Isolation and Characterization of the Bacteriophage Yucatan Specific to Bacillus Licheniformis

Isolation and Characterization of the Bacteriophage Yucatan Specific to Bacillus Licheniformis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Isolation and Characterization of the Bacteriophage Yucatan Specific to Bacillus Licheniformis Daniel Bravo-Pérez, Jean Pierre González-Gómez, Ernestina Castro-Longoria, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9557867/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Bacteriophages have been proposed as a biological alternative for reducing bacterial pathogens in diverse fields, including the food industry, healthcare, and agriculture, considered as promising tools to counteract antibiotic resistance. In this study, we present//provide the morphological and genetic characterization of a novel bacteriophage isolated from the digestive system of Bemisia tabaci (whitefly) Biotype A, collected from Capsicum chinense (habanero pepper). Its bacteriolytic activity, thermostability, pH tolerance, and host range were assessed. Host range analysis revealed that among the bacteria tested, Bacillus licheniformis served as the host for this bacteriophage. Transmission electron microscopy images showed virions with a head–tail structure, characterized by a long, contractile tail and an icosahedral head. Characterization results indicated that bacteriolytic activity of the phage begins at the first hour of contact with its host. Among the evaluated parameters, the optimal temperature for maximum activity was 20°C, while the optimal pH was 7.0. Lytic activity was observed exclusively against Bacillus licheniformis strains. The newly identified phage possesses a double-stranded DNA genome of 147,395 bp, predicting a total of 266 protein-coding sequences, with a GC content of 38.98%. It lacks host virulence-modifying genes and antibiotic resistance genes and exhibits a strictly virulent life cycle. Based on viral taxonomy criteria, this phage was classified as a novel species within the family Herelleviridae , with its closest relative being phage SIOphi. The proposed name for this isolate is Bacillus phage Yucatan . insect symbionts Bacillus lytic phage genomic analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Insects represent the animal group with the greatest number of species worldwide. Owing to their wide distribution, they are commonly associated with diverse microorganisms, including bacteria, viruses, fungi, protozoa, nematodes, and multicellular parasites (Mondal et al. 2023 ). Permanent associations between bacteria and higher organisms are referred to as symbiosis, with the type of symbiosis depending on the host, the symbiont, and the environmental conditions (Arora et al. 2017). Thus, bacterial identification is important to determine roles related to hosts’ biology including nutrition, development, defense against pathogens, community interactions, and survival in hostile environments through toxin metabolism (Rupawate et al. 2023 ). Bemisia tabaci is one of the world's most important pests, as it directly attacks agricultural products and is the main vector of plant viruses, such as Geminiviruses. B. tabaci is a species complex with genetically distinct lineages, host types, and rapid spread. Biotype A is found in the Americas, while biotypes B (MEAM1) and Q (MED) are present worldwide (De Barro et al. 2011 ). Therefore, biotype A is of paramount importance for studies related to endemic species in the Americas, specifically Mexico and Capsicum annuum plants. Several studies have isolated and identified bacterial symbionts from Bemisia tabaci . Ateyyat et al. (2023) isolated and identified culturable bacteria reporting B. licheniformis in both nymphs and adults. Similarly, Pujar et al. ( 2024 ) isolated bacterial colonies from Bemisia tabaci where the predominant bacterial species identified were Bacillus licheniformis , Bacillus pumilus , and Bacillus safensis , among others. The examples suggest that B. licheniformis is an important component of the Bemisia tabaci microbiome. Bacillus licheniformis belongs to the family Bacillaceae , whose members are characterized by their ability to form endospores, conferring high resistance to heat, radiation, chemicals, and desiccation, and enabling survival under adverse conditions for extended periods (He et al. 2023 ). B. licheniformis is predominantly found in soil and is a rod-shaped, Gram-positive, motile, and facultatively anaerobic species (Logan et al. 2015). It is part of the Bacillus subtilis group, which comprises two subspecies ( B. subtilis subsp. subtilis and B. subtilis subsp. spizizenii ) and twelve species ( B. mojavensis , B. valismortis , B. amyloliquefaciens , B. atrophaeus , B. pumilus , B. licheniformis , B. sonorensis , B. aquimaris , B. oleronius , B. sporothermodurans , B. carboniphilus , and B. endophyticus ) (Mandic-Mulec et al. 2015 ). Although B. licheniformis isolates are mainly derived from soil, their sources of isolation are diverse, including insects, bird feathers, internal plant tissues, animal leather, milk, paper, and cardboard (Logan et al. 2015). In insects, B. licheniformis has been isolated from the digestive systems of both nymphs and adults of whitefly species ( Bemisia tabaci , B. argentifolii ) (Ateyyat et al. 2023; Davidson et al. 2000 ; Pujar et al. 2024 ). Some studies suggest that, due to the characteristics of the insect microbiota, including B. licheniformis , host survival against insecticides is enhanced by the production of pesticide-degrading enzymes, the microbiome acting as a type of defense (Fan et al. 2025 ). Once the symbiotic bacteria of the insect are identified and their importance in biological development is recognized, one strategy that has been employed to disrupt insect development is the elimination of these bacteria through antibiotic treatment, resulting in reduced fertility and survival rates (Bai et al. 2019 ; Koga et al. 2007 ). However, the emergence of antibiotic resistance among bacteria poses a significant challenge for their management, highlighting the need for alternative approaches. One such alternative, used for over a century and now being widely reconsidered, is phage therapy (Sawa et al. 2024 ). Bacteriophages, or phages, are viruses that specifically infect and replicate within bacterial cells. They are recognized as the most abundant biological entities on Earth, exhibiting diverse sizes, morphologies, and genomic organizations (Kasman et al. 2022). Tailed phages are considered key components in shaping the structure, dynamics, and interactions of microbial communities across different habitats. They also exert a significant impact on human health and the food industry (Nami et al. 2021 ). Structurally, phages consist of nucleic acid enclosed within a protein shell, the capsid, which protects the genetic material and mediates its delivery into the host cell (Rees et al. 2024). Phages are highly specific, often infecting only a single bacterial species or even particular strains within that species. Although phages represent a promising alternative for biocontrol, it is essential to characterize them both biologically and genetically. Such characterization allows the determination of host range, resistance to environmental stresses, and verification of the absence of undesirable genes in their genomes, particularly those associated with host virulence modification or antibiotic resistance. This knowledge is fundamental for understanding the potential of phages to regulate bacterial populations and for tailoring their application to be both precise and effective. Furthermore, molecular analysis provides a robust foundation for their taxonomic classification (Elois et al. 2023 ; Jo et al. 2023 ). In this study, we present the morphological and genomic characterization of a novel bacteriophage related to SIOphi, isolated from the digestive system of Bemisia tabaci (whitefly) Biotype A, collected from habanero. This phage demonstrated in vitro effectiveness in controlling cultured Bacillus licheniformis . Materials and Methods Sampling Adult specimens of the whitefly Bemisia tabaci were collected from habanero pepper ( Capsicum chinense Jacq.) in greenhouses at the Scientific Research Center of Yucatán (21°01′47″ N, 89°38′20″ W). The sampled whiteflies corresponded to biotype A, as reported before (Bravo-Pérez et al. 2024 ). Specimens were collected in small jars with mesh lids containing 70% ethanol and transported to the laboratory for processing. The jars were stored at 4°C and processed on the same day. Isolation of intestinal bacteria To ensure that only intestinal bacteria were obtained and to prevent external contamination, insects were first cooled at 4°C for 15 minutes. They were then washed in a Petri dish under a stereoscope within a laminar flow hood using 70% ethanol for 5 minutes, with gentle agitation every minute. Subsequently, the insects were rinsed three times in phosphate-buffered saline (PBS, pH 7.4). From the final rinse, 100 µL of buffer was spread onto trypticase soy agar (TSA; BIOXON®) plates to confirm the absence of residual bacteria. In addition, sanitized whole insects were deposited on TSA plates to verify the absence of bacteria or other microorganisms. The abdomens of 50 insects were carefully removed and placed in a 1.5 mL microcentrifuge tube containing 200 µL of sterile PBS, then macerated with a sterile pestle for 30 seconds (Apte-Deshpande et al. 2012 ). From the macerated mixture, 100 µL were taken to prepare dilutions (10⁻¹ to 10⁻²), which were plated on TSA medium by spread plating: 100 µL of the 10⁻¹ dilution (two replicates), 100 µL of the 10⁻² dilution (two replicates), and 100 µL of the initial macerate (one replicate). Plates were incubated at 37°C for 48 h. Colonies were selected based on differential morphology, suspended in 100 µL of sterile distilled water, and individually spread onto TSA plates. After 72 h, colonies were cultured in 5 mL of trypticase soy broth (TSB; BIOXON®) for subsequent DNA extraction. Bacterial growth was monitored by spectrophotometry, until an optical density of 0.8 at 600 nm was reached. Bacterial DNA Extraction DNA extraction was carried out using a cetyltrimethylammonium bromide (CTAB)-based protocol (Sambrook and Russell 2001 ; Tito et al. 2015 ). DNA concentration and purity were assessed with a Nanodrop 2000c spectrophotometer (Thermo Scientific, USA), while integrity was confirmed by visualization on a 1.5% agarose gel under UV illumination. PCR amplification, cloning and sequencing Bacterial DNA was amplified by PCR using a T100™ thermal cycler (Bio-Rad, Germany). Degenerate primers 1494r (5′-TAC GGY TAC CTT GTT AGC ACT T-3′) and 27f (5′-AGA GTT TGA TCM TGG CTC AG-3′) were used//employed to amplify a ~ 1542 bp fragment corresponding to the near complete 16S rRNA gene region. PCR reactions were performed in a final volume of 25 µL, containing 17.7 µL of water, 1 µL of MgCl 2 (50 mM), 0.5 µL of dNTPs (10 mM), 0.6 µL of each primer (20 pmol), 0.1 µL of GoTaq® DNA Polymerase (Promega), and 2 µL of DNA template. Cycling conditions were as follows: initial denaturation at 94°C for 6 min (1 cycle); 35 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 45 s, and extension at 72°C for 1 min, followed by a final extension at 72°C for 4 min. Amplified products were analyzed by electrophoresis on a 1.5% agarose gel stained with ethidium bromide and visualized under UV illumination. PCR products (25 µL) were subsequently purified using the Wizard® SV Gel and PCR Clean-Up System kit according to the manufacturer’s instructions. The PCR products were ligated into the pGEM-T Easy cloning vector (Promega®) according to the manufacturer’s instructions. The ligation products were subsequently used to transform competent Escherichia coli TOP10 cells. Five clones from each candidate strain were selected and submitted to Macrogen (South Korea) for sequencing. Sample enrichment Bacteria isolated from the intestinal microbiota of whiteflies were individually cultured on TSA plates. A single colony from each isolate was subsequently inoculated into TSB and incubated for 24 h at 37°C. After incubation, each isolate was processed, and 1 mL of each isolate was combined in 50 mL Corning tubes to create a bacterial pool consisting of Bacillus licheniformis , Streptomyces nigra , Solibacillus silvestris , Solibacillus isronensis , and Paenibacillus lautus . Each isolate was inoculated with 5 mL of insect macerate and incubated under aerobic conditions at 37°C for 18–24 h. After incubation, the tubes were vortexed and centrifuged at 13,800 rpm for 10 min at 4°C. The supernatant was collected and filtered through nitrocellulose membranes with a pore size of 0.22 µm; this filtrate was designated as the “lysate.” Identification of bacteriophages by the “spot” test To detect the presence of lytic bacteriophages against each bacterial isolate, a spot test was performed using the double agar layer technique with the lysates obtained from each sample. Briefly, 1 mL of bacteria in the exponential growth phase was mixed with 3 mL of 0.8% (w/v) TSB-agarose, preheated and liquefied at 45°C, and the mixture was poured onto TSA plates. After drying, 5 µL of each lysate was applied to the soft agar layer. Once the plates had dried, they were inverted and incubated at 37°C for 18–24 h. This procedure was repeated for each bacterial isolate and corresponding lysate in which bacteriophage presence was suspected. The appearance of bacterial cell clearance zones in lysis plaques at the site of inoculation indicated the presence of lytic bacteriophages against the tested bacteria (Clokie and Kropinski 2009 ). Purification of bacteriophage For bacteriophage propagation, the double agar layer technique was used. The strain Bacillus licheniformis , isolated from the abdomen of whiteflies, produced lysis plaques and was therefore used as the host bacterium for bacteriophage isolation. This strain was cultured in TSB at 37°C for 24 h and evaluations were conducted using B. licheniformis in the logarithmic growth phase (5 h). Subsequently, 1 mL of bacterial culture and 100 µL of lysate containing bacteriophages were mixed with 3 mL of 0.8% (w/v) TSB-agarose, preheated and liquefied at 45°C. The mixture was homogenized, poured onto TSA plates, allowed to dry, and incubated at 37°C for 18–24 h. After incubation, plaques were selected based on size and clarity and transferred to 1.5 mL Eppendorf tubes containing 1 mL of 1.5% meat extract (BIOXON®). The double agar layer and single-plaque recovery procedure was repeated four times to obtain unique and purified phages (Clokie et al. 2009). Propagation of bacteriophages For bacteriophage propagation, the methodology described by Carey-Smith et al. (Carey-Smith et al. 2006 ) was followed with modifications. Briefly, 1 mL of the host strain in the exponential growth phase was mixed with 100 µL of purified bacteriophage in a test tube containing 3 mL of 0.8% (w/v) TSB-agarose liquefied at 45°C. The mixture was poured onto TSA plates and allowed to dry. Plates were incubated at 37°C for 18–24 h. Once bacteriophage replication was evident, the soft agar layer was recovered using a sterile cell scraper and transferred to 1.5 mL tubes containing meat extract. The final eluate was centrifuged at 8,500 rpm for 10 min at 4°C to remove bacterial cells and culture residues. The supernatant was then filtered through nitrocellulose membranes with a pore size of 0.45 µm and stored at 4°C, protected from light. Concentration and titration of bacteriophage For phage concentration and titration, 40 mL of bacteriophage propagated in the previous step was centrifuged at 40,000 rpm for 2 h. The supernatant was discarded, and the pellet was resuspended in 10 mL of meat extract buffer, followed by filtration through a 0.22 µm AcrodiscR syringe filter. Phage titers were determined using a standard plaque assay method (Clokie and Kropinski, 2009 ). Briefly, serial dilutions (10⁻¹ to 10⁻¹ 0 ) of the concentrated bacteriophage were prepared in 1.5% meat extract buffer, and the double agar layer technique was performed. Dilutions yielding between 30 and 300 countable plaques were selected, and the phage concentration was calculated accordingly. Transmission Electron Microscope Phage morphology was examined by transmission electron microscopy (TEM) using a Hitachi H7500 instrument (Hitachi Ltd., Tokyo, Japan) operated at 100 keV to visualize uranile acetate stained preparations (Luo et al. 2012 ). Purified phage particles were suspended in 2% (w/v) phosphotungstic acid (pH 7.2) and applied onto the surface of a carbon-coated, glow-discharged copper grid (200 mesh). Host range test The host range was determined using the spot test (Clokie and Kropinski, 2009 ). For this evaluation, five bacterial species isolated from the digestive system of the whitefly were tested. Bacteriolytic activity The bacteriophage infection kinetics assay was performed following the methodology described by Haq et al. ( 2012 ). Briefly, 1 mL of a Bacillus licheniformis culture was inoculated into flasks containing 50 mL of TSB broth, to which 1 mL of bacteriophage suspension in PBS (titer: 7 × 10 8 pfu/mL) was added. Cultures were incubated at 25°C with continuous shaking. A control group without bacteriophage inoculation was included. Optical density at 600 nm (OD 600 ) was measured at hourly intervals for 9 h. All assays were performed in triplicate. Thermal and pH stability Thermal stability tests were performed to evaluate the heat resistance of the phage at pH 7, with room temperature (∼23°C) serving as the control. Phage stability was assessed using a hot plate with a beaker of water and a thermometer. Briefly, phage stocks (1 × 10 8 pfu/mL in 1.5% meat extract buffer) were incubated separately for 60 min at temperatures ranging from 20 to 60°C, with intervals of 10ºC between each point. For pH stability, phage suspensions (1 × 10 8 pfu/mL) were added to meat extract buffer adjusted to different pH values (range 3–10, with intervals of 1 pH unit between each point) and incubated at 37°C for 24 h. Following incubation, surviving phages were quantified using the double agar layer method. All experiments were conducted in triplicate. Phage DNA extraction DNA extraction from bacteriophages was performed using the methodology described by Sambrook and Russell ( 2001 ), with modifications. Briefly, 1 mL of (titer: 8 × 10 8 pfu/mL) phage suspension was placed in a 1.5 mL microcentrifuge tube, to which 2 U of DNase I (Sigma-Aldrich, USA) and 2 U of RNase A (Sigma-Aldrich, USA) were added. The mixture was incubated at 37°C for 30 min. Following incubation, 40 µL of EDTA (0.5 M, pH 8.0; Sigma, USA), 25 µL of proteinase K (20 mg/mL; Qiagen, Germany), and 50 µL of sodium dodecyl sulfate (SDS; 10%; Sigma, USA) were added, and the tubes were mixed by inversion 5–10 times. Samples were then incubated at 56°C for 2 h. After incubation, an equal volume of phenol (Sigma-Aldrich, USA) was added and mixed by inversion until fully emulsified, followed by centrifugation at 3,500 rpm for 10 min at 25°C. The aqueous phase was transferred to a fresh 1.5 mL microcentrifuge tube, and an equal volume of phenol-chloroform (1:1, v/v; Sigma-Aldrich, USA) was added. Centrifugation was repeated under the same conditions three times. The final aqueous phase was transferred to a new 1.5 mL tube, and 200 µL of 3 M sodium acetate, together with absolute ethanol were added until the tube was full. Samples were incubated at -20°C overnight. The resulting pellet was centrifuged at 13,000 rpm for 30 min, the supernatant discarded, and the pellet washed with 70% ethanol. A second centrifugation was performed at 13,000 rpm for 15 min. The supernatant was removed, and the pellet was air-dried at room temperature before resuspension in 100 µL of nuclease-free water. DNA concentration and purity were assessed. Genome sequencing and analysis DNA sequencing was conducted at the Centro de Investigación en Alimentación y Desarrollo (CIAD), Mazatlán Unit, using the MiniSeq sequencing system (Illumina, Inc.) with a 2 × 150 bp paired-end protocol (300 cycles). Libraries were prepared using Nextera adapters, barcoded, and pooled with additional samples, and sequenced on a MiniSeq Mid Output Kit (300 cycles). Raw FASTQ reads were quality-trimmed using fastp v0.22.0 with the default settings (Chen et al., 2018 ), and de novo assembly was performed with SPAdes v3.15.5 (Bankevich et al. 2012 ). A minimum coverage cutoff of 10x was applied for contig retention, resulting in a single contig with an average coverage of 193.7x. Genome annotation was conducted using Pharokka v1.7.3 (Bouras et al. 2023 ) to identify structural and functional features, which was also employed for genome annotation and for the identification of virulence genes in the VFDB database and antibiotic resistance genes in the CARD database. Manual curation (BLASTp and domain-based validation) was then performed to confirm and refine Pharokka functional assignments; conflicts were resolved by prioritizing domain-supported hits and phage hallmark gene context, while low-confidence proteins were annotated as hypothetical. Bacphlip v0.9.6 (Hockenberry and Wilke 2021 ) was used to predict the lifestyle (virulent or temperate) of the isolated phage, while Taxmyphage v0.3.3 (Millard et al. 2024 ) was applied to determine phage taxonomy. A heat map was generated with VIRIDIC v1.0 (Turner et al. 2021 ) to refine the phage classification obtained from the phylogenetic tree. VIRIDIC was used to calculate the intergenomic similarity matrix with the most closely related phages reported in GenBank belonging to Siophivirus genus. In addition, phage sequences from the closest nine genus of Bastillevirinae subfamily were used to determine their taxonomic placement. Results Bacterial identification A total of nine bacterial colonies were isolated based on their color, shape, and size. Five colonies exhibited irregular circular morphology, with colors ranging from whitish to creamy gray, a convex surface, and a dry appearance after several days. Colonies labeled MB9 and MB10 were translucent and transparent, respectively, displaying irregular growth patterns. Streptomyces nigra formed colonies with a grayish-white, powdery appearance that hardened over time and developed a reddish-brown pigmentation around the colony. The remaining isolates included Solibacillus silvestris and Solibacillus isronensis , which produced dark beige circular colonies with convex surfaces and rounded edges, as well as Paenibacillus lautus , which formed light yellow circular colonies with convex morphology. Based on sequence analysis, five colonies were identified as Bacillus licheniformis , while the remaining isolates corresponded to Streptomyces nigra , Solibacillus silvestris , Solibacillus isronensis , and Paenibacillus lautus (Table 1 ). All isolates shared > 96% nucleotide identity with previously reported sequences. Table 1 Identification of the bacteria isolated from the digestive system of Bemisia tabaci Sample Organism Percentage of nucleotide identity Lysis plaques GenBank Accession No. MB2 Bacillus licheniformis 98.84 + PV399969 MB3 Streptomyces nigra 98.30 - PV399970 MB4 Bacillus licheniformis 99.32 + PV399971 MB7 Bacillus licheniformis 96.08 + PV399972 MB8 Solibacillus silvestris 97.70 - PV399973 MB9 Bacillus licheniformis 99.64 + PV399974 MB10 Bacillus licheniformis 99.46 + PV399975 MB11 Solibacillus isronensis 99.41 - PV399976 MB12 Paenibacillus lautus 97.48 - PV399977 Phage identification Among the bacterial isolates obtained from the digestive system of whiteflies ( B. licheniformis , S. nigra , S. silvestris , S. isronensis , and P. lautus ), only B. licheniformis tested positive for the presence of lysis plaques in the spot test (Table 1 ). Three lysates were evaluated: the first derived from macerated whitefly insects, the second from sooty mold on host leaves, and the third from macerated whitefly egg masses. Bacteriophages were detected exclusively in the first lysate (Fig. 1 a). Transmission electron microscopy revealed that the phage virions possess an icosahedral head (Fig. 1 b). Host range Host range analysis was performed on B. licheniformis , S. nigra , S. silvestris , S. isronensis , and P. lautus (Table 1 ). The phage produced visible plaques on all five B. licheniformis strains (MB2, MB4, MB7, MB9, and MB10), indicating that these strains were susceptible to infection. The plaques were round, translucent, and exhibited well-defined boundaries on double-layer agar plates. In contrast, no lysis plaques were observed on the other bacterial species isolated from the digestive system of whiteflies. Bacteriolytic activity The growth kinetics of Bacillus licheniformis revealed a lag phase during the first hour, followed by an exponential (logarithmic) phase that extended until the fifth hour. The stationary phase was maintained throughout the 9-hour evaluation period. In contrast, when assessing the bacteriolytic activity of the isolated phage (7 × 10 8 PFU/mL) over the same period, the lag phase of B. licheniformis was prolonged until the fourth hour. Notably, a stationary phase was not observed; instead, bacterial growth began to decline at the eighth hour, immediately following the exponential phase (Fig. 2 ). Thermal and pH stability The results demonstrated that the Yucatan phage retained 98.38% activity relative to the control at 20°C for 60 min, but progressively decreased with increasing temperature, reaching approximately 50% at 60°C (Fig. 3 A). The phage remained stable when stored at 4°C and for up to three months at -20°C and − 80°C (data not shown). Maximum activity was observed at pH 7; however, phage viability declined under more acidic or alkaline conditions. At pH 3, no activity was detected, while at pH 4, activity was reduced to 25%, and at pH 10, activity decreased to 20% (Fig. 3 B). Genomic characterization A 147,395 bp phage genome was obtained through sequencing (GenBank accession number PV261948). BLAST analysis revealed that the phage with the highest coverage (97%) and nucleotide identity (96.57%) was phage SIOphi. The highest average nucleotide indentity value was observed between our phage and SIOphi (94.6%), followed by phages KKP_4047 and KKP_4048 (92.5%), KKP_4050 (92%), and KKP_4049 (91.8%) (Fig. 4 ). These results and taxmyphage program classified Bacillus phage vB_Blim_Yucatan as a new species within the genus Siophivirus , subfamily Bastillevirinae , family Herelleviridae from class Caudoviricetes . Members of this family are bacterial viruses that infect hosts belonging to the Firmicutes phylum (Barylski et al. 2020 ). Genomic analysis revealed that the Yucatan phage genome contains no virulence or antibiotic resistance genes. The Bacphlip v0.9.6 program predicted a lytic lifestyle with 95% confidence. Analysis with Pharokka identified 266 protein-coding sequences (CDS), of which 188 encode proteins with unknown or hypothetical functions. Based on predicted functions, the remaining 69 CDS encoding known proteins were classified into functional groups: transcription regulation (4 CDS), nucleotide and DNA/RNA metabolism (24 CDS), lysis (2 CDS), moron/auxiliary metabolic genes and host takeover (7 CDS), integration and excision (1 CDS), head and packaging (17 CDS), and others (9 CDS) (Fig. 5 ). DISCUSSION Members of the family Herelleviridae are bacterial viruses that infect hosts within the phylum Firmicutes . Their genomes consist of linear dsDNA of approximately 125 to 170 kb, and some members posses terminally redundant ends or terminal repeats; additionally, certain members encode tRNAs. Virions display a head-and-tail morphology characterized by an icosahedral head and a long, contractile tail. Notably, the genome of Bacillus phage SPO1 contains thymidine replaced by 5-hydroxymethyluridine, and DNA modifications have also been reported in other members of the family (Barylski et al., 2020 ). The Yucatan phage, virulent against Bacillus licheniformis and isolated in the present study, represents a new species within the Herelleviridae family. Its genome is 147,395 bp in length, predicting a total of 266 protein-coding sequences, and has a GC content of 38.98%. Comparative analysis revealed 94.6% homology with its closest relative, phage SIOphi, which according to ICTV is currently the only recognized species of the genus Siophivirus . Phage SIOphi possesses a genome of 146,698 bp, encodes 206 predicted unique protein-coding sequences, lacks tRNAs and terminal repeats, and infects Bacillus subtilis isolated from soil samples (Krasowska et al. 2015 ). In addition, SIOphi was reported alongside three other phages infecting B. subtilis , with genome sizes ranging from 153,882 to 156,577 bp, encoding between 256 and 270 protein-coding genes, no tRNA genes, and GC contents of 38.6–38.7%, lower than that of their host B. subtilis (~ 43%) (Magness et al. 2023 ). By contrast, B. licheniformis has a GC content of ~ 46.2% (Rey et al. 2004 ), which is also higher than that of the Yucatan phage. The Herelleviridae family comprises virions with isometric icosahedral heads measuring 85–100 nm in diameter. The heads display capsomeres, with capsid subunits arranged in pentons and hexons. Uncontracted tails range from 130 to 185 nm in length, featuring a base plate of approximately 60 nm and a small collar [40]. Electron microscopy revealed that the Yucatan phage is morphologically similar to phages SIOphi and fHSpt3 (Krasowska et al. 2015 ; Midha et al. 2024 ). According to the morphological characteristics of this phage type and the latest classification by the International Committee on Taxonomy of Viruses (ICTV), the Yucatan phage exhibits the morphology typical of the class Caudoviricetes , which includes tailed phages. Within this class, the families with the highest number of sequences reported in GenBank are Autographiviridae , Herelleviridae , and Demerecviridae . Phages with this morphology are characterized by linear double-stranded DNA genomes (Zhu et al. 2022 ). Although bacteriophage classification has traditionally relied on morphological criteria and only rarely on molecular data (Ackermann 2009 ), Bacillus phage Yucatan is described here both morphologically and molecularly, making it one of the few phages of the genus Siophivirus to be characterized in both ways. Temperature plays a crucial role in bacteriophage survival, attachment efficiency, and latency period duration (Olson et al. 2004 ). Phage Yucatan remained active at the highest temperature tested (60°C) for 60 min. These results are consistent with those reported by Melo, who observed that phage SIOphi retained approximately 43% activity at 60°C after 180 min, but lost activity at 70°C within 30 min and at 80°C within 2 min. In general, members of the former family Myoviridae (now Herelleviridae ) (Barylski et al. 2020 ) and Siphoviridae are considered resistant to elevated temperatures (Jończyk et al. 2011 ). The acidity and alkalinity of the environment are important factors influencing phage stability (Melo et al. 2019 ). The Yucatan phage exhibited its highest activity at pH 7. These results are consistent with those reported by Krasowska et al. ( 2015 ), who found that phage SIOphi displayed maximum activity between pH 6 and 8, with no activity at pH 3 or 10. In contrast, the Yucatan phage retained 20% activity at pH 10. The application of bacteriophages in biotechnological processes requires detailed knowledge of their biological characteristics, including host range, latency period, growth dynamics, and resistance to stress conditions such as temperature and pH. The evaluated properties of the Yucatan phage presented in this study demonstrate its potential to suppress Bacillus licheniformis populations. Genomic analysis revealed two proteins involved in bacterial lysis: holin and endolysin, the holin-endolysin system is employed by most double-stranded DNA and tailed phages infecting Gram-positive hosts, facilitating the release of progeny virions during the final stages of the lytic cycle (Wu et al. 2021 ), and these proteins are also present in phage SIOphi. Holins are small membrane proteins that oligomerize to form pores in the cytoplasmic membrane, thereby enabling the release of endolysins into the periplasm (Wang et al. 2000 ; Young 2013 ). Endolysins are peptidoglycan hydrolases that degrade the bacterial cell wall, with lysis occurring due to osmotic pressure differences between the cell and its environment. During the lysis process, endolysins are synthesized and accumulate in the cytoplasm at the late stage of phage infection, when proteins cannot cross the cytoplasmic membrane (Schmelcher et al. 2012 ). Although ICTV currently recognizes phage SIOphi as the sole species of the genus Siophivirus , Midha et al. 2024 described Bacillus phage Fhstp3 as a member of this genus. Phage Fhstp3 was isolated from wastewater, infects B. subtilis , and possesses a genome of 150,187 bp with 221 protein-coding sequences. It lacks virulence and antibiotic resistance genes and exhibits a virulent life cycle, with 78.97% confidence according to Phage AI (Tynecki et al. 2020 ). For a phage to be considered suitable for phage therapy, host range is a critical characteristic. Ideally, a therapeutic phage should be specific to a single bacterial species to avoid off-target effects, yet broad enough within that species to infect multiple strains (Hyman 2019 ). The phage characterized in this study was evaluated against five independent Bacillus licheniformis isolated from the abdomen homogenate of the whitefly, and it exhibited lytic activity against all of them. Genomic analysis confirmed that the phage does not harbor antibiotic resistance genes, virulence factors, CRISPR elements, or tRNAs, all of which support its suitability as a potential biocontrol agent targeting B. licheniformis in the whitefly gut. The Yucatan phage showed reduced activity under pH conditions outside the neutral range; however, it can still be considered a promising candidate for suppressing B. licheniformis populations in the digestive system of Bemisia tabaci . The pH of most insect digestive systems typically ranges from 6 to 8, although exceptions exist, for example, blue flies ( Calliphora ) exhibit highly acidic gut conditions, while lepidopterans maintain alkaline environments between pH 8 and 10 (Engel et al. 2013). Previous studies using oral antibiotic treatments to eliminate gut symbionts have reported suppression or elimination of these bacteria, resulting in higher mortality rates, slower growth, and reduced body size (Bai et al. 2019 ; Koga et al. 2007 ; Rupawate et al. 2023 ). The use of bacteriophages to suppress bacterial populations within insect microbiomes as an alternative to antibiotics has been scarcely studied. Xu et al. ( 2016 ) evaluated phage BiBurk16MC_R against Burkholderia but obtained limited results, probably due to the blockage of the connection between the anterior and posterior abdominal regions, it suggests that the initial colonization by the symbiont programs the ontogeny of the midgut, providing a protected residence against microbial antagonists. Therefore, further evaluation of the Yucatan phage across different developmental instars of Bemisia tabaci , using both contact and ingestion methods, is necessary to determine its effectiveness as a biocontrol agent. Declarations Funding. PhD scholarship # 733809 for Daniel Bravo Pérez from Consejo Nacional de Ciencia y Tecnología (Conacyt). Data availability All raw data are available from the corresponding author upon request. Declarations Conflict of interest: The authors have no relevant financial or non-financial interests to disclose. Author Contribution DBP, CCHQ and OAMV conceived the project, DBP performed the majority of the experiments, JPGG contributed to the bioinformatic analysis, YMG contributed to molecular whiteflies identification, ECL contributed to electron microscopy experiments, JPGG, LRP and CHZ contributed to the analysis of the data, DBP, OAMV and CHZ wrote the manuscript, and all authors contributed to the final version. All authors gave their final approval of the version to be published and pledge accountability for the quality and reliability of the work. References Ackermann HW (2009) Phage classification and characterization. Methods Mol Biol 501:127–140. https://doi.org/10.1007/978-1-60327-164-6_13 Arora AK, Douglas AE (2017) Hype or opportunity? Using microbial symbionts in novel strategies for insect pest control. 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Front Microbiol 14:1146390. https://doi.org/10.3389/fmicb.2023.1146390 Sambrook J, Russell D (2001) Molecular Cloning: A Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Sawa T, Moriyama K, Kinoshita M (2024) Current status of bacteriophage therapy for severe bacterial infections. J Intensive Care 12:44. https://doi.org/10.1186/s40560-024-00759-7 Schmelcher M, Donovan DM, Loessner MJ (2012) Bacteriophage endolysins as novel antimicrobials. Future Microbiol 7(10):1147–1171. https://doi.org/10.2217/fmb.12.97 Tynecki P, Guziński A, Kazimierczak J, Jadczuk M, Dastych J, Onisko A (2020) PhageAI - Bacteriophage Life Cycle Recognition with Machine Learning and Natural Language Processing. bioRxiv. https://doi.org/10.1101/2020.07.11.198606 Tito TM, Rodrigues NMB, Coelho SMO, de Souza MMS, Zonta E, Coelho IS (2015) Choice of DNA extraction protocols from Gram negative and positive bacteria and directly from the soil. Afr J Microbiol Res 9(12):863–871. https://doi.org/10.5897/AJMR2014.7259 Turner D, Kropinski AM, Adriaenssens EM (2021) A Roadmap for Genome-Based Phage Taxonomy. Viruses 13(3):506. https://doi.org/10.3390/v13030506 Wang IN, Smith DL, Young R (2000) Holins: The Protein Clocks of Bacteriophage Infections. Annu Rev Microbiol 54:799–825. https://doi.org/10.1146/annurev.micro.54.1.799 Wu Z, Zhang Y, Xu X, Ahmed T, Yang Y, Loh B, Leptihn S, Yan C, Chen J, Li B (2021) The Holin-Endolysin Lysis System of the OP2-Like Phage X2 Infecting Xanthomonas oryzae pv. oryzae . Viruses 13(10):1949. https://doi.org/10.3390/v13101949 Xu Y, Buss EA, Boucias DG (2016) Impacts of Antibiotic and Bacteriophage Treatments on the Gut-Symbiont-Associated Blissus insularis (Hemiptera: Blissidae). Insects 7(4):61. https://doi.org/10.3390/insects7040061 Young R (2013) Phage lysis: do we have the hole story yet? Curr Opin Microbiol 6(6):790–797. https://doi.org/10.1016/j.mib.2013.08.008 Zhu Y, Shang J, Peng C, Sun Y (2022) Phage family classification under Caudoviricetes: A review of current tools using the latest ICTV classification framework. Front Microbiol 16(13):1032186. https://doi.org/10.3389/fmicb.2022.1032186 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 18 May, 2026 Reviewers agreed at journal 04 May, 2026 Reviewers invited by journal 04 May, 2026 Editor assigned by journal 29 Apr, 2026 Submission checks completed at journal 29 Apr, 2026 First submitted to journal 28 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9557867","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":637282698,"identity":"c846ca68-bfca-4efd-8c24-fd1573d63ddf","order_by":0,"name":"Daniel Bravo-Pérez","email":"","orcid":"","institution":"Centro de Investigación Científica de Yucatán (CICY)","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Bravo-Pérez","suffix":""},{"id":637282699,"identity":"217d7366-6848-4255-878e-e4c4e8d34b90","order_by":1,"name":"Jean Pierre González-Gómez","email":"","orcid":"","institution":"A.C. (CIAD)","correspondingAuthor":false,"prefix":"","firstName":"Jean","middleName":"Pierre","lastName":"González-Gómez","suffix":""},{"id":637282703,"identity":"59200b75-3a69-48ee-8bbe-1183725bdc8d","order_by":2,"name":"Ernestina Castro-Longoria","email":"","orcid":"","institution":"Center for Scientific Research and Higher Education at Ensenada","correspondingAuthor":false,"prefix":"","firstName":"Ernestina","middleName":"","lastName":"Castro-Longoria","suffix":""},{"id":637282706,"identity":"538a38fb-3daf-4c1b-a529-dc5849c17a67","order_by":3,"name":"Cristóbal Chaidez-Quiroz","email":"","orcid":"","institution":"A.C. 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B.Viability of Yucatan phage at different pH.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9557867/v1/cbb44174af2c8fa7314d5d60.jpg"},{"id":109205980,"identity":"743752fc-5576-49df-836c-e33b6db7ffbb","added_by":"auto","created_at":"2026-05-13 15:10:03","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":47690,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap of genomic sequence similarity (right) and alignment metrics (left) generated by VIRIDIC between \u003cem\u003eBacillus\u003c/em\u003e phage Yucatan, the phages with the highest similarity, and phages other genera.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9557867/v1/dd7ace36d1242aa66904eca4.jpg"},{"id":109205927,"identity":"d5b0d109-3125-4775-95eb-09b58ef79084","added_by":"auto","created_at":"2026-05-13 15:09:32","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":55731,"visible":true,"origin":"","legend":"\u003cp\u003eGenome map of the Yucatan phage. The phage genome was represented in a circular shape. Different colors represent different gene functions.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9557867/v1/1be1295848c2ce0ebff4866e.jpg"},{"id":109207297,"identity":"dd401896-08fe-46ee-8ee9-50b745d6b263","added_by":"auto","created_at":"2026-05-13 15:19:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":891253,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9557867/v1/35d52ceb-a24a-4c1e-95af-211494afe4ea.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eIsolation and Characterization of the Bacteriophage Yucatan Specific to Bacillus Licheniformis\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInsects represent the animal group with the greatest number of species worldwide. Owing to their wide distribution, they are commonly associated with diverse microorganisms, including bacteria, viruses, fungi, protozoa, nematodes, and multicellular parasites (Mondal et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Permanent associations between bacteria and higher organisms are referred to as symbiosis, with the type of symbiosis depending on the host, the symbiont, and the environmental conditions (Arora et al. 2017). Thus, bacterial identification is important to determine roles related to hosts\u0026rsquo; biology including nutrition, development, defense against pathogens, community interactions, and survival in hostile environments through toxin metabolism (Rupawate et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eBemisia tabaci\u003c/em\u003e is one of the world's most important pests, as it directly attacks agricultural products and is the main vector of plant viruses, such as Geminiviruses. \u003cem\u003eB. tabaci\u003c/em\u003e is a species complex with genetically distinct lineages, host types, and rapid spread. Biotype A is found in the Americas, while biotypes B (MEAM1) and Q (MED) are present worldwide (De Barro et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Therefore, biotype A is of paramount importance for studies related to endemic species in the Americas, specifically Mexico and \u003cem\u003eCapsicum annuum\u003c/em\u003e plants. Several studies have isolated and identified bacterial symbionts from \u003cem\u003eBemisia tabaci\u003c/em\u003e. Ateyyat et al. (2023) isolated and identified culturable bacteria reporting \u003cem\u003eB. licheniformis\u003c/em\u003e in both nymphs and adults. Similarly, Pujar et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) isolated bacterial colonies from \u003cem\u003eBemisia tabaci\u003c/em\u003e where the predominant bacterial species identified were \u003cem\u003eBacillus licheniformis\u003c/em\u003e, \u003cem\u003eBacillus pumilus\u003c/em\u003e, and \u003cem\u003eBacillus safensis\u003c/em\u003e, among others. The examples suggest that \u003cem\u003eB. licheniformis\u003c/em\u003e is an important component of the \u003cem\u003eBemisia tabaci\u003c/em\u003e microbiome.\u003c/p\u003e \u003cp\u003e \u003cem\u003eBacillus licheniformis\u003c/em\u003e belongs to the family \u003cem\u003eBacillaceae\u003c/em\u003e, whose members are characterized by their ability to form endospores, conferring high resistance to heat, radiation, chemicals, and desiccation, and enabling survival under adverse conditions for extended periods (He et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). \u003cem\u003eB. licheniformis\u003c/em\u003e is predominantly found in soil and is a rod-shaped, Gram-positive, motile, and facultatively anaerobic species (Logan et al. 2015). It is part of the \u003cem\u003eBacillus subtilis\u003c/em\u003e group, which comprises two subspecies (\u003cem\u003eB. subtilis\u003c/em\u003e subsp. \u003cem\u003esubtilis\u003c/em\u003e and \u003cem\u003eB. subtilis\u003c/em\u003e subsp. \u003cem\u003espizizenii\u003c/em\u003e) and twelve species (\u003cem\u003eB. mojavensis\u003c/em\u003e, \u003cem\u003eB. valismortis\u003c/em\u003e, \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e, \u003cem\u003eB. atrophaeus\u003c/em\u003e, \u003cem\u003eB. pumilus\u003c/em\u003e, \u003cem\u003eB. licheniformis\u003c/em\u003e, \u003cem\u003eB. sonorensis\u003c/em\u003e, \u003cem\u003eB. aquimaris\u003c/em\u003e, \u003cem\u003eB. oleronius\u003c/em\u003e, \u003cem\u003eB. sporothermodurans\u003c/em\u003e, \u003cem\u003eB. carboniphilus\u003c/em\u003e, and \u003cem\u003eB. endophyticus\u003c/em\u003e) (Mandic-Mulec et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Although \u003cem\u003eB. licheniformis\u003c/em\u003e isolates are mainly derived from soil, their sources of isolation are diverse, including insects, bird feathers, internal plant tissues, animal leather, milk, paper, and cardboard (Logan et al. 2015). In insects, \u003cem\u003eB. licheniformis\u003c/em\u003e has been isolated from the digestive systems of both nymphs and adults of whitefly species (\u003cem\u003eBemisia tabaci\u003c/em\u003e, \u003cem\u003eB. argentifolii\u003c/em\u003e) (Ateyyat et al. 2023; Davidson et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Pujar et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Some studies suggest that, due to the characteristics of the insect microbiota, including \u003cem\u003eB. licheniformis\u003c/em\u003e, host survival against insecticides is enhanced by the production of pesticide-degrading enzymes, the microbiome acting as a type of defense (Fan et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Once the symbiotic bacteria of the insect are identified and their importance in biological development is recognized, one strategy that has been employed to disrupt insect development is the elimination of these bacteria through antibiotic treatment, resulting in reduced fertility and survival rates (Bai et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Koga et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). However, the emergence of antibiotic resistance among bacteria poses a significant challenge for their management, highlighting the need for alternative approaches. One such alternative, used for over a century and now being widely reconsidered, is phage therapy (Sawa et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBacteriophages, or phages, are viruses that specifically infect and replicate within bacterial cells. They are recognized as the most abundant biological entities on Earth, exhibiting diverse sizes, morphologies, and genomic organizations (Kasman et al. 2022). Tailed phages are considered key components in shaping the structure, dynamics, and interactions of microbial communities across different habitats. They also exert a significant impact on human health and the food industry (Nami et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Structurally, phages consist of nucleic acid enclosed within a protein shell, the capsid, which protects the genetic material and mediates its delivery into the host cell (Rees et al. 2024). Phages are highly specific, often infecting only a single bacterial species or even particular strains within that species. Although phages represent a promising alternative for biocontrol, it is essential to characterize them both biologically and genetically. Such characterization allows the determination of host range, resistance to environmental stresses, and verification of the absence of undesirable genes in their genomes, particularly those associated with host virulence modification or antibiotic resistance. This knowledge is fundamental for understanding the potential of phages to regulate bacterial populations and for tailoring their application to be both precise and effective. Furthermore, molecular analysis provides a robust foundation for their taxonomic classification (Elois et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Jo et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In this study, we present the morphological and genomic characterization of a novel bacteriophage related to SIOphi, isolated from the digestive system of \u003cem\u003eBemisia tabaci\u003c/em\u003e (whitefly) Biotype A, collected from habanero. This phage demonstrated \u003cem\u003ein vitro\u003c/em\u003e effectiveness in controlling cultured \u003cem\u003eBacillus licheniformis\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSampling\u003c/h2\u003e \u003cp\u003eAdult specimens of the whitefly \u003cem\u003eBemisia tabaci\u003c/em\u003e were collected from habanero pepper (\u003cem\u003eCapsicum chinense\u003c/em\u003e Jacq.) in greenhouses at the Scientific Research Center of Yucat\u0026aacute;n (21\u0026deg;01\u0026prime;47\u0026Prime; N, 89\u0026deg;38\u0026prime;20\u0026Prime; W). The sampled whiteflies corresponded to biotype A, as reported before (Bravo-P\u0026eacute;rez et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Specimens were collected in small jars with mesh lids containing 70% ethanol and transported to the laboratory for processing. The jars were stored at 4\u0026deg;C and processed on the same day.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIsolation of intestinal bacteria\u003c/h3\u003e\n\u003cp\u003eTo ensure that only intestinal bacteria were obtained and to prevent external contamination, insects were first cooled at 4\u0026deg;C for 15 minutes. They were then washed in a Petri dish under a stereoscope within a laminar flow hood using 70% ethanol for 5 minutes, with gentle agitation every minute. Subsequently, the insects were rinsed three times in phosphate-buffered saline (PBS, pH 7.4). From the final rinse, 100 \u0026micro;L of buffer was spread onto trypticase soy agar (TSA; BIOXON\u0026reg;) plates to confirm the absence of residual bacteria. In addition, sanitized whole insects were deposited on TSA plates to verify the absence of bacteria or other microorganisms.\u003c/p\u003e \u003cp\u003eThe abdomens of 50 insects were carefully removed and placed in a 1.5 mL microcentrifuge tube containing 200 \u0026micro;L of sterile PBS, then macerated with a sterile pestle for 30 seconds (Apte-Deshpande et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). From the macerated mixture, 100 \u0026micro;L were taken to prepare dilutions (10⁻\u0026sup1; to 10⁻\u0026sup2;), which were plated on TSA medium by spread plating: 100 \u0026micro;L of the 10⁻\u0026sup1; dilution (two replicates), 100 \u0026micro;L of the 10⁻\u0026sup2; dilution (two replicates), and 100 \u0026micro;L of the initial macerate (one replicate). Plates were incubated at 37\u0026deg;C for 48 h. Colonies were selected based on differential morphology, suspended in 100 \u0026micro;L of sterile distilled water, and individually spread onto TSA plates. After 72 h, colonies were cultured in 5 mL of trypticase soy broth (TSB; BIOXON\u0026reg;) for subsequent DNA extraction. Bacterial growth was monitored by spectrophotometry, until an optical density of 0.8 at 600 nm was reached.\u003c/p\u003e\n\u003ch3\u003eBacterial DNA Extraction\u003c/h3\u003e\n\u003cp\u003eDNA extraction was carried out using a cetyltrimethylammonium bromide (CTAB)-based protocol (Sambrook and Russell \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Tito et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). DNA concentration and purity were assessed with a Nanodrop 2000c spectrophotometer (Thermo Scientific, USA), while integrity was confirmed by visualization on a 1.5% agarose gel under UV illumination.\u003c/p\u003e\n\u003ch3\u003ePCR amplification, cloning and sequencing\u003c/h3\u003e\n\u003cp\u003eBacterial DNA was amplified by PCR using a T100\u0026trade; thermal cycler (Bio-Rad, Germany). Degenerate primers 1494r (5\u0026prime;-TAC GGY TAC CTT GTT AGC ACT T-3\u0026prime;) and 27f (5\u0026prime;-AGA GTT TGA TCM TGG CTC AG-3\u0026prime;) were used//employed to amplify a\u0026thinsp;~\u0026thinsp;1542 bp fragment corresponding to the near complete 16S rRNA gene region. PCR reactions were performed in a final volume of 25 \u0026micro;L, containing 17.7 \u0026micro;L of water, 1 \u0026micro;L of MgCl\u003csub\u003e2\u003c/sub\u003e (50 mM), 0.5 \u0026micro;L of dNTPs (10 mM), 0.6 \u0026micro;L of each primer (20 pmol), 0.1 \u0026micro;L of GoTaq\u0026reg; DNA Polymerase (Promega), and 2 \u0026micro;L of DNA template. Cycling conditions were as follows: initial denaturation at 94\u0026deg;C for 6 min (1 cycle); 35 cycles of denaturation at 94\u0026deg;C for 30 s, annealing at 58\u0026deg;C for 45 s, and extension at 72\u0026deg;C for 1 min, followed by a final extension at 72\u0026deg;C for 4 min. Amplified products were analyzed by electrophoresis on a 1.5% agarose gel stained with ethidium bromide and visualized under UV illumination. PCR products (25 \u0026micro;L) were subsequently purified using the Wizard\u0026reg; SV Gel and PCR Clean-Up System kit according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cp\u003eThe PCR products were ligated into the pGEM-T Easy cloning vector (Promega\u0026reg;) according to the manufacturer\u0026rsquo;s instructions. The ligation products were subsequently used to transform competent \u003cem\u003eEscherichia coli\u003c/em\u003e TOP10 cells. Five clones from each candidate strain were selected and submitted to Macrogen (South Korea) for sequencing.\u003c/p\u003e\n\u003ch3\u003eSample enrichment\u003c/h3\u003e\n\u003cp\u003eBacteria isolated from the intestinal microbiota of whiteflies were individually cultured on TSA plates. A single colony from each isolate was subsequently inoculated into TSB and incubated for 24 h at 37\u0026deg;C. After incubation, each isolate was processed, and 1 mL of each isolate was combined in 50 mL Corning tubes to create a bacterial pool consisting of \u003cem\u003eBacillus licheniformis\u003c/em\u003e, \u003cem\u003eStreptomyces nigra\u003c/em\u003e, \u003cem\u003eSolibacillus silvestris\u003c/em\u003e, \u003cem\u003eSolibacillus isronensis\u003c/em\u003e, and \u003cem\u003ePaenibacillus lautus\u003c/em\u003e. Each isolate was inoculated with 5 mL of insect macerate and incubated under aerobic conditions at 37\u0026deg;C for 18\u0026ndash;24 h. After incubation, the tubes were vortexed and centrifuged at 13,800 rpm for 10 min at 4\u0026deg;C. The supernatant was collected and filtered through nitrocellulose membranes with a pore size of 0.22 \u0026micro;m; this filtrate was designated as the \u0026ldquo;lysate.\u0026rdquo;\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of bacteriophages by the \u0026ldquo;spot\u0026rdquo; test\u003c/h2\u003e \u003cp\u003eTo detect the presence of lytic bacteriophages against each bacterial isolate, a spot test was performed using the double agar layer technique with the lysates obtained from each sample. Briefly, 1 mL of bacteria in the exponential growth phase was mixed with 3 mL of 0.8% (w/v) TSB-agarose, preheated and liquefied at 45\u0026deg;C, and the mixture was poured onto TSA plates. After drying, 5 \u0026micro;L of each lysate was applied to the soft agar layer. Once the plates had dried, they were inverted and incubated at 37\u0026deg;C for 18\u0026ndash;24 h. This procedure was repeated for each bacterial isolate and corresponding lysate in which bacteriophage presence was suspected. The appearance of bacterial cell clearance zones in lysis plaques at the site of inoculation indicated the presence of lytic bacteriophages against the tested bacteria (Clokie and Kropinski \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePurification of bacteriophage\u003c/h3\u003e\n\u003cp\u003eFor bacteriophage propagation, the double agar layer technique was used. The strain \u003cem\u003eBacillus licheniformis\u003c/em\u003e, isolated from the abdomen of whiteflies, produced lysis plaques and was therefore used as the host bacterium for bacteriophage isolation. This strain was cultured in TSB at 37\u0026deg;C for 24 h and evaluations were conducted using \u003cem\u003eB. licheniformis\u003c/em\u003e in the logarithmic growth phase (5 h). Subsequently, 1 mL of bacterial culture and 100 \u0026micro;L of lysate containing bacteriophages were mixed with 3 mL of 0.8% (w/v) TSB-agarose, preheated and liquefied at 45\u0026deg;C. The mixture was homogenized, poured onto TSA plates, allowed to dry, and incubated at 37\u0026deg;C for 18\u0026ndash;24 h. After incubation, plaques were selected based on size and clarity and transferred to 1.5 mL Eppendorf tubes containing 1 mL of 1.5% meat extract (BIOXON\u0026reg;). The double agar layer and single-plaque recovery procedure was repeated four times to obtain unique and purified phages (Clokie et al. 2009).\u003c/p\u003e\n\u003ch3\u003ePropagation of bacteriophages\u003c/h3\u003e\n\u003cp\u003eFor bacteriophage propagation, the methodology described by Carey-Smith et al. (Carey-Smith et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) was followed with modifications. Briefly, 1 mL of the host strain in the exponential growth phase was mixed with 100 \u0026micro;L of purified bacteriophage in a test tube containing 3 mL of 0.8% (w/v) TSB-agarose liquefied at 45\u0026deg;C. The mixture was poured onto TSA plates and allowed to dry. Plates were incubated at 37\u0026deg;C for 18\u0026ndash;24 h. Once bacteriophage replication was evident, the soft agar layer was recovered using a sterile cell scraper and transferred to 1.5 mL tubes containing meat extract. The final eluate was centrifuged at 8,500 rpm for 10 min at 4\u0026deg;C to remove bacterial cells and culture residues. The supernatant was then filtered through nitrocellulose membranes with a pore size of 0.45 \u0026micro;m and stored at 4\u0026deg;C, protected from light.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eConcentration and titration of bacteriophage\u003c/h2\u003e \u003cp\u003eFor phage concentration and titration, 40 mL of bacteriophage propagated in the previous step was centrifuged at 40,000 rpm for 2 h. The supernatant was discarded, and the pellet was resuspended in 10 mL of meat extract buffer, followed by filtration through a 0.22 \u0026micro;m AcrodiscR syringe filter. Phage titers were determined using a standard plaque assay method (Clokie and Kropinski, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Briefly, serial dilutions (10⁻\u0026sup1; to 10⁻\u0026sup1;\u003csup\u003e0\u003c/sup\u003e) of the concentrated bacteriophage were prepared in 1.5% meat extract buffer, and the double agar layer technique was performed. Dilutions yielding between 30 and 300 countable plaques were selected, and the phage concentration was calculated accordingly.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTransmission Electron Microscope\u003c/h2\u003e \u003cp\u003ePhage morphology was examined by transmission electron microscopy (TEM) using a Hitachi H7500 instrument (Hitachi Ltd., Tokyo, Japan) operated at 100 keV to visualize uranile acetate stained preparations (Luo et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Purified phage particles were suspended in 2% (w/v) phosphotungstic acid (pH 7.2) and applied onto the surface of a carbon-coated, glow-discharged copper grid (200 mesh).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHost range test\u003c/h2\u003e \u003cp\u003eThe host range was determined using the spot test (Clokie and Kropinski, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). For this evaluation, five bacterial species isolated from the digestive system of the whitefly were tested.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eBacteriolytic activity\u003c/h2\u003e \u003cp\u003eThe bacteriophage infection kinetics assay was performed following the methodology described by Haq et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Briefly, 1 mL of a \u003cem\u003eBacillus licheniformis\u003c/em\u003e culture was inoculated into flasks containing 50 mL of TSB broth, to which 1 mL of bacteriophage suspension in PBS (titer: 7 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e pfu/mL) was added. Cultures were incubated at 25\u0026deg;C with continuous shaking. A control group without bacteriophage inoculation was included. Optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) was measured at hourly intervals for 9 h. All assays were performed in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eThermal and pH stability\u003c/h2\u003e \u003cp\u003eThermal stability tests were performed to evaluate the heat resistance of the phage at pH 7, with room temperature (\u0026sim;23\u0026deg;C) serving as the control. Phage stability was assessed using a hot plate with a beaker of water and a thermometer. Briefly, phage stocks (1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e pfu/mL in 1.5% meat extract buffer) were incubated separately for 60 min at temperatures ranging from 20 to 60\u0026deg;C, with intervals of 10\u0026ordm;C between each point. For pH stability, phage suspensions (1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e pfu/mL) were added to meat extract buffer adjusted to different pH values (range 3\u0026ndash;10, with intervals of 1 pH unit between each point) and incubated at 37\u0026deg;C for 24 h. Following incubation, surviving phages were quantified using the double agar layer method. All experiments were conducted in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePhage DNA extraction\u003c/h2\u003e \u003cp\u003eDNA extraction from bacteriophages was performed using the methodology described by Sambrook and Russell (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), with modifications. Briefly, 1 mL of (titer: 8 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e pfu/mL) phage suspension was placed in a 1.5 mL microcentrifuge tube, to which 2 U of DNase I (Sigma-Aldrich, USA) and 2 U of RNase A (Sigma-Aldrich, USA) were added. The mixture was incubated at 37\u0026deg;C for 30 min. Following incubation, 40 \u0026micro;L of EDTA (0.5 M, pH 8.0; Sigma, USA), 25 \u0026micro;L of proteinase K (20 mg/mL; Qiagen, Germany), and 50 \u0026micro;L of sodium dodecyl sulfate (SDS; 10%; Sigma, USA) were added, and the tubes were mixed by inversion 5\u0026ndash;10 times. Samples were then incubated at 56\u0026deg;C for 2 h. After incubation, an equal volume of phenol (Sigma-Aldrich, USA) was added and mixed by inversion until fully emulsified, followed by centrifugation at 3,500 rpm for 10 min at 25\u0026deg;C. The aqueous phase was transferred to a fresh 1.5 mL microcentrifuge tube, and an equal volume of phenol-chloroform (1:1, v/v; Sigma-Aldrich, USA) was added. Centrifugation was repeated under the same conditions three times. The final aqueous phase was transferred to a new 1.5 mL tube, and 200 \u0026micro;L of 3 M sodium acetate, together with absolute ethanol were added until the tube was full. Samples were incubated at -20\u0026deg;C overnight. The resulting pellet was centrifuged at 13,000 rpm for 30 min, the supernatant discarded, and the pellet washed with 70% ethanol. A second centrifugation was performed at 13,000 rpm for 15 min. The supernatant was removed, and the pellet was air-dried at room temperature before resuspension in 100 \u0026micro;L of nuclease-free water. DNA concentration and purity were assessed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eGenome sequencing and analysis\u003c/h2\u003e \u003cp\u003eDNA sequencing was conducted at the Centro de Investigaci\u0026oacute;n en Alimentaci\u0026oacute;n y Desarrollo (CIAD), Mazatl\u0026aacute;n Unit, using the MiniSeq sequencing system (Illumina, Inc.) with a 2 \u0026times; 150 bp paired-end protocol (300 cycles). Libraries were prepared using Nextera adapters, barcoded, and pooled with additional samples, and sequenced on a MiniSeq Mid Output Kit (300 cycles). Raw FASTQ reads were quality-trimmed using fastp v0.22.0 with the default settings (Chen et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and \u003cem\u003ede novo\u003c/em\u003e assembly was performed with SPAdes v3.15.5 (Bankevich et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). A minimum coverage cutoff of 10x was applied for contig retention, resulting in a single contig with an average coverage of 193.7x.\u003c/p\u003e \u003cp\u003eGenome annotation was conducted using Pharokka v1.7.3 (Bouras et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) to identify structural and functional features, which was also employed for genome annotation and for the identification of virulence genes in the VFDB database and antibiotic resistance genes in the CARD database. Manual curation (BLASTp and domain-based validation) was then performed to confirm and refine Pharokka functional assignments; conflicts were resolved by prioritizing domain-supported hits and phage hallmark gene context, while low-confidence proteins were annotated as hypothetical. Bacphlip v0.9.6 (Hockenberry and Wilke \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) was used to predict the lifestyle (virulent or temperate) of the isolated phage, while Taxmyphage v0.3.3 (Millard et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) was applied to determine phage taxonomy. A heat map was generated with VIRIDIC v1.0 (Turner et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) to refine the phage classification obtained from the phylogenetic tree. VIRIDIC was used to calculate the intergenomic similarity matrix with the most closely related phages reported in GenBank belonging to \u003cem\u003eSiophivirus\u003c/em\u003e genus. In addition, phage sequences from the closest nine genus of \u003cem\u003eBastillevirinae\u003c/em\u003e subfamily were used to determine their taxonomic placement.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eBacterial identification\u003c/h2\u003e \u003cp\u003eA total of nine bacterial colonies were isolated based on their color, shape, and size. Five colonies exhibited irregular circular morphology, with colors ranging from whitish to creamy gray, a convex surface, and a dry appearance after several days. Colonies labeled MB9 and MB10 were translucent and transparent, respectively, displaying irregular growth patterns. \u003cem\u003eStreptomyces nigra\u003c/em\u003e formed colonies with a grayish-white, powdery appearance that hardened over time and developed a reddish-brown pigmentation around the colony. The remaining isolates included \u003cem\u003eSolibacillus silvestris\u003c/em\u003e and \u003cem\u003eSolibacillus isronensis\u003c/em\u003e, which produced dark beige circular colonies with convex surfaces and rounded edges, as well as \u003cem\u003ePaenibacillus lautus\u003c/em\u003e, which formed light yellow circular colonies with convex morphology.\u003c/p\u003e \u003cp\u003eBased on sequence analysis, five colonies were identified as \u003cem\u003eBacillus licheniformis\u003c/em\u003e, while the remaining isolates corresponded to \u003cem\u003eStreptomyces nigra\u003c/em\u003e, \u003cem\u003eSolibacillus silvestris\u003c/em\u003e, \u003cem\u003eSolibacillus isronensis\u003c/em\u003e, and \u003cem\u003ePaenibacillus lautus\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All isolates shared\u0026thinsp;\u0026gt;\u0026thinsp;96% nucleotide identity with previously reported sequences.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIdentification of the bacteria isolated from the digestive system of \u003cem\u003eBemisia tabaci\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOrganism\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePercentage\u003c/p\u003e \u003cp\u003eof nucleotide identity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLysis plaques\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGenBank\u003c/p\u003e \u003cp\u003eAccession No.\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMB2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eBacillus licheniformis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e98.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePV399969\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMB3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eStreptomyces nigra\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e98.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePV399970\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMB4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eBacillus licheniformis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePV399971\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMB7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eBacillus licheniformis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e96.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePV399972\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMB8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSolibacillus silvestris\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e97.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePV399973\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMB9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eBacillus licheniformis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePV399974\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMB10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eBacillus licheniformis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePV399975\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMB11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSolibacillus isronensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePV399976\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMB12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePaenibacillus lautus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e97.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePV399977\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e\u003c/h2\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003ePhage identification\u003c/h2\u003e \u003cp\u003eAmong the bacterial isolates obtained from the digestive system of whiteflies (\u003cem\u003eB. licheniformis\u003c/em\u003e, \u003cem\u003eS. nigra\u003c/em\u003e, \u003cem\u003eS. silvestris\u003c/em\u003e, \u003cem\u003eS. isronensis\u003c/em\u003e, and \u003cem\u003eP. lautus\u003c/em\u003e), only \u003cem\u003eB. licheniformis\u003c/em\u003e tested positive for the presence of lysis plaques in the spot test (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Three lysates were evaluated: the first derived from macerated whitefly insects, the second from sooty mold on host leaves, and the third from macerated whitefly egg masses. Bacteriophages were detected exclusively in the first lysate (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Transmission electron microscopy revealed that the phage virions possess an icosahedral head (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eHost range\u003c/h2\u003e \u003cp\u003eHost range analysis was performed on \u003cem\u003eB. licheniformis\u003c/em\u003e, \u003cem\u003eS. nigra\u003c/em\u003e, \u003cem\u003eS. silvestris\u003c/em\u003e, \u003cem\u003eS. isronensis\u003c/em\u003e, and \u003cem\u003eP. lautus\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The phage produced visible plaques on all five \u003cem\u003eB. licheniformis\u003c/em\u003e strains (MB2, MB4, MB7, MB9, and MB10), indicating that these strains were susceptible to infection. The plaques were round, translucent, and exhibited well-defined boundaries on double-layer agar plates. In contrast, no lysis plaques were observed on the other bacterial species isolated from the digestive system of whiteflies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e\u003c/h2\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eBacteriolytic activity\u003c/h2\u003e \u003cp\u003eThe growth kinetics of \u003cem\u003eBacillus licheniformis\u003c/em\u003e revealed a lag phase during the first hour, followed by an exponential (logarithmic) phase that extended until the fifth hour. The stationary phase was maintained throughout the 9-hour evaluation period. In contrast, when assessing the bacteriolytic activity of the isolated phage (7 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e PFU/mL) over the same period, the lag phase of \u003cem\u003eB. licheniformis\u003c/em\u003e was prolonged until the fourth hour. Notably, a stationary phase was not observed; instead, bacterial growth began to decline at the eighth hour, immediately following the exponential phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eThermal and pH stability\u003c/h2\u003e \u003cp\u003eThe results demonstrated that the Yucatan phage retained 98.38% activity relative to the control at 20\u0026deg;C for 60 min, but progressively decreased with increasing temperature, reaching approximately 50% at 60\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The phage remained stable when stored at 4\u0026deg;C and for up to three months at -20\u0026deg;C and \u0026minus;\u0026thinsp;80\u0026deg;C (data not shown). Maximum activity was observed at pH 7; however, phage viability declined under more acidic or alkaline conditions. At pH 3, no activity was detected, while at pH 4, activity was reduced to 25%, and at pH 10, activity decreased to 20% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003e\u003c/h2\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eGenomic characterization\u003c/h2\u003e \u003cp\u003eA 147,395 bp phage genome was obtained through sequencing (GenBank accession number PV261948). BLAST analysis revealed that the phage with the highest coverage (97%) and nucleotide identity (96.57%) was phage SIOphi. The highest average nucleotide indentity value was observed between our phage and SIOphi (94.6%), followed by phages KKP_4047 and KKP_4048 (92.5%), KKP_4050 (92%), and KKP_4049 (91.8%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These results and taxmyphage program classified \u003cem\u003eBacillus\u003c/em\u003e phage vB_Blim_Yucatan as a new species within the genus \u003cem\u003eSiophivirus\u003c/em\u003e, subfamily \u003cem\u003eBastillevirinae\u003c/em\u003e, family \u003cem\u003eHerelleviridae\u003c/em\u003e from class \u003cem\u003eCaudoviricetes\u003c/em\u003e. Members of this family are bacterial viruses that infect hosts belonging to the \u003cem\u003eFirmicutes\u003c/em\u003e phylum (Barylski et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGenomic analysis revealed that the Yucatan phage genome contains no virulence or antibiotic resistance genes. The Bacphlip v0.9.6 program predicted a lytic lifestyle with 95% confidence. Analysis with Pharokka identified 266 protein-coding sequences (CDS), of which 188 encode proteins with unknown or hypothetical functions. Based on predicted functions, the remaining 69 CDS encoding known proteins were classified into functional groups: transcription regulation (4 CDS), nucleotide and DNA/RNA metabolism (24 CDS), lysis (2 CDS), moron/auxiliary metabolic genes and host takeover (7 CDS), integration and excision (1 CDS), head and packaging (17 CDS), and others (9 CDS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eMembers of the family \u003cem\u003eHerelleviridae\u003c/em\u003e are bacterial viruses that infect hosts within the phylum \u003cem\u003eFirmicutes\u003c/em\u003e. Their genomes consist of linear dsDNA of approximately 125 to 170 kb, and some members posses terminally redundant ends or terminal repeats; additionally, certain members encode tRNAs. Virions display a head-and-tail morphology characterized by an icosahedral head and a long, contractile tail. Notably, the genome of \u003cem\u003eBacillus\u003c/em\u003e phage SPO1 contains thymidine replaced by 5-hydroxymethyluridine, and DNA modifications have also been reported in other members of the family (Barylski et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe Yucatan phage, virulent against \u003cem\u003eBacillus licheniformis\u003c/em\u003e and isolated in the present study, represents a new species within the \u003cem\u003eHerelleviridae\u003c/em\u003e family. Its genome is 147,395 bp in length, predicting a total of 266 protein-coding sequences, and has a GC content of 38.98%. Comparative analysis revealed 94.6% homology with its closest relative, phage SIOphi, which according to ICTV is currently the only recognized species of the genus \u003cem\u003eSiophivirus\u003c/em\u003e. Phage SIOphi possesses a genome of 146,698 bp, encodes 206 predicted unique protein-coding sequences, lacks tRNAs and terminal repeats, and infects \u003cem\u003eBacillus subtilis\u003c/em\u003e isolated from soil samples (Krasowska et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition, SIOphi was reported alongside three other phages infecting \u003cem\u003eB. subtilis\u003c/em\u003e, with genome sizes ranging from 153,882 to 156,577 bp, encoding between 256 and 270 protein-coding genes, no tRNA genes, and GC contents of 38.6\u0026ndash;38.7%, lower than that of their host \u003cem\u003eB. subtilis\u003c/em\u003e (~\u0026thinsp;43%) (Magness et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). By contrast, \u003cem\u003eB. licheniformis\u003c/em\u003e has a GC content of ~\u0026thinsp;46.2% (Rey et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), which is also higher than that of the Yucatan phage.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eHerelleviridae\u003c/em\u003e family comprises virions with isometric icosahedral heads measuring 85\u0026ndash;100 nm in diameter. The heads display capsomeres, with capsid subunits arranged in pentons and hexons. Uncontracted tails range from 130 to 185 nm in length, featuring a base plate of approximately 60 nm and a small collar [40]. Electron microscopy revealed that the Yucatan phage is morphologically similar to phages SIOphi and fHSpt3 (Krasowska et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Midha et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAccording to the morphological characteristics of this phage type and the latest classification by the International Committee on Taxonomy of Viruses (ICTV), the Yucatan phage exhibits the morphology typical of the class \u003cem\u003eCaudoviricetes\u003c/em\u003e, which includes tailed phages. Within this class, the families with the highest number of sequences reported in GenBank are \u003cem\u003eAutographiviridae\u003c/em\u003e, \u003cem\u003eHerelleviridae\u003c/em\u003e, and \u003cem\u003eDemerecviridae\u003c/em\u003e. Phages with this morphology are characterized by linear double-stranded DNA genomes (Zhu et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough bacteriophage classification has traditionally relied on morphological criteria and only rarely on molecular data (Ackermann \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), \u003cem\u003eBacillus\u003c/em\u003e phage Yucatan is described here both morphologically and molecularly, making it one of the few phages of the genus \u003cem\u003eSiophivirus\u003c/em\u003e to be characterized in both ways. Temperature plays a crucial role in bacteriophage survival, attachment efficiency, and latency period duration (Olson et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Phage Yucatan remained active at the highest temperature tested (60\u0026deg;C) for 60 min. These results are consistent with those reported by Melo, who observed that phage SIOphi retained approximately 43% activity at 60\u0026deg;C after 180 min, but lost activity at 70\u0026deg;C within 30 min and at 80\u0026deg;C within 2 min. In general, members of the former family \u003cem\u003eMyoviridae\u003c/em\u003e (now \u003cem\u003eHerelleviridae\u003c/em\u003e) (Barylski et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and \u003cem\u003eSiphoviridae\u003c/em\u003e are considered resistant to elevated temperatures (Jończyk et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe acidity and alkalinity of the environment are important factors influencing phage stability (Melo et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The Yucatan phage exhibited its highest activity at pH 7. These results are consistent with those reported by Krasowska et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), who found that phage SIOphi displayed maximum activity between pH 6 and 8, with no activity at pH 3 or 10. In contrast, the Yucatan phage retained 20% activity at pH 10.\u003c/p\u003e \u003cp\u003eThe application of bacteriophages in biotechnological processes requires detailed knowledge of their biological characteristics, including host range, latency period, growth dynamics, and resistance to stress conditions such as temperature and pH. The evaluated properties of the Yucatan phage presented in this study demonstrate its potential to suppress \u003cem\u003eBacillus licheniformis\u003c/em\u003e populations.\u003c/p\u003e \u003cp\u003eGenomic analysis revealed two proteins involved in bacterial lysis: holin and endolysin, the holin-endolysin system is employed by most double-stranded DNA and tailed phages infecting Gram-positive hosts, facilitating the release of progeny virions during the final stages of the lytic cycle (Wu et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and these proteins are also present in phage SIOphi. Holins are small membrane proteins that oligomerize to form pores in the cytoplasmic membrane, thereby enabling the release of endolysins into the periplasm (Wang et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Young \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Endolysins are peptidoglycan hydrolases that degrade the bacterial cell wall, with lysis occurring due to osmotic pressure differences between the cell and its environment. During the lysis process, endolysins are synthesized and accumulate in the cytoplasm at the late stage of phage infection, when proteins cannot cross the cytoplasmic membrane (Schmelcher et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough ICTV currently recognizes phage SIOphi as the sole species of the genus \u003cem\u003eSiophivirus\u003c/em\u003e, Midha et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e described \u003cem\u003eBacillus\u003c/em\u003e phage Fhstp3 as a member of this genus. Phage Fhstp3 was isolated from wastewater, infects \u003cem\u003eB. subtilis\u003c/em\u003e, and possesses a genome of 150,187 bp with 221 protein-coding sequences. It lacks virulence and antibiotic resistance genes and exhibits a virulent life cycle, with 78.97% confidence according to Phage AI (Tynecki et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor a phage to be considered suitable for phage therapy, host range is a critical characteristic. Ideally, a therapeutic phage should be specific to a single bacterial species to avoid off-target effects, yet broad enough within that species to infect multiple strains (Hyman \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The phage characterized in this study was evaluated against five independent \u003cem\u003eBacillus licheniformis\u003c/em\u003e isolated from the abdomen homogenate of the whitefly, and it exhibited lytic activity against all of them. Genomic analysis confirmed that the phage does not harbor antibiotic resistance genes, virulence factors, CRISPR elements, or tRNAs, all of which support its suitability as a potential biocontrol agent targeting \u003cem\u003eB. licheniformis\u003c/em\u003e in the whitefly gut.\u003c/p\u003e \u003cp\u003eThe Yucatan phage showed reduced activity under pH conditions outside the neutral range; however, it can still be considered a promising candidate for suppressing \u003cem\u003eB. licheniformis\u003c/em\u003e populations in the digestive system of \u003cem\u003eBemisia tabaci\u003c/em\u003e. The pH of most insect digestive systems typically ranges from 6 to 8, although exceptions exist, for example, blue flies (\u003cem\u003eCalliphora\u003c/em\u003e) exhibit highly acidic gut conditions, while lepidopterans maintain alkaline environments between pH 8 and 10 (Engel et al. 2013). Previous studies using oral antibiotic treatments to eliminate gut symbionts have reported suppression or elimination of these bacteria, resulting in higher mortality rates, slower growth, and reduced body size (Bai et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Koga et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Rupawate et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe use of bacteriophages to suppress bacterial populations within insect microbiomes as an alternative to antibiotics has been scarcely studied. Xu et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) evaluated phage BiBurk16MC_R against \u003cem\u003eBurkholderia\u003c/em\u003e but obtained limited results, probably due to the blockage of the connection between the anterior and posterior abdominal regions, it suggests that the initial colonization by the symbiont programs the ontogeny of the midgut, providing a protected residence against microbial antagonists. Therefore, further evaluation of the Yucatan phage across different developmental instars of \u003cem\u003eBemisia tabaci\u003c/em\u003e, using both contact and ingestion methods, is necessary to determine its effectiveness as a biocontrol agent.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding.\u003c/h2\u003e \u003cp\u003ePhD scholarship # 733809 for Daniel Bravo P\u0026eacute;rez from Consejo Nacional de Ciencia y Tecnolog\u0026iacute;a (Conacyt).\u003c/p\u003e \u003cp\u003eData availability All raw data are available from the corresponding author upon request.\u003c/p\u003e \u003cp\u003eDeclarations Conflict of interest: The authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eDBP, CCHQ and OAMV conceived the project, DBP performed the majority of the experiments, JPGG contributed to the bioinformatic analysis, YMG contributed to molecular whiteflies identification, ECL contributed to electron microscopy experiments, JPGG, LRP and CHZ contributed to the analysis of the data, DBP, OAMV and CHZ wrote the manuscript, and all authors contributed to the final version. 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Front Microbiol 16(13):1032186. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2022.1032186\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2022.1032186\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biol","sideBox":"Learn more about [Biologia](http://link.springer.com/journal/11756)","snPcode":"11756","submissionUrl":"https://www.editorialmanager.com/biol/default2.aspx","title":"Biologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"insect, symbionts, Bacillus, lytic phage, genomic analysis","lastPublishedDoi":"10.21203/rs.3.rs-9557867/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9557867/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBacteriophages have been proposed as a biological alternative for reducing bacterial pathogens in diverse fields, including the food industry, healthcare, and agriculture, considered as promising tools to counteract antibiotic resistance. In this study, we present//provide the morphological and genetic characterization of a novel bacteriophage isolated from the digestive system of \u003cem\u003eBemisia tabaci\u003c/em\u003e (whitefly) Biotype A, collected from \u003cem\u003eCapsicum chinense\u003c/em\u003e (habanero pepper). Its bacteriolytic activity, thermostability, pH tolerance, and host range were assessed. Host range analysis revealed that among the bacteria tested, \u003cem\u003eBacillus licheniformis\u003c/em\u003e served as the host for this bacteriophage. Transmission electron microscopy images showed virions with a head\u0026ndash;tail structure, characterized by a long, contractile tail and an icosahedral head. Characterization results indicated that bacteriolytic activity of the phage begins at the first hour of contact with its host. Among the evaluated parameters, the optimal temperature for maximum activity was 20\u0026deg;C, while the optimal pH was 7.0. Lytic activity was observed exclusively against \u003cem\u003eBacillus licheniformis\u003c/em\u003e strains. The newly identified phage possesses a double-stranded DNA genome of 147,395 bp, predicting a total of 266 protein-coding sequences, with a GC content of 38.98%. It lacks host virulence-modifying genes and antibiotic resistance genes and exhibits a strictly virulent life cycle. Based on viral taxonomy criteria, this phage was classified as a novel species within the family \u003cem\u003eHerelleviridae\u003c/em\u003e, with its closest relative being phage SIOphi. The proposed name for this isolate is \u003cem\u003eBacillus phage Yucatan\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Isolation and Characterization of the Bacteriophage Yucatan Specific to Bacillus Licheniformis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-13 05:27:12","doi":"10.21203/rs.3.rs-9557867/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-18T11:56:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"47641988953409676420148552644090957625","date":"2026-05-04T23:42:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-05-04T06:04:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-30T03:31:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-30T03:31:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biologia","date":"2026-04-28T19:33:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biol","sideBox":"Learn more about [Biologia](http://link.springer.com/journal/11756)","snPcode":"11756","submissionUrl":"https://www.editorialmanager.com/biol/default2.aspx","title":"Biologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8aa988a2-0e58-4383-b96e-f060a054b3db","owner":[],"postedDate":"May 13th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-18T11:56:12+00:00","index":33,"fulltext":""},{"type":"reviewerAgreed","content":"47641988953409676420148552644090957625","date":"2026-05-04T23:42:10+00:00","index":24,"fulltext":""},{"type":"reviewersInvited","content":"19","date":"2026-05-04T06:04:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-30T03:31:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-30T03:31:25+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-13T05:27:12+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-13 05:27:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9557867","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9557867","identity":"rs-9557867","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}