported in Bacillus subtilis [37]. In our study, B. zanthoxyli showed both amylase and cellulase activity, but B. tropicus was observed to produce amylase only. A study reported by Mohapatra et al. [38] demonstrated that 35% of strains of Bacillus were capable of producing large amounts of protease, amylase, and carboxymethylcellulase. In this study, 10WR (B. zanthoxyli) was able to show amylase, protease, and cellulase enzymatic activity. Hence, it was capable of producing enzymes that play an important role in organic matter decomposition and nutrient cycling and can be the maximum industrial enzyme producer. A previous study [36] revealed that the highest production for all three enzymes was obtained from Bacillus strains capable of growth at a 5% NaCl. However, in our study, B. zanthoxyli grew lower at 5% NaCl but was capable of producing amylase, protease, and cellulase simultaneously.
In the antibiotic susceptibility test, ciprofloxacin, tetramycin, vancomycin, and chloramphenicol were displayed 100% susceptibility against all the tested strains. However, 6RW (B. tropicus), 11RWL (B. thuringiensis), 18RW (B. altitudinis), and 22RW (F. barbaricus) have showed resistance against ampicillin. The B. tropicus was the only strain that showed resistance towards two antibiotics, ampicillin and rifampicin, which are the third and second generation antibiotics, respectively. Hence, this bacterium carries antibiotic resistance genes, and there is a risk that these genes could transfer to pathogenic bacteria, rendering them resistant to antibiotics. In previous studies, isolated B. cereus strains from clinical blood cultures were found to be susceptible to vancomycin (30 µg), chloramphenicol (30 µg) and ciprofloxacin (5 µg) [39].
In our investigation, the WGS data revealed that the desire bacteria was B. altitudinis. The bacteria have many potential biopesticide activities, as a biocontrol agent for Grape downy mildew, a biocontrol agent for potato common scab, and a biocontrol agent for the postharvest prevention of sweet potato black rot [40–42]. The generated FASTQ read data were assembled through the BV-BRC website and Galaxy server by SPAdes [43], whereas Rabha and coworkers [44] used SOAPdenovo for assembling the WGS data of B. thuringiensis. However, Prokka [45] is the most common online functional annotation platform for the prokaryotic genome and has been considered by many researchers [46, 47]. The annotated data have been examined by QUEST and RUST, but some researchers suggested use of their systems [48], and BUSCO [49].
In the previous studies of mosquitocidal activity, Geetha with colleagues [31] reported that B. amyloliquefaciens has bactericidal activity against A. stephensi, C. quinquefasciatus and A. aegypti. In another study, Abinaya and coworkers [50] investigated mosquitocidal activity and reported that Bacillus licheniformis showed bactericidal activity against A. stephensi and A. aegypti. Moreover, a previous study [51, 52] demonstrated that B. sphaericus has antagonistic activity against A. stephensi and A. aegypti. However, in our study, 18RW (B. altitudinis) was showed highly lethal effect to mosquito larvae, causing 77% larval mortality within 48 h, and had no effect on aquatic organisms. Therefore, B. altitudinis can be a potential mosquitoes controlling biological agent.
Conclusions
In our study, among the all tested isolates, the 18RW (B. altitudinis) was exhibited excellent mosquito larvicidal activity with mortality rate of 77% mosquito larvae after 48 h of observation along with antimicrobial activity against some pathogenic microbes. Beside this, the isolate was susceptible to multiple antibiotics and had no lethal effect on A. salina larvae. The overall findings of the study proved the B. altitudinis as a highly promising biopesticide for the both mosquito’s larvae (C. quinquefasciatus and A. aegypti) and the pathogenic bacteria (S. aureus and S. typhi). Moreover, the study revealed, the biopesticide is environmentally safe and this is the first report to prove that B. altitudinis is a highly potential mosquito-controlling agent.
Limitations
B. altitudinis is only effective against the larvae stage of mosquitoes that cannot inhibit or kill other developmental stages of mosquitoes. Moreover, the study have performed in the laboratory scale and the larvae were collected from a distinct region. The large scale study must be needed to establish the biopesticide commercially that was not conducted in our project.
Acknowledgements
The authors are grateful to Bangladesh Council of Scientific and Industrial Research (BCSIR) to provide financial and technical support.
Authors’ contributions
Md. Saddam Hossain designed and planned the research project, and the manuscript was edited primarily by him. All the experiments were observed by him and performed according to his guidelines. Motahara Farhan Anjum is the co-first author of the manuscript, who performed all the experimental works and wrote the first draft of the manuscript. Md. Asik Rabbani assisted Motahara Farhan Anjum in all the experiments of the project and helped to write the manuscript. Md. Rakibul Hasan helped to conduct the mosquito's larvae lethality tests. Md Mehadi Hasan Sohag edited the final manuscript and provided some guidelines for the experiments conducted in this study. Nishat Tasnim helped complete the research experiments and edited the manuscript. Debabrata Karmakar assisted in the preparation of different chemicals and edited the manuscript. Sharmin Akter helped to edit the draft manuscript and helped with sample collection and transportation. Md. Masudur Rahman helped to collect samples from mangrove forests and edited the manuscript. Md. Rezaul Karim provided guidelines and technical support to complete the research project.
Funding
The project was funded by BCSIR, Dhaka, Bangladesh, under the R&D program of the fiscal year 2023-24.
Data availability
Accession number: PRJNA1207607Uploaded link: https://submit.ncbi.nlm.nih.gov/subs/wgs/SUB14994820/overview.
Declarations
Ethical approval and consent to participate
In our study, we did not conduct any human or animal related experiments. On the other hand, the R&D was approved by the higher authority of BCSIR.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Md. Saddam Hossain and Motahara Farhan Anjum contributed equally to this work.
References
- 1.WHO. A global brief on vector-borne diseases. 2014. www.who.int
- 2.Mamun MA, Misti JM, Griffiths MD, Gozal D. The dengue epidemic in Bangladesh: risk factors and actionable items. Lancet. 2019;394(10215):2149–50. 10.1016/S0140-6736(19)32524-3. [DOI] [PubMed] [Google Scholar]
- 3.Mahmood R, Benzadid MS, Weston S, Hossain A, Ahmed T, Mitra DK, Ahmed S. Dengue outbreak 2019: clinical and laboratory profiles of dengue virus infection in Dhaka city. Heliyon. 2021;7(6). 10.1016/j.heliyon.2021.e07183. [DOI] [PMC free article] [PubMed]
- 4.Anwar S, Mourosi JT, Khan MF, Ullah MO, Vanakker OM, Hosen MJ. Chikungunya outbreak in Bangladesh (2017): Clinical and hematological findings. PLoS Neglected Tropical Diseases. 2020;14(2). 10.1371/journal.pntd.0007466. [DOI] [PMC free article] [PubMed]
- 5.Rahman M, Khan S, Sultan M, Islam M. Characterization of Bacillus spharicus binary proteins for biological control of Culex quinquefasciatus mosquitoes: a review. Int J Biosci. 2012;2:1–13 https://www.cabidigitallibrary.org/10.5555/20123123913. [Google Scholar]
- 6.Sánchez-Bayo F. Insecticides mode of action in relation to their toxicity to non target organisms. J Environ Anal Toxicol. 2012;S4:S4-002. 10.4172/2161-0525.S4-002. [Google Scholar]
- 7.Araújo MF, Castanheira EM, Sousa SF. The buzz on insecticides: a review of uses, molecular structures, targets, adverse effects, and alternatives. Molec. 2023;28(8):3641. 10.3390/molecules28083641. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Benelli G, et al. Mosquito control with green nanopesticides: towards the One Health approach? A review of non-target effects. Environmental Science and Pollution Research. 2018;25(11):10184–206. 10.1007/s11356-017-9752-4. [DOI] [PubMed] [Google Scholar]
- 9.Vryzas Z. Pesticide fate in soil-sediment-water environment in relation to contamination preventing actions. Curr Opinion Environment Sci Health. 2018;4:5–9. 10.1016/j.coesh.2018.03.001. [Google Scholar]
- 10.Tudi M, et al. Agriculture development, pesticide application and its impact on the environment. Int J Environ Res Public Health. 2021;18(3):1112. 10.3390/ijerph18031112. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Ahmad M, et al. Biochar as a sorbent for contaminant management in soil and water: a review. Chemosphere. 2014;99:19–33. 10.1016/j.chemosphere.2013.10.071. [DOI] [PubMed] [Google Scholar]
- 12.Karunamoorthi K, Sabesan S. Insecticide resistance in insect vectors of disease with special reference to mosquitoes: a potential threat to global public health. 2013;2(1):4-18.
- 13.Benelli G. Plant-mediated biosynthesis of nanoparticles as an emerging tool against mosquitoes of medical and veterinary importance: a review. Parasitol Res. 2016;115(1):23–34. 10.1007/s00436-015-4800-9. [DOI] [PubMed] [Google Scholar]
- 14.Vlaiculescu A, Varrone C. Sustainable and eco-friendly alternatives to reduce the use of pesticides. In Pesticides in the natural environment. 2022;329–364. 10.1016/B978-0-323-90489-6.00014-8.
- 15.Poopathi S, Abidha S. Mosquitocidal bacterial toxins (Bacillus sphaericus and Bacillus thuringiensis serovar israelensis): Mode of action, cytopathological effects and mechanism of resistance. J Physiol Pathophysiol. 2010;1(3):22–38. [Google Scholar]
- 16.El-Kawokgy TM, Hussein HA, Aly NA, Mohamed SA. Highly toxic and broad-spectrum insecticidal local Bacillus strains engineered using protoplast fusion. Canadian J Microbiol. 2015;61(1):38–47. 10.1139/cjm-2014-053. [DOI] [PubMed] [Google Scholar]
- 17.Habiba G, Imen K, Rabeh C, Lina L. A comparative study between biological and chemical control against domestic mosquito larvae. Biodiversitas. 2022b;23(12):6456–62. 10.13057/biodiv/d231244. [Google Scholar]
- 18.Vaghela NR, Gohel SD. The Rhizospheric Alkali-Halotolerant Bacillus sp. KhSb-159 Enhanced Growth Parameters of the Mung Bean Crop. Curr Microbiol. 2025;82(3):114. 10.1007/s00284-025-04087-3. [DOI] [PubMed] [Google Scholar]
- 19.Hossain MS, et al. Isolation, Screening, and Molecular Characterization of Rhizosphere Derived Potential Biofertilizer from Different Crops Land for Sustainable Agriculture and Environment. Appl Environ Biotechnol. 2023;8(2):29–43. 10.26789/AEB.2023.02.004. [Google Scholar]
- 20.Puttawong K, Beesa N, Kasem S, Jindapunnapat K, Chinnasri B, Sasnarukkit A. Potential of Bacillus spp. against root-knot nematode, Meloidogyne enterolobii parasitizing chili (Capsicum annuum L.). Crop Protection. 2024;184:106780. 10.1016/j.cropro.2024.106780. [Google Scholar]
- 21.Balouiri M, et al. Antifungal activity of Bacillus spp. isolated from Calotropis procera AIT. Rhizosphere against Candida albicans. Asian J Pham Clin Res. 2015;8:213–7. [Google Scholar]
- 22.Sadhu S, Saha P, Sen SK, Mayilraj S, Maiti TK. Production, purification and characterization of a novel thermotolerant endoglucanase (CMCase) from Bacillus strain isolated from cow dung. Springer Plus. 2013;2:1–10. 10.1186/2193-1801-2-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Sharma AK, Sharma V, Saxena J, Yadav B, Alam A, Prakash A. Isolation and screening of extracellular protease enzyme from bacterial and fungal isolates of soil. Int J Sci Res Environ Sci. 2015;3(9):0334–40. 10.12983/ijsres-2015-p0334-034. [Google Scholar]
- 24.Hudzicki J. Kirby-Bauer disk diffusion susceptibility test protocol. Am Soc Microbiol. 2009;15(1):1–23. [Google Scholar]
- 25.Dehus H. Investigating urban mosquito ecology and mosquito management. "Master’s thesis, The Ohio State University. 2025. http://rave.ohiolink.edu/etdc/view?acc_num=osu1744801795993685.
- 26.Reegan AD, Patil PB, Pushpalatha E, Mariappan T, Dasgupta SK, Mathur A, Kocher DK, Bharti M, Mishra P, Chandrasekaran N, Suman DS. Mosquito Control: Part I: Chemicals Including Repellents, Non-chemical, and Biological. InMosquitoes of India (pp. 137–169). CRC Press.
- 27.Amutha V, Aiswarya D, Deepak P, Selvaraj R, Tamilselvan C, Perumal P, Balasubramani G. Toxicity potential evaluation of ethyl acetate extract of Cymodocea serrulata against the mosquito vectors vis-a-vis zebrafish embryos and Artemia salina cysts. South Afr J Botany. 2023;152:230–9. 10.1016/j.sajb.2022.12.005. [Google Scholar]
- 28.Bomfim RR, Araujo AA, Cuadros-Orellana S, Melo MG, Quintans-Junior LJ, Cavalcanti SC, de Bioprocessos LDE. Larvicidal activity of Cladonia substellata extract and usnic acid against Aedes aegypti and Artemia salina. Latin Am J Pharma. 2009;28(4):580–4. [Google Scholar]
- 29.Ondov BD, Treangen TJ, Melsted P, Mallonee AB, Bergman NH, Koren S, Phillippy AM. Mash: fast genome and metagenome distance estimation using MinHash. Genome Biol. 2016;17:1–14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004;32(5):1792–7. 10.1093/nar/gkh340. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Geetha I, Manonmani AM, Prabakaran G. Bacillus amyloliquefaciens: A mosquitocidal bacterium from mangrove forests of Andaman & Nicobar islands. India Acta Tropica. 2011;120(3):155–9. 10.1016/J.ACTATROPICA.2011.07.006. [DOI] [PubMed] [Google Scholar]
- 32.Kunnikuruvan A, Vijayakumar A, Sivaprakasam M, Padmanaban H, Mandodan S, Gangmei K, Lukose J, Bora B, Gupta B, Ashokkumar M, Balakrishnan V. Enhanced Mosquito Larvicidal Efficacy and Dehairing Properties of Bacillus thuringiensis Serovar israelensis Strain VCRC-B649 Isolated from Malabar Coast, India. Curr Microbiol. 2025;82(2):93. 10.1007/s00284-025-04070-y. [DOI] [PubMed] [Google Scholar]
- 33.Katak RM, et al. Larvicidal activities against Aedes aegypti of supernatant and pellet fractions from cultured Bacillus spp. Isolated from amazonian microenvironments. Tropical Med Infectious Dis. 2021;6(2):104. 10.3390/TROPICALMED6020104/S1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Yeşilyurt A, Biryol S, Soydinç A, İşık S, Usta M. Determination of Antimicrobial Effects of Secondary Metabolites of Different Bacteria Belonging to the Genus Bacillus. Afyon Kocatepe Üniversitesi Fen Ve Mühendislik Bilimleri Dergisi. 2024;24(1):1–7.
- 35.Zhang X, Gao J, Zhao F, Zhao Y, Li Z. Characterization of a salt-tolerant bacterium Bacillus sp from a membrane bioreactor for saline wastewater treatment. J Environment Sci. 2014;26(6):1369–74. 10.1016/S1001-0742(13)60613-0. [DOI] [PubMed] [Google Scholar]
- 36.Avşar C, Koyuncu H, Aras ES. Isolation and molecular characterization of Bacillus spp. isolated from soil for production of industrial enzymes. Biol Chem Res. 2017;3(9):72–86. [Google Scholar]
- 37.Hakim A, Rumzum BF, Iqbal A, Hossain ET, Ahmed J, Kalam AA. Production and partial characterization of dehairing alkaline protease from Bacillus subtilis AKAL7 and Exiguobacterium indicum AKAL11 by using organic. Cell ComA. 2018;4:646. 10.1016/j.heliyon.2018. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Mohapatra BR, Bapuji M, Sree AJAB. Production of industrial enzymes (amylase, carboxymethylcellulase and protease) by bacteria isolated from marine sedentary organisms. Acta Biotechnolog. 2003;23(1):75–84. 10.1002/abio.200390011. [Google Scholar]
- 39.Weber DJ, Saviteer SM, Rutala WA, Thomann CA. In vitro susceptibility of Bacillus spp to selected antimicrobial agents. Antimicrob AgentsChemother. 1988;32(5):642–5. 10.1128/AAC.32.5.642. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.Zeng Q, Xie J, Li Y, Gao T, Zhang X, Wang Q. Comprehensive genomic analysis of the endophytic Bacillus altitudinis strain GLB197, a potential biocontrol agent of grape downy mildew. Front Gene. 2021;12:729603. 10.3389/fgene.2021.729603. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41.Li B, et al. Bacillus altitudinis strain AMCC 101304: a novel potential biocontrol agent for potato common scab. Biocont Sci Technol. 2019;29(10):1009–22. 10.1080/09583157.2019.1641791. [Google Scholar]
- 42.Zhang YJ, Cao XY, Chen YJ, Cong H, Wang YM, Jiang JH, Li LD. Potential utility of endophytic Bacillus altitudinis strain P32-3 as a biocontrol agent for the postharvest prevention of sweet potato black rot. Biological Control. 2023;186:105350. 10.1016/j.biocontrol.2023.105350. [Google Scholar]
- 43.Bankevich et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. Journal of computational biology. 2012;19(5):455–477. 10.1089/cmb.2012.0021. [DOI] [PMC free article] [PubMed]
- 44.Rabha M, Das D, Konwar T, Acharjee S, Sarmah BK. Whole genome sequencing of a novel Bacillus thuringiensis isolated from Assam soil. BMC Microbiol. 2023;23(1):91. 10.1186/s12866-023-02821-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 45.Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinform. 2014;30(14):2068–9. 10.1093/bioinformatics/btu153. [DOI] [PubMed] [Google Scholar]
- 46.Zhao X, Zervas A, Hendriks M, Rajkovic A, Van Overbeek L, Hendriksen NB, Uyttendaele M. Identification and characterization of Bacillus thuringiensis and other Bacillus cereus group isolates from spinach by whole genome sequencing. Front Microbiol. 2022;13:1030921. 10.3389/fmicb.2022.1030921. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 47.Nathar S, Rajmichael R, Jeyaraj PC, Nagarajan H, Mathimaran A, Kingsley JD, Jeyaraman J. Exploring Nocardia’s ecological spectrum and novel therapeutic frontiers through whole-genome sequencing: unraveling drug resistance and virulence factors. Arch Microbiol. 2024;206(2):76. 10.1007/s00203-023-03799-z. [DOI] [PubMed] [Google Scholar]
- 48.Gurevich A, Saveliev V, Vyahhi N, Tesler G. QUAST: quality assessment tool for genome assemblies. Bioinform. 2013;29(8):1072–5. 10.1093/bioinformatics/btt086. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 49.Manni M, Berkeley MR, Seppey M, Simão FA, Zdobnov EM. BUSCO update: novel and streamlined workflows along with broader and deeper phylogenetic coverage for scoring of eukaryotic, prokaryotic, and viral genomes. Mole Biol Evol. 2021;38(10):4647–54. 10.1093/molbev/msab199. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 50.Abinaya M, et al. Structural characterization of Bacillus licheniformis Dahb1 exopolysaccharide—antimicrobial potential and larvicidal activity on malaria and Zika virus mosquito vectors. Environ Sci Poll Res. 2018;25(19):18604–19. 10.1007/S11356-018-2002-6/METRICS. [DOI] [PubMed] [Google Scholar]
- 51.Subramaniam J, Kovendan K, Mahesh KP, Murugan K, Walton W. Mosquito larvicidal activity of Aloe vera (Family: Liliaceae) leaf extract and Bacillus sphaericus, against Chikungunya vector, Aedes aegypti. Saudi J Biol Sci. 2012;19(4):503–9. 10.1016/J.SJBS.2012.07.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 52.Ragavendran C, Govindaraj A, Kamaraj C, Natarajan D, Malafaia G, Alrefaei AF, Almutairi MH. Fusarium begoniae metabolites: a promising larvicidal, pupicidal potential, histopathological alterations and detoxifications enzyme profiles of medically important mosquito vector Aedes aegypti, Culex quinquefasciatus and Anopheles stephensi. 3 Biotech. 2024;14(10):226. 10.1007/s13205-024-04061-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Accession number: PRJNA1207607Uploaded link: https://submit.ncbi.nlm.nih.gov/subs/wgs/SUB14994820/overview.