Description and Characterization of Indigenous Isolates of the Symbiotic Bacterium Photorhabdus luminescens (Thomas and Poinar)

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Prabhuraj, Saroja N. R, Nagaraj M. N., Arunkumar Hosamani¹, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6982668/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract This study aimed to identify and characterize Photorhabdus luminescens , a bacterial endosymbiont of the entomopathogenic nematode Heterorhabditis spp., collected from various crop ecosystems in Raichur, Karnataka. Both phenotypic and genotypic analyses were employed to confirm bacterial identity. Three distinct bacterial strains named UASR22PL1, UASR22PL2, and UASR22PL3 were isolated by streaking haemolymph obtained from Galleria mellonella larvae infected with Heterorhabditis spp. All strains exhibited Gram-negative bacillus morphology characteristic of P. luminescens . After 72 hours of incubation, bacterial colonies appeared smooth, small, round, glistening, and convex with entire margins. They developed a red pigmentation with a bluish hue, and over time, transitioned from primary to secondary form due to prolonged static incubation of the culture. The phylogenetic tree constructed further confirmed that the isolated strains belonged to the species P. luminescens , establishing a close group of P. luminescens strain KZ2R1.. Known for their potent insecticidal activity, these three native isolates have potential applications in biocontrol strategies for managing significant agricultural pests. Photorhabdus luminescens Heterorhabditis indica Symbiotic Bacteria Biological Control Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Entomopathogenic nematodes (EPNs), which are obligatory insect parasites that live in soil, foliage, and hidden places, are members of the Heterorhabditidae and Steinernematidae families. They have the ability to significantly reduce a variety of agricultural pests. These nematodes are present across all continents (Grewal et al. 2005 ) except Antarctica (Hominick 2002 ). In India, fifteen species of EPNs have been documented, comprising three species of Heterorhabditis and twelve of Steinernema (Bhat et al. 2021 ). Naturally occurring in soil, these nematodes locate their insect hosts through chemical cues such as CO₂ and vibrational signals (Kaya & Gaugler 1993 ). Due to increasing demands for food security and safety, alongside the need for effective and affordable pest control solutions, EPNs are among the most extensively used biocontrol agents (Lizzy 2020 ). A bacterial symbiont that has garnered attention in recent years is Photorhabdus luminescens , which lives in association with Heterorhabditis spp. This facultatively anaerobic bacterium, classified under Enterobacteriaceae in the gamma group of purple bacteria, is motile, Gram-negative, bioluminescent, non-spore-forming, and capable of producing diverse toxins and enzymes (Akhurst, 1980). Photorhabdus bacteria exhibit two phenotypic variants: the primary form (Form I) commonly found in symbiosis with nematodes, and the secondary form (Form II), which usually arises in artificial cultures and rarely in natural insect hosts during the later stages of nematode development (Akhurst 1980). These two forms show distinct morphological and physiological differences. Currently, three primary Photorhabdus species are recognized— P. luminescens , P. temperata , and P. asymbiotica —along with seven subspecies identified within P. luminescens (Singh et al. 2012 ). Advanced biochemical and molecular techniques, such as 16S rRNA gene amplification, and phenotypic traits are necessary for the identification of these species and subspecies (An & Grewal 2010 ). Although a lot of research have focused on isolating Heterorhabditis species for biocontrol use in Karnataka (Prabhuraj et al. 2000 ; Subbanna et al. 2008 ; Uma 2015 ), comprehensive studies on their bacterial symbionts are lacking. Proper identification and evaluation of these bacteria are critical to understanding their role in pest management. This study addresses that gap by isolating and characterizing P. luminescens strains from H. indica . Materials and Methods Study Period and Location The present research involving the isolation and identification of P. luminescens isolates was undertaken at the Department of Entomology, College of Agriculture, Raichur, during the years 2021 to 2023. The study focused on both morphological and molecular characterization of bacterial strains isolated from Heterorhabditis spp. collected in the Raichur region. Isolation of Entomopathogenic Nematode ( Heterorhabditis indica ) from Soil Entomopathogenic nematodes were extracted directly from soil samples collected from a horticultural nursery and mulberry field at the College of Agriculture, Raichur. A modified baiting method as described by Prabhuraj et al . (2000) was employed. This technique utilized 2.5 mL plastic vials, which had holes punctured on their flat sides and were sealed with 80-mesh brass screens. Each vial was loaded with two to three late-instar Galleria mellonella larvae and a moist piece of blotting paper to facilitate nematode entry. Chlorpyriphos (0.04%) was applied around the traps to deter ants. After burying the traps 15 cm deep in soil, they were retrieved after four days. Dead larvae were then transferred to White’s traps to collect infective juveniles (IJs). Laboratory Maintenance of Heterorhabditis indica Infective juveniles obtained from field-isolated nematodes served as the foundational culture. A nematode suspension was prepared through serial dilution to ensure each milliliter contained approximately 100 infective juveniles. This suspension (1 mL) was evenly distributed over filter paper inside 10 cm Petri dishes. Five healthy G. mellonella larvae were placed in each dish, which was sealed and enclosed in polythene covers to facilitate infection. Infected larvae were identifiable by a characteristic brick-red coloration—an indication of Heterorhabditis infection. These cadavers were transferred to individual White’s traps to harvest fresh IJs. Isolation of Pure Cultures of Photorhabdus luminescens Selective media, including Nutrient Agar (NA) and Nutrient Bromothymol Blue Agar (NBTA), were used for isolation, culture, and analysis of P. luminescens . The protocol developed by Akhurst (1980) was followed. Five final-instar larvae of G. mellonella were infected by exposing them to ~100 IJs per larva inside Petri plates lined with moist filter paper. After 72 hours post-infection, the dead larvae were sterilized by immersing them in 95% ethanol, briefly igniting, and rinsing with sterile water. The cadavers were dissected under aseptic conditions in a watch glass using sterilized tools. A loopful of haemolymph was streaked onto NA and NBTA plates, which were sealed with parafilm and incubated in darkness at 28°C for 24–48 hours in a BOD incubator. Initial colonies representing the primary form of P. luminescens were identified based on morphological characteristics, and subculturing was carried out to obtain pure cultures. Phenotypic Characterization of P. luminescens Both primary and secondary morphological forms of the bacterium were analyzed based on colony appearance, edge structure, elevation, consistency, surface texture, and pigmentation. These observations were carried out following standard microbiological procedures as described by Aneja (2003). Data were recorded from the second day post-inoculation up to twenty days. Test for Bioluminescence Plates containing 12- to 24-hour-old cultures of P. luminescens were placed under a UV transilluminator (BIO-BEE, wavelength 306–365 nm) to evaluate bioluminescence. The emitted glow under UV exposure was recorded as a phenotypic marker. Molecular Characterization via 16S rRNA Analysis Genomic DNA was extracted from cultures of the three Photorhabdus isolates—UASR22PL1, UASR22PL2, and UASR22PL3—using the Nucleospin Tissue XS kit (Catalogue No. 740901.50). Extracted DNA was stored in 1.5 mL Eppendorf tubes at -20°C with TE buffer and RNase inhibitor. DNA concentration was determined using a Nanodrop spectrophotometer, and 100–300 ng was used for PCR amplification. The 16S rRNA gene was partially amplified using universal primers 27f (AGAGTTTGATCMTGGCTCAG) and 1525r (AAGGAGGTGATCCAGCC), which are standard for Photorhabdus identification (Weisburg et al. 1991). Amplification was performed using an Eppendorf Master Cycler Gradient (Model 5331) with the following thermal profile: initial denaturation at 95°C for 5 minutes; 35 cycles at 94°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute; followed by a final extension at 72°C for 10 minutes. PCR products were sequenced externally by Barcode Biosciences Pvt. Ltd., Bangalore. Resulting sequences were deposited in the GenBank database and assembled using BioEdit software. Phylogenetic Tree Construction Phylogenetic analysis was conducted using the maximum parsimony method with default parameters in ClustalW (Thompson et al. 1997). Heuristic search strategies including stepwise addition and tree-bisection-reconnection (TBR) were applied. Bootstrapping with 1000 replicates (Felsenstein 1993) was used to evaluate branch support. Tree construction was performed using MEGA 11 software (Tamura et al. 2007), accessible at http://megasoftware.net. The evolutionary tree was inferred through the Neighbor-Joining method. Bootstrap values from 1000 replicates are shown alongside branches, and branches with <50% bootstrap support were collapsed. Evolutionary distances were calculated using the Maximum Composite Likelihood method and are expressed as base substitutions per site. The dataset included nine nucleotide sequences with ambiguous positions removed by pairwise deletion. Final analysis was performed using MEGA11. Results and Discussion Cultural Characterization Three bacterial strains symbiotically associated with Heterorhabditis indica were successfully isolated from soil samples taken from the horticulture nursery and mulberry plot of the University of Agricultural Sciences, Raichur (Fig 1). These isolates were labeled with unique codes—UASR22PL1, UASR22PL2, and UASR22PL3—where "PL" stands for Photorhabdus luminescens , "UASR" refers to the institution, "22" denotes the year of collection, and the final digit distinguishes the individual isolate. Each isolate exhibited two phenotypic forms: the primary (1°) and secondary (2°) cell types. Initially, the colonies appeared in the primary form—small, circular, smooth-edged, glossy, and convex with complete margins. These colonies developed an opaque appearance with coloration ranging from pink to deep brick red overlaid with a bluish hue within the first three days post-inoculation. As incubation progressed, the colony shapes became irregular, flattened, and surrounded by clear zones, a transformation visible from the fourth to ninth day. These cultural traits were consistent with prior descriptions of P. luminescens as a symbiont of H. indica , as cited in works by Akhurst (1980), Boemare and Akhurst (1988), Babic et al. (2000), Nagesh et al. (2001), Sharad Mohan et al. (2004), Singh et al. (2012), Vishruth et al. (2014) and Uma (2015). By the tenth day after inoculation, the colonies began to shift toward the secondary form, which lacked the characteristic surrounding clear zone. For isolates UASR22PL1 and UASR22PL3, the colonies transitioned to a transparent appearance that later turned dark red or brown. In UASR22PL2, this transition occurred around the twelfth day. The secondary form could be visually identified by a watery outer layer and an oily red core. These forms remained morphologically stable thereafter. The progression and time taken for the transition from primary to secondary form closely matched earlier observations (Bleakley and Nealson 1988; Vishruth et al. 2014; Hazir et al. 2004; An and Grewal 2011; Uma 2009; Uma 2015). When cultured on NBTA medium, the bacterial colonies absorbed the bromothymol blue pigment from the media and changed color within 3–4 days. The primary forms developed red-centered colonies overlaid with a blue tint and were encircled by a clear halo due to the reduction of TTC (Triphenyl Tetrazolium Chloride) and bromothymol blue absorption. In contrast, secondary forms did not absorb bromothymol blue and thus formed dark red colonies without the surrounding clear zones (Akhurst 1980; Boemare and Akhurst 1988). This transformation is thought to result from nutrient depletion in the medium, which alters not only the morphology but also the physiological properties such as biochemical activity, antibiotic production, and antagonistic behavior (Akhurst 1980). Morphological Characteristics Microscopic examination revealed that both forms—primary and secondary—of all three P. luminescens isolates were rod-shaped, flagellated, Gram-negative, and motile. They were either strictly aerobic or facultatively anaerobic. The isolates took up simple stains readily. When grown on Nutrient Agar, all colonies emitted bioluminescence when exposed to ultraviolet light, as observed under a UV lamp. Notably, the primary forms exhibited stronger luminescence than the secondary forms (Table 2, Figure 2). The observed bioluminescent property, coupled with morphological traits, reinforced that all three bacterial isolates were indeed P. luminescens , consistent with earlier studies by Bleakley and Nealson (1988), Nagesh et al. (2001), Shahina et al. (2004), Peel et al. (1999), Han and Ehlers (2001), and Rashid et al. (2014). Molecular Characterization and Phylogenetic Tree Analysis The PCR amplification of the 16S rRNA gene region for all three isolates yielded products approximately 1500 base pairs in length. This was confirmed by the appearance of a single, bright DNA band on 1% agarose gel electrophoresis (Fig 3). The sequences of the amplified products were submitted to GenBank with the following accession numbers: UASR22PL1 – OQ472600, UASR22PL2 – OQ473650, and UASR22PL3–OQ472608 BLAST analysis of the sequences via NCBI’s database revealed that the phylogenetic dendrogram divided the Photorhabdus isolates into two clusters. Group A1 included UASR22PL1 and UASR22PL2, which displayed 64% similarity between themselves and showed 99% identity with P. luminescens strain KZ2R1 (as per Uma, 2015). The UASR22PL3 isolate, classified under Group A2, showed 99% similarity with A1 isolates and a 95.79% similarity to P. luminescens strain CAU4886 (Ren & Zhao, 2017) (Table 3, Fig. 4). Thus, the Photorhabdus strains isolated in this study were conclusively identified as P. luminescens , closely related to the KZ2R1 strain. These findings are consistent with prior reports which used 16S rRNA sequence-based phylogenies to differentiate groups within the Photorhabdus genus (Szallas et al . 1997; Brunel et al . 1997; Liu et al. 1997; Saux et al. 1999; Hazir et al . 2004). The 16S sequences from this study were aligned with homologous Photorhabdus sequences from GenBank for accurate placement in the phylogenetic tree. Insecticidal Relevance The bacterium P. luminescens maintains a mutualistic relationship with Heterorhabditis nematodes. Upon nematode penetration into the host insect’s body cavity, the bacteria are released into the hemocoel, where they initiate septicemia by releasing a suite of toxins and enzymes—including chitinases, lipases, and proteases. These bio-convert the host tissue into nutrients for both the nematode and the bacterium (Akhurst 1980). This mode of action highlights the organism’s potential as a highly efficient microbial insecticide. Hence, the thorough characterization of Photorhabdus strains from various habitats is essential for selecting the most potent isolates for biocontrol programs. Conclusion This study successfully isolated and characterized three bacterial strains—UASR22PL1, UASR22PL2, and UASR22PL3—associated with the entomopathogenic nematode Heterorhabditis spp. These isolates were obtained from soil samples through an in-situ trapping technique. Detailed morphological and molecular analyses confirmed their identity as Photorhabdus luminescens , a symbiotic bacterium known for its biocontrol capabilities. The findings suggest that these native strains possess promising insecticidal properties and could be harnessed for biological control of lepidopteran and hemipteran pests prevalent in the region. Abbreviations EPN – Entomopathogenic Nematodes BOD – Biological Oxygen Demand PCR – Polymerase Chain Reaction UV – Ultraviolet TAE buffer – Tris-Acetate-EDTA Buffer DNA – Deoxyribonucleic Acid NCBI – National Center for Biotechnology Information MEGA – Molecular Evolutionary Genetics Analysis NBTA – Nutrient Bromothymol Blue Agar NA – Nutrient Agar bp – Base Pair TTC – Triphenyl Tetrazolium Chloride IJs – Infective Juveniles Declarations All authors participated in the conceptualization and design of the research. Akshatha S. conducted the experimental work, collected the data, and carried out the analysis. The initial draft of the manuscript was written by Akshatha S. and Prabhuraj A., with all co-authors providing feedback on earlier versions. The final manuscript was reviewed and approved by all authors. We confirm that this work is original, has not been previously published, and is not under consideration for publication elsewhere. There are no conflicts of interest associated with this research. This study was supported financially by the Karnataka Science and Technology Promotion Society (KSTePS), Department of Science and Technology, Government of Karnataka. No experiments involving human or animal subjects were conducted in this research. Author Contribution All authors participated in the conceptualization and design of the research. Akshatha S. conducted the experimental work, collected the data, and carried out the analysis. The initial draft of the manuscript was written by Akshatha S. and Prabhuraj A., with all co-authors providing feedback on earlier versions. The final manuscript was reviewed and approved by all authors. Acknowledgement This study was supported financially by the Karnataka Science and Technology Promotion Society (KSTePS), Department of Science and Technology, Government of Karnataka. 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Nucleic Acids Res 25:4876–4882 Uma GP (2015) Characterization of extracellular secretions of symbiotic bacterium Photorhabdus luminescens . Ph.D. Thesis, Univ. Agric. Sci., Raichur, India Vishruth K, Vendan T, Nagaraj S (2014) Isolation and characterization of entomopathogenic symbiotic bacterium Photorhabdus luminescens of Heterorhabditis indica from soils of five agro-climatic zones of Karnataka. Biosci Biotechnol Res Asia 11(1):129–139 Weisburg GW, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173:697–703 Tables Table 1. Colony characteristics of Primary and secondary forms of P. luminescens on NBTA media Sl. No. Isolate Days after Inoculation Form Edge Elevation Consistency Clear zone Surface color 1. UASR22PL1 1 Circular Entire Convex Opaque No Clear zone Smooth and Glistening Dark Brick red over laid by blue 4 Circular Entire Concave Opaque Clear zone Smooth and Glistening Central red core overlaid by light blue color 8 Irregular Undulated Flat Opaque Clear zone Rough and Glistening Brick red to dark red color 12 Irregular Undulated Flat Transparent Clear zone Rough and Glistening Dark red to brownish blue over laid by another layer 2. UASR22PL2 1 Circular Entire Convex Opaque No Clear zone Smooth and Glistening Light Pink 4 Circular Entire Concave Opaque Clear zone Smooth and Glistening Dark Pink 8 Irregular Undulated Flat Transparent Clear zone Rough and Glistening Dark Brick red over laid by blue 12 Irregular Undulated Flat Transparent Clear zone Rough and Glistening Dark Brick red over laid by blue 3. UASR22PL3 1 Circular Entire Convex Opaque No Clear zone Smooth and Glistening Central dark pink over laid by light pink layer 4 Irregular Undulated Sunken Transparent Clear zone Rough and Glistening Dark pink to light pink core and outer layer pink to bluish 8 Irregular Undulated Flat Transparent Clear zone Rough and Glistening Pink layer turn to red color 12 Irregular Undulated Flat Transparent Clear zone Rough and Glistening Red color Table 2. Morphological characteristics of primary and secondary forms of P. luminescens Test UASR22PL1 UASR22PL2 UASR22PL3 Primary form Secondary form Primary form Secondary form Primary form Secondary form Simple staining + ve + ve + ve + ve + ve + ve Gram staining - ve - ve - ve - ve - ve - ve Motility + ve + ve + ve + ve + ve + ve Bioluminescence + ve + ve + ve + ve + ve + ve Shape Medium to long rod Medium to long rod Medium to long rod Medium to long rod Medium to long rod Medium to long rod Table 3. Identification of P. luminescens based on sequence similarities from NCBI Genebank data and their accession numbers Sl. No. Isolates Accession number NCBI Identity Similarity Accession no. of NCBI Identity 1 P. luminescens UASR22PL-1 OQ472600 Photorhabdus luminescens KZ2R1 99% KP224438 2 P. luminescens UASR22PL-2 OQ473650 Photorhabdus luminescens strain KZ2R1 99% KP224438 3 P. luminescens UASR22PL-3 OQ472608 Photorhabdus luminescens strain CAU9727 95.79% MF429509.1 Additional Declarations No competing interests reported. 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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-6982668","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":494615516,"identity":"c0ae8e50-0d23-492c-8ff8-d20a5c5e4615","order_by":0,"name":"Akshatha S","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDklEQVRIiWNgGAWjYDACHgY2IHmAgZ+9AUhXADEzcwMRWhIOMEj2HADyzoC0MBKpxeBGAgMDYxtIiIAW/p7jzx78/HFHnuFG8jPJr/Nqo/nbgVp+VGzDqUXibI+5YU/CM8PGnmdm0rLbjufOOMzYwNhz5jZua87zsEnwJBxmbGZPMJOW3HYstwGohZmxDbcW+fPszyT/JBy2b2NI/yYtOedY7nxCWgzONphJA21J7OHIMZP82FCTu4GQFsMzZ8ykZdIOJ8/gOVNszXDsQO5GoJaD+Pwidyb9meQbm8O2+4+3b7z5o6Yud975wwcf/KjA431kwMzDcBjMOECceiBg/MFQR7TiUTAKRsEoGDkAALKYYtsOhE5qAAAAAElFTkSuQmCC","orcid":"","institution":"University of Agricultural Sciences Raichur","correspondingAuthor":true,"prefix":"","firstName":"Akshatha","middleName":"","lastName":"S","suffix":""},{"id":494615517,"identity":"081c62b5-bb51-4d67-82b5-35202f42033d","order_by":1,"name":"A. Prabhuraj","email":"","orcid":"","institution":"University of Agricultural Sciences Raichur","correspondingAuthor":false,"prefix":"","firstName":"A.","middleName":"","lastName":"Prabhuraj","suffix":""},{"id":494615518,"identity":"4ae94436-17a8-408d-babf-e9264658050c","order_by":2,"name":"Saroja N. R","email":"","orcid":"","institution":"University of Agricultural Sciences Raichur","correspondingAuthor":false,"prefix":"","firstName":"Saroja","middleName":"N.","lastName":"R","suffix":""},{"id":494615519,"identity":"f0719d31-6992-42b2-b30e-fecebc65d4fc","order_by":3,"name":"Nagaraj M. N.","email":"","orcid":"","institution":"University of Agricultural Sciences Raichur","correspondingAuthor":false,"prefix":"","firstName":"Nagaraj","middleName":"M.","lastName":"N.","suffix":""},{"id":494615520,"identity":"ae18bf04-94e7-475e-a704-171f365d93ec","order_by":4,"name":"Arunkumar Hosamani¹","email":"","orcid":"","institution":"University of Agricultural Sciences Raichur","correspondingAuthor":false,"prefix":"","firstName":"Arunkumar","middleName":"","lastName":"Hosamani¹","suffix":""},{"id":494615521,"identity":"19a0c8db-aca8-4b1d-ba0a-88234f01119e","order_by":5,"name":"Shivaleela Shivaleela","email":"","orcid":"","institution":"University of Agricultural Sciences Raichur","correspondingAuthor":false,"prefix":"","firstName":"Shivaleela","middleName":"","lastName":"Shivaleela","suffix":""}],"badges":[],"createdAt":"2025-06-26 10:53:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6982668/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6982668/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88269882,"identity":"2659851e-f8da-4181-a437-9b6b4fe5e46b","added_by":"auto","created_at":"2025-08-04 16:56:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2230685,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIsolation of\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e Photorhabdus luminescens \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003efrom Entomopathogenic nematode\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e (H. indica)\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6982668/v1/7fcebdd27540c8c238245adb.png"},{"id":88269883,"identity":"21120c65-b354-46cc-b18c-e8f6feee35a3","added_by":"auto","created_at":"2025-08-04 16:56:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":986885,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological characters of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. luminescens \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(A: Gram’s staining, B: Bioluminescence)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6982668/v1/7962945bf0856951d3e63d94.png"},{"id":88269884,"identity":"0a477549-bfff-4cec-88c6-674404143045","added_by":"auto","created_at":"2025-08-04 16:56:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":375827,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGel doc image showing bands at 1500 bp in 1.0 per cent agarose gel (gDNA and 16S Amplicon QC data)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6982668/v1/56b57a0cf0c2413b75ebfba0.png"},{"id":88269885,"identity":"0fb9127b-4f37-4613-8005-7b110d09f27a","added_by":"auto","created_at":"2025-08-04 16:56:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":410734,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDendrogram showing the phylogeny of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. luminescens\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e isolates based on 16S rRNA analysis using neighborhood joining method\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6982668/v1/77c69b1603b0e16fb03a7d9a.png"},{"id":88273158,"identity":"51104aad-1552-4691-95a5-17943d7e6683","added_by":"auto","created_at":"2025-08-04 17:28:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6394913,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6982668/v1/834ceef1-9e08-405b-b828-39df7cac79b3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Description and Characterization of Indigenous Isolates of the Symbiotic Bacterium Photorhabdus luminescens (Thomas and Poinar)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEntomopathogenic nematodes (EPNs), which are obligatory insect parasites that live in soil, foliage, and hidden places, are members of the Heterorhabditidae and Steinernematidae families. They have the ability to significantly reduce a variety of agricultural pests.\u003c/p\u003e\u003cp\u003eThese nematodes are present across all continents (Grewal et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) except Antarctica (Hominick \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). In India, fifteen species of EPNs have been documented, comprising three species of \u003cem\u003eHeterorhabditis\u003c/em\u003e and twelve of \u003cem\u003eSteinernema\u003c/em\u003e (Bhat et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Naturally occurring in soil, these nematodes locate their insect hosts through chemical cues such as CO₂ and vibrational signals (Kaya \u0026amp; Gaugler \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). Due to increasing demands for food security and safety, alongside the need for effective and affordable pest control solutions, EPNs are among the most extensively used biocontrol agents (Lizzy \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eA bacterial symbiont that has garnered attention in recent years is \u003cem\u003ePhotorhabdus luminescens\u003c/em\u003e, which lives in association with \u003cem\u003eHeterorhabditis\u003c/em\u003e spp. This facultatively anaerobic bacterium, classified under Enterobacteriaceae in the gamma group of purple bacteria, is motile, Gram-negative, bioluminescent, non-spore-forming, and capable of producing diverse toxins and enzymes (Akhurst, 1980).\u003c/p\u003e\u003cp\u003e\u003cem\u003ePhotorhabdus\u003c/em\u003e bacteria exhibit two phenotypic variants: the primary form (Form I) commonly found in symbiosis with nematodes, and the secondary form (Form II), which usually arises in artificial cultures and rarely in natural insect hosts during the later stages of nematode development (Akhurst 1980). These two forms show distinct morphological and physiological differences.\u003c/p\u003e\u003cp\u003eCurrently, three primary \u003cem\u003ePhotorhabdus\u003c/em\u003e species are recognized\u0026mdash;\u003cem\u003eP. luminescens\u003c/em\u003e, \u003cem\u003eP. temperata\u003c/em\u003e, and \u003cem\u003eP. asymbiotica\u003c/em\u003e\u0026mdash;along with seven subspecies identified within \u003cem\u003eP. luminescens\u003c/em\u003e (Singh et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Advanced biochemical and molecular techniques, such as 16S rRNA gene amplification, and phenotypic traits are necessary for the identification of these species and subspecies (An \u0026amp; Grewal \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Although a lot of research have focused on isolating \u003cem\u003eHeterorhabditis\u003c/em\u003e species for biocontrol use in Karnataka (Prabhuraj et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Subbanna et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Uma \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), comprehensive studies on their bacterial symbionts are lacking. Proper identification and evaluation of these bacteria are critical to understanding their role in pest management. This study addresses that gap by isolating and characterizing \u003cem\u003eP. luminescens\u003c/em\u003e strains from \u003cem\u003eH. indica\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003ch3\u003e\u003cstrong\u003eStudy Period and Location\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe present research involving the isolation and identification of \u003cem\u003eP. luminescens\u003c/em\u003e isolates was undertaken at the Department of Entomology, College of Agriculture, Raichur, during the years 2021 to 2023. The study focused on both morphological and molecular characterization of bacterial strains isolated from \u003cem\u003eHeterorhabditis\u003c/em\u003e spp. collected in the Raichur region.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eIsolation of Entomopathogenic Nematode (\u003c/strong\u003e\u003cem\u003eHeterorhabditis indica\u003c/em\u003e\u003cstrong\u003e) from Soil\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eEntomopathogenic nematodes were extracted directly from soil samples collected from a horticultural nursery and mulberry field at the College of Agriculture, Raichur. A modified baiting method as described by Prabhuraj \u003cem\u003eet al\u003c/em\u003e. (2000) was employed. This technique utilized 2.5 mL plastic vials, which had holes punctured on their flat sides and were sealed with 80-mesh brass screens. Each vial was loaded with two to three late-instar \u003cem\u003eGalleria mellonella\u003c/em\u003e larvae and a moist piece of blotting paper to facilitate nematode entry. Chlorpyriphos (0.04%) was applied around the traps to deter ants. After burying the traps 15 cm deep in soil, they were retrieved after four days. Dead larvae were then transferred to White\u0026rsquo;s traps to collect infective juveniles (IJs).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLaboratory Maintenance of\u0026nbsp;\u003c/strong\u003e\u003cem\u003eHeterorhabditis indica\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eInfective juveniles obtained from field-isolated nematodes served as the foundational culture. A nematode suspension was prepared through serial dilution to ensure each milliliter contained approximately 100 infective juveniles. This suspension (1 mL) was evenly distributed over filter paper inside 10 cm Petri dishes. Five healthy \u003cem\u003eG. mellonella\u003c/em\u003e larvae were placed in each dish, which was sealed and enclosed in polythene covers to facilitate infection. Infected larvae were identifiable by a characteristic brick-red coloration\u0026mdash;an indication of \u003cem\u003eHeterorhabditis\u003c/em\u003e infection. These cadavers were transferred to individual White\u0026rsquo;s traps to harvest fresh IJs.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eIsolation of Pure Cultures of\u0026nbsp;\u003c/strong\u003e\u003cem\u003ePhotorhabdus luminescens\u003c/em\u003e\u003c/h3\u003e\n\u003cp\u003eSelective media, including Nutrient Agar (NA) and Nutrient Bromothymol Blue Agar (NBTA), were used for isolation, culture, and analysis of \u003cem\u003eP. luminescens\u003c/em\u003e. The protocol developed by Akhurst (1980) was followed. Five final-instar larvae of \u003cem\u003eG. mellonella\u003c/em\u003e were infected by exposing them to ~100 IJs per larva inside Petri plates lined with moist filter paper. After 72 hours post-infection, the dead larvae were sterilized by immersing them in 95% ethanol, briefly igniting, and rinsing with sterile water. The cadavers were dissected under aseptic conditions in a watch glass using sterilized tools. A loopful of haemolymph was streaked onto NA and NBTA plates, which were sealed with parafilm and incubated in darkness at 28\u0026deg;C for 24\u0026ndash;48 hours in a BOD incubator. Initial colonies representing the primary form of \u003cem\u003eP. luminescens\u003c/em\u003e were identified based on morphological characteristics, and subculturing was carried out to obtain pure cultures.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003ePhenotypic Characterization of\u0026nbsp;\u003c/strong\u003e\u003cem\u003eP. luminescens\u003c/em\u003e\u003c/h3\u003e\n\u003cp\u003eBoth primary and secondary morphological forms of the bacterium were analyzed based on colony appearance, edge structure, elevation, consistency, surface texture, and pigmentation. These observations were carried out following standard microbiological procedures as described by Aneja (2003). Data were recorded from the second day post-inoculation up to twenty days.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eTest for Bioluminescence\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003ePlates containing 12- to 24-hour-old cultures of \u003cem\u003eP. luminescens\u003c/em\u003e were placed under a UV transilluminator (BIO-BEE, wavelength 306\u0026ndash;365 nm) to evaluate bioluminescence. The emitted glow under UV exposure was recorded as a phenotypic marker.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eMolecular Characterization via 16S rRNA Analysis\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eGenomic DNA was extracted from cultures of the three \u003cem\u003ePhotorhabdus\u003c/em\u003e isolates\u0026mdash;UASR22PL1, UASR22PL2, and UASR22PL3\u0026mdash;using the Nucleospin Tissue XS kit (Catalogue No. 740901.50). Extracted DNA was stored in 1.5 mL Eppendorf tubes at -20\u0026deg;C with TE buffer and RNase inhibitor. DNA concentration was determined using a Nanodrop spectrophotometer, and 100\u0026ndash;300 ng was used for PCR amplification. The 16S rRNA gene was partially amplified using universal primers 27f (AGAGTTTGATCMTGGCTCAG) and 1525r (AAGGAGGTGATCCAGCC), which are standard for \u003cem\u003ePhotorhabdus\u003c/em\u003e identification (Weisburg et al. 1991). Amplification was performed using an Eppendorf Master Cycler Gradient (Model 5331) with the following thermal profile: initial denaturation at 95\u0026deg;C for 5 minutes; 35 cycles at 94\u0026deg;C for 1 minute, 55\u0026deg;C for 1 minute, and 72\u0026deg;C for 1 minute; followed by a final extension at 72\u0026deg;C for 10 minutes. PCR products were sequenced externally by Barcode Biosciences Pvt. Ltd., Bangalore. Resulting sequences were deposited in the GenBank database and assembled using BioEdit software.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003ePhylogenetic Tree Construction\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003ePhylogenetic analysis was conducted using the maximum parsimony method with default parameters in ClustalW (Thompson et al. 1997). Heuristic search strategies including stepwise addition and tree-bisection-reconnection (TBR) were applied. Bootstrapping with 1000 replicates (Felsenstein 1993) was used to evaluate branch support. Tree construction was performed using MEGA 11 software (Tamura et al. 2007), accessible at http://megasoftware.net.\u003c/p\u003e\n\u003cp\u003eThe evolutionary tree was inferred through the Neighbor-Joining method. Bootstrap values from 1000 replicates are shown alongside branches, and branches with \u0026lt;50% bootstrap support were collapsed. Evolutionary distances were calculated using the Maximum Composite Likelihood method and are expressed as base substitutions per site. The dataset included nine nucleotide sequences with ambiguous positions removed by pairwise deletion. Final analysis was performed using MEGA11.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003ch3\u003e\u003cstrong\u003eCultural Characterization\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThree bacterial strains symbiotically associated with \u003cem\u003eHeterorhabditis indica\u003c/em\u003e were successfully isolated from soil samples taken from the horticulture nursery and mulberry plot of the University of Agricultural Sciences, Raichur (Fig 1). These isolates were labeled with unique codes\u0026mdash;UASR22PL1, UASR22PL2, and UASR22PL3\u0026mdash;where \u0026quot;PL\u0026quot; stands for \u003cem\u003ePhotorhabdus luminescens\u003c/em\u003e, \u0026quot;UASR\u0026quot; refers to the institution, \u0026quot;22\u0026quot; denotes the year of collection, and the final digit distinguishes the individual isolate.\u003c/p\u003e\n\u003cp\u003eEach isolate exhibited two phenotypic forms: the primary (1\u0026deg;) and secondary (2\u0026deg;) cell types. Initially, the colonies appeared in the primary form\u0026mdash;small, circular, smooth-edged, glossy, and convex with complete margins. These colonies developed an opaque appearance with coloration ranging from pink to deep brick red overlaid with a bluish hue within the first three days post-inoculation. As incubation progressed, the colony shapes became irregular, flattened, and surrounded by clear zones, a transformation visible from the fourth to ninth day.\u003c/p\u003e\n\u003cp\u003eThese cultural traits were consistent with prior descriptions of \u003cem\u003eP. luminescens\u003c/em\u003e as a symbiont of \u003cem\u003eH. indica\u003c/em\u003e, as cited in works by Akhurst (1980), Boemare and Akhurst (1988), Babic et al. (2000), Nagesh et al. (2001), Sharad Mohan et al. (2004), Singh et al. (2012), Vishruth et al. (2014) and Uma (2015).\u003c/p\u003e\n\u003cp\u003eBy the tenth day after inoculation, the colonies began to shift toward the secondary form, which lacked the characteristic surrounding clear zone. For isolates UASR22PL1 and UASR22PL3, the colonies transitioned to a transparent appearance that later turned dark red or brown. In UASR22PL2, this transition occurred around the twelfth day. The secondary form could be visually identified by a watery outer layer and an oily red core. These forms remained morphologically stable thereafter. The progression and time taken for the transition from primary to secondary form closely matched earlier observations (Bleakley and Nealson 1988; Vishruth et al. 2014; Hazir et al. 2004; An and Grewal 2011; Uma 2009; Uma 2015).\u003c/p\u003e\n\u003cp\u003eWhen cultured on NBTA medium, the bacterial colonies absorbed the bromothymol blue pigment from the media and changed color within 3\u0026ndash;4 days. The primary forms developed red-centered colonies overlaid with a blue tint and were encircled by a clear halo due to the reduction of TTC (Triphenyl Tetrazolium Chloride) and bromothymol blue absorption. In contrast, secondary forms did not absorb bromothymol blue and thus formed dark red colonies without the surrounding clear zones (Akhurst 1980; Boemare and Akhurst 1988). This transformation is thought to result from nutrient depletion in the medium, which alters not only the morphology but also the physiological properties such as biochemical activity, antibiotic production, and antagonistic behavior (Akhurst 1980).\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eMorphological Characteristics\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eMicroscopic examination revealed that both forms\u0026mdash;primary and secondary\u0026mdash;of all three \u003cem\u003eP. luminescens\u003c/em\u003e isolates were rod-shaped, flagellated, Gram-negative, and motile. They were either strictly aerobic or facultatively anaerobic. The isolates took up simple stains readily. When grown on Nutrient Agar, all colonies emitted bioluminescence when exposed to ultraviolet light, as observed under a UV lamp. Notably, the primary forms exhibited stronger luminescence than the secondary forms (Table 2, Figure 2).\u003c/p\u003e\n\u003cp\u003eThe observed bioluminescent property, coupled with morphological traits, reinforced that all three bacterial isolates were indeed \u003cem\u003eP. luminescens\u003c/em\u003e, consistent with earlier studies by Bleakley and Nealson (1988), Nagesh et al. (2001), Shahina et al. (2004), Peel et al. (1999), Han and Ehlers (2001), and Rashid et al. (2014).\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eMolecular Characterization and Phylogenetic Tree Analysis\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe PCR amplification of the 16S rRNA gene region for all three isolates yielded products approximately 1500 base pairs in length. This was confirmed by the appearance of a single, bright DNA band on 1% agarose gel electrophoresis (Fig 3). The sequences of the amplified products were submitted to GenBank with the following accession numbers: UASR22PL1 \u0026ndash; OQ472600, UASR22PL2 \u0026ndash; OQ473650, and UASR22PL3\u0026ndash;OQ472608\u0026nbsp;\u003cbr\u003eBLAST analysis of the sequences via NCBI\u0026rsquo;s database revealed that the phylogenetic dendrogram divided the \u003cem\u003ePhotorhabdus\u003c/em\u003e isolates into two clusters. Group A1 included UASR22PL1 and UASR22PL2, which displayed 64% similarity between themselves and showed 99% identity with \u003cem\u003eP. luminescens\u003c/em\u003e strain KZ2R1 (as per Uma, 2015). The UASR22PL3 isolate, classified under Group A2, showed 99% similarity with A1 isolates and a 95.79% similarity to \u003cem\u003eP. luminescens\u003c/em\u003e strain CAU4886 (Ren \u0026amp; Zhao, 2017) (Table 3, Fig. 4).\u003c/p\u003e\n\u003cp\u003eThus, the \u003cem\u003ePhotorhabdus\u003c/em\u003e strains isolated in this study were conclusively identified as \u003cem\u003eP. luminescens\u003c/em\u003e, closely related to the KZ2R1 strain. These findings are consistent with prior reports which used 16S rRNA sequence-based phylogenies to differentiate groups within the \u003cem\u003ePhotorhabdus\u003c/em\u003e genus (Szallas \u003cem\u003eet al\u003c/em\u003e. 1997; Brunel \u003cem\u003eet al\u003c/em\u003e. 1997; Liu \u003cem\u003eet al.\u003c/em\u003e 1997; Saux \u003cem\u003eet al.\u003c/em\u003e 1999; Hazir \u003cem\u003eet al\u003c/em\u003e. 2004). The 16S sequences from this study were aligned with homologous \u003cem\u003ePhotorhabdus\u003c/em\u003e sequences from GenBank for accurate placement in the phylogenetic tree.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eInsecticidal Relevance\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe bacterium \u003cem\u003eP. luminescens\u003c/em\u003e maintains a mutualistic relationship with \u003cem\u003eHeterorhabditis\u003c/em\u003e nematodes. Upon nematode penetration into the host insect\u0026rsquo;s body cavity, the bacteria are released into the hemocoel, where they initiate septicemia by releasing a suite of toxins and enzymes\u0026mdash;including chitinases, lipases, and proteases. These bio-convert the host tissue into nutrients for both the nematode and the bacterium (Akhurst 1980). This mode of action highlights the organism\u0026rsquo;s potential as a highly efficient microbial insecticide. Hence, the thorough characterization of \u003cem\u003ePhotorhabdus\u003c/em\u003e strains from various habitats is essential for selecting the most potent isolates for biocontrol programs.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study successfully isolated and characterized three bacterial strains\u0026mdash;UASR22PL1, UASR22PL2, and UASR22PL3\u0026mdash;associated with the entomopathogenic nematode \u003cem\u003eHeterorhabditis\u003c/em\u003e spp. These isolates were obtained from soil samples through an in-situ trapping technique. Detailed morphological and molecular analyses confirmed their identity as \u003cem\u003ePhotorhabdus luminescens\u003c/em\u003e, a symbiotic bacterium known for its biocontrol capabilities. The findings suggest that these native strains possess promising insecticidal properties and could be harnessed for biological control of lepidopteran and hemipteran pests prevalent in the region.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cul\u003e\n \u003cli\u003eEPN \u0026ndash; Entomopathogenic Nematodes\u003c/li\u003e\n \u003cli\u003eBOD \u0026ndash; Biological Oxygen Demand\u003c/li\u003e\n \u003cli\u003ePCR \u0026ndash; Polymerase Chain Reaction\u003c/li\u003e\n \u003cli\u003eUV \u0026ndash; Ultraviolet\u003c/li\u003e\n \u003cli\u003eTAE buffer \u0026ndash; Tris-Acetate-EDTA Buffer\u003c/li\u003e\n \u003cli\u003eDNA \u0026ndash; Deoxyribonucleic Acid\u003c/li\u003e\n \u003cli\u003eNCBI \u0026ndash; National Center for Biotechnology Information\u003c/li\u003e\n \u003cli\u003eMEGA \u0026ndash; Molecular Evolutionary Genetics Analysis\u003c/li\u003e\n \u003cli\u003eNBTA \u0026ndash; Nutrient Bromothymol Blue Agar\u003c/li\u003e\n \u003cli\u003eNA \u0026ndash; Nutrient Agar\u003c/li\u003e\n \u003cli\u003ebp \u0026ndash; Base Pair\u003c/li\u003e\n \u003cli\u003eTTC \u0026ndash; Triphenyl Tetrazolium Chloride\u003c/li\u003e\n \u003cli\u003eIJs \u0026ndash; Infective Juveniles\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Declarations","content":"\u003cp\u003eAll authors participated in the conceptualization and design of the research. Akshatha S. conducted the experimental work, collected the data, and carried out the analysis. The initial draft of the manuscript was written by Akshatha S. and Prabhuraj A., with all co-authors providing feedback on earlier versions. The final manuscript was reviewed and approved by all authors.\u003c/p\u003e\u003cp\u003eWe confirm that this work is original, has not been previously published, and is not under consideration for publication elsewhere. There are no conflicts of interest associated with this research.\u003c/p\u003e\u003cp\u003eThis study was supported financially by the Karnataka Science and Technology Promotion Society (KSTePS), Department of Science and Technology, Government of Karnataka. No experiments involving human or animal subjects were conducted in this research.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors participated in the conceptualization and design of the research. Akshatha S. conducted the experimental work, collected the data, and carried out the analysis. The initial draft of the manuscript was written by Akshatha S. and Prabhuraj A., with all co-authors providing feedback on earlier versions. The final manuscript was reviewed and approved by all authors.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis study was supported financially by the Karnataka Science and Technology Promotion Society (KSTePS), Department of Science and Technology, Government of Karnataka. No experiments involving human or animal subjects were conducted in this research\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAkhurst RJ (1982) Antibiotic activity of \u003cem\u003eXenorhabdus\u003c/em\u003e spp. bacteria symbiotically associated with insect pathogenic nematodes of the families Heterorhabditidae and Steinernematidae. \u003cem\u003eJ Gen Microbiol\u003c/em\u003e 128:3061\u0026ndash;3065\u003c/li\u003e\n \u003cli\u003eAdams BJ, Fodor A, Koppenh\u0026ouml;fer HS, Stackebrandt E, Stock SP, Klein MG (2006) Reprint of Biodiversity and systematics of nematode bacterium entomopathogens. \u003cem\u003eBiol Control\u003c/em\u003e 38:4\u0026ndash;21\u003c/li\u003e\n \u003cli\u003eAn R, Grewal PS (2010) \u003cem\u003ePhotorhabdus temperata\u003c/em\u003e subsp. \u003cem\u003estackebrandtii\u003c/em\u003e subsp. nov. (Enterobacteriales: Enterobacteriaceae). \u003cem\u003eCurr Microbiol\u003c/em\u003e 61(4):291\u0026ndash;297\u003c/li\u003e\n \u003cli\u003eAn R, Grewal PS (2011) \u003cem\u003ePhotorhabdus luminescens\u003c/em\u003e subsp. \u003cem\u003ekleinii\u003c/em\u003e subsp. nov. (Enterobacteriales: Enterobacteriaceae). \u003cem\u003eCurr Microbiol\u003c/em\u003e 62(2):539\u0026ndash;543\u003c/li\u003e\n \u003cli\u003eAneja KR (2003) \u003cem\u003eExperiments in Microbiology, Plant Pathology and Biotechnology\u003c/em\u003e. New Age International (P) Ltd, pp. 245\u0026ndash;275\u003c/li\u003e\n \u003cli\u003eBabic I, Fischer LM, Giraud E, Boemare N (2000) Occurrence of natural dixenic associations between the symbiont \u003cem\u003ePhotorhabdus luminescens\u003c/em\u003e and bacteria related to \u003cem\u003eOchrobactrum\u003c/em\u003e spp. in tropical entomopathogenic \u003cem\u003eHeterorhabditis\u003c/em\u003e spp. (Nematoda, Rhabditida). \u003cem\u003eMicrobiol\u003c/em\u003e 146(3):709\u0026ndash;718\u003c/li\u003e\n \u003cli\u003eBhat AH, Chaubey AK, Shokoohi E, Machado RJ (2021) Molecular and phenotypic characterization of \u003cem\u003eHeterorhabditis indica\u003c/em\u003e (Nematoda: Rhabditida) nematodes isolated during a survey of agricultural soils in Western Uttar Pradesh, India. \u003cem\u003eActa Parasitol\u003c/em\u003e 66(1):236\u0026ndash;252\u003c/li\u003e\n \u003cli\u003eBleakley B, Nealson KH (1988) Characterization of primary and secondary forms of \u003cem\u003eXenorhabdus luminescens\u003c/em\u003e strain Hm. \u003cem\u003eFEMS Microbiol Ecol\u003c/em\u003e 53:241\u0026ndash;250\u003c/li\u003e\n \u003cli\u003eBoemare NE, Akhurst RJ (1988) Biochemical and physiological characterization of colony form variants in \u003cem\u003eXenorhabdus\u003c/em\u003e spp. (Enterobacteriaceae). \u003cem\u003eJ Gen Microbiol\u003c/em\u003e 134:751\u0026ndash;761\u003c/li\u003e\n \u003cli\u003eBurges HD (1998) Formulation of mycoinsecticides. In: Burges HD (ed) \u003cem\u003eFormulation of Microbial Biopesticides\u003c/em\u003e. Kluwer Academic Publishers, Dordrecht, pp. 131\u0026ndash;185\u003c/li\u003e\n \u003cli\u003eBrunel B, Givaudan A-L, Akhurst RJ, Boemare N (1997) Fast and accurate identification of \u003cem\u003eXenorhabdus\u003c/em\u003e and \u003cem\u003ePhotorhabdus\u003c/em\u003e species by restriction analysis of PCR-amplified 16S rRNA genes. \u003cem\u003eAppl Environ Microbiol\u003c/em\u003e 63:574\u0026ndash;580\u003c/li\u003e\n \u003cli\u003eEhlers RU, Deseo KV, Stackebrandt E (1991) Identification of \u003cem\u003eSteinernema\u003c/em\u003e spp. (Nematoda) and symbiotic bacteria, \u003cem\u003eXenorhabdus\u003c/em\u003e spp. from Italian and German soils. \u003cem\u003eNematologica\u003c/em\u003e 37:360\u0026ndash;366\u003c/li\u003e\n \u003cli\u003eFelsenstein J (1993) \u003cem\u003ePHYLIP \u0026ndash; Phylogeny Inference Package\u003c/em\u003e, version 3.5. 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CABI Publishing\u003c/li\u003e\n \u003cli\u003eHan R, Ehlers R (2001) Effect of \u003cem\u003ePhotorhabdus luminescens\u003c/em\u003e phase variants on the in vivo and in vitro development and reproduction of the entomopathogenic nematodes \u003cem\u003eHeterorhabditis bacteriophora\u003c/em\u003e and \u003cem\u003eSteinernema carpocapsae\u003c/em\u003e. \u003cem\u003eFEMS Microbiol Ecol\u003c/em\u003e 35(3):239\u0026ndash;247\u003c/li\u003e\n \u003cli\u003eHazir S, Stackebrandt E, Lang E, Schumann P, Ehlers RU, Keskin N (2004) Two new subspecies of \u003cem\u003ePhotorhabdus luminescens\u003c/em\u003e isolated from \u003cem\u003eHeterorhabditis bacteriophora\u003c/em\u003e: \u003cem\u003ePhotorhabdus luminescens\u003c/em\u003e subsp. \u003cem\u003ekayaii\u003c/em\u003e subsp. nov. and \u003cem\u003eP. luminescens\u003c/em\u003e subsp. \u003cem\u003ethracensis\u003c/em\u003e subsp. nov. \u003cem\u003eSyst Appl Microbiol\u003c/em\u003e 27:36\u0026ndash;42\u003c/li\u003e\n \u003cli\u003eHominick WM (2002) Biogeography. In: CABI Publishing eBooks, pp. 115\u0026ndash;143. https://doi.org/10.1079/9780851995670.0115\u003c/li\u003e\n \u003cli\u003eKaya HK, Gaugler R (1993) Entomopathogenic nematodes. \u003cem\u003eAnnu Rev Entomol\u003c/em\u003e 38:181\u0026ndash;206\u003c/li\u003e\n \u003cli\u003eLizzy AM (2020) \u003cem\u003eBeauveria\u003c/em\u003e. In: Beneficial Microbes in Agro-Ecology, pp. 727\u0026ndash;748\u003c/li\u003e\n \u003cli\u003eLiu J, Berry R, Poinar G, Moldenke A (1997) Phylogeny of \u003cem\u003ePhotorhabdus\u003c/em\u003e and \u003cem\u003eXenorhabdus\u003c/em\u003e species and strains as determined by comparison of partial 16S rRNA gene sequences. \u003cem\u003eInt J Syst Bacteriol\u003c/em\u003e 47:948\u0026ndash;954\u003c/li\u003e\n \u003cli\u003eNagesh M, Hussaini SS, Singh SP (2001) Isolation and characterization of symbiotic bacteria from \u003cem\u003eHeterorhabditis\u003c/em\u003e spp. and \u003cem\u003eSteinernema carpocapsae\u003c/em\u003e. \u003cem\u003ePest Manag Hortic Ecosyst\u003c/em\u003e 8:38\u0026ndash;42\u003c/li\u003e\n \u003cli\u003ePrabhuraj A (1997) Faunastic studies on entomopathogenic nematodes families Steinernematidae and Heterorhabditidae and their potential as bio-control agents of white grubs and caterpillar pests. Ph.D. Thesis, Univ. Agric. Sci., Bangalore, India\u003c/li\u003e\n \u003cli\u003ePrabhuraj A, Viraktamath CA, Kumar ARV (2000) Modified technique for the isolation of insect parasitic nematodes. \u003cem\u003eJ Biol Control\u003c/em\u003e 14:83\u0026ndash;85\u003c/li\u003e\n \u003cli\u003ePeel MM, Alfredson DA, Gerrard JG, Davis JM, Robson JM, McDougall RJ, Scullie BL, Akhurst RJ (1999) Isolation, identification, and molecular characterization of strains of \u003cem\u003ePhotorhabdus luminescens\u003c/em\u003e from infected humans in Australia. \u003cem\u003eJ Clin Microbiol\u003c/em\u003e 37:3647\u0026ndash;3653\u003c/li\u003e\n \u003cli\u003eRashid P, Santhosh EJ, Devasahayam S, Jacob TK (2014) Natural occurrence of entomopathogenic nematodes associated with ginger (\u003cem\u003eZingiber officinale\u003c/em\u003e) ecosystem in India. \u003cem\u003eIndian J Nematol\u003c/em\u003e 44(2):238\u0026ndash;246\u003c/li\u003e\n \u003cli\u003eRen F, Zhao L (2017) National Center for Biotechnology Information (NCBI). https://www.ncbi.nlm.nih.gov/nuccore/MF429509.1/\u003c/li\u003e\n \u003cli\u003eSaux FM, Mauleon H, Constant P, Brunel B, Boemare N (1998) PCR-ribotyping of \u003cem\u003eXenorhabdus\u003c/em\u003e and \u003cem\u003ePhotorhabdus\u003c/em\u003e isolates from the Caribbean region in relation to the taxonomy and geographic distribution of their nematode hosts. \u003cem\u003eAppl Environ Microbiol\u003c/em\u003e 64:4246\u0026ndash;4254\u003c/li\u003e\n \u003cli\u003eSz\u0026aacute;ll\u0026aacute;s E, Koch C, Fodor A, Burghardt J, Buss O, Szentirmai A, Nealson KH, Stackebrandt E (1997) Phylogenetic evidence for the taxonomic heterogeneity of \u003cem\u003ePhotorhabdus luminescens\u003c/em\u003e. \u003cem\u003eInt J Syst Bacteriol\u003c/em\u003e 47:402\u0026ndash;407\u003c/li\u003e\n \u003cli\u003eSharad Mohan, Sirohi A, Gaur HS (2004) Successful management of mango mealy bug, \u003cem\u003eDrosicha mangiferae\u003c/em\u003e by \u003cem\u003ePhotorhabdus luminescens\u003c/em\u003e, a symbiotic bacterium from entomopathogenic nematode \u003cem\u003eHeterorhabditis indica\u003c/em\u003e. \u003cem\u003eInt J Nematol\u003c/em\u003e 14:195\u0026ndash;198\u003c/li\u003e\n \u003cli\u003eShahina F, Manzar H, Tabassum KA (2004) Symbiotic bacteria \u003cem\u003eXenorhabdus\u003c/em\u003e and \u003cem\u003ePhotorhabdus\u003c/em\u003e associated with entomopathogenic nematodes in Pakistan. \u003cem\u003ePak J Nematol\u003c/em\u003e 22(2):117\u0026ndash;128\u003c/li\u003e\n \u003cli\u003eSingh S, Eric M, Floyd I, Leonard HD (2012) Characterization of \u003cem\u003ePhotorhabdus\u003c/em\u003e growth for the rearing of the beneficial nematode \u003cem\u003eHeterorhabditis bacteriophora\u003c/em\u003e. \u003cem\u003eIndian J Microbiol\u003c/em\u003e 52:325\u0026ndash;331\u003c/li\u003e\n \u003cli\u003eSubbanna ARNS, Krishnayya KV, Arjuna R, Sudheeri JM, Srinivasa R (2008) Occurrence of entomopathogenic nematodes in different soils of Guntur district of Andhra Pradesh. \u003cem\u003eIndian J Nematol\u003c/em\u003e 38(2):218\u0026ndash;222\u003c/li\u003e\n \u003cli\u003eTamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. \u003cem\u003eMol Biol Evol\u003c/em\u003e 24:1596\u0026ndash;1599\u003c/li\u003e\n \u003cli\u003eThompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. \u003cem\u003eNucleic Acids Res\u003c/em\u003e 25:4876\u0026ndash;4882\u003c/li\u003e\n \u003cli\u003eUma GP (2015) Characterization of extracellular secretions of symbiotic bacterium \u003cem\u003ePhotorhabdus luminescens\u003c/em\u003e. Ph.D. Thesis, Univ. Agric. Sci., Raichur, India\u003c/li\u003e\n \u003cli\u003eVishruth K, Vendan T, Nagaraj S (2014) Isolation and characterization of entomopathogenic symbiotic bacterium \u003cem\u003ePhotorhabdus luminescens\u003c/em\u003e of \u003cem\u003eHeterorhabditis indica\u003c/em\u003e from soils of five agro-climatic zones of Karnataka. \u003cem\u003eBiosci Biotechnol Res Asia\u003c/em\u003e 11(1):129\u0026ndash;139\u003c/li\u003e\n \u003cli\u003eWeisburg GW, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. \u003cem\u003eJ Bacteriol\u003c/em\u003e 173:697\u0026ndash;703\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1. \u0026nbsp;Colony characteristics of Primary and secondary forms of \u003cem\u003eP. luminescens\u0026nbsp;\u003c/em\u003eon NBTA media\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 4px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSl. No.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIsolate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDays after\u0026nbsp;\u003cbr\u003e\u0026nbsp;Inoculation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eForm\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003ch1\u003eEdge\u003c/h1\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eElevation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eConsistency\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eClear\u0026nbsp;\u003cbr\u003e\u0026nbsp;zone\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSurface\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ecolor\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 4px;\"\u003e\n \u003cp\u003e1.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eUASR22PL1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003eCircular\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003eEntire\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003eConvex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eOpaque\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003eNo Clear zone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eSmooth and Glistening\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003eDark Brick red over laid by blue\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 4px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003eCircular\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003eEntire\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003eConcave\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eOpaque\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003eClear zone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eSmooth and Glistening\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003eCentral red core overlaid by light blue color\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 4px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003eIrregular\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003eUndulated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003eFlat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eOpaque\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003eClear zone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eRough and Glistening\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003eBrick red to dark red color\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 4px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003eIrregular\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003eUndulated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003eFlat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eTransparent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003eClear zone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eRough and Glistening\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003eDark red to brownish blue over laid by another layer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 4px;\"\u003e\n \u003cp\u003e2.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eUASR22PL2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003eCircular\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003eEntire\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003eConvex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eOpaque\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003eNo Clear zone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eSmooth and Glistening\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003eLight Pink\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 4px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003eCircular\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003eEntire\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003eConcave\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eOpaque\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003eClear zone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eSmooth and Glistening\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003eDark Pink\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 4px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003eIrregular\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003eUndulated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003eFlat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eTransparent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003eClear zone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eRough and Glistening\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003eDark Brick red over laid by blue\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 4px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003eIrregular\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003eUndulated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003eFlat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eTransparent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003eClear zone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eRough and Glistening\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003eDark Brick red over laid by blue\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 4px;\"\u003e\n \u003cp\u003e3.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eUASR22PL3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003eCircular\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003eEntire\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003eConvex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eOpaque\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003eNo Clear zone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eSmooth and Glistening\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003eCentral dark pink over laid by light pink layer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 4px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003eIrregular\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003eUndulated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003eSunken\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eTransparent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003eClear zone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eRough and Glistening\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003eDark pink to light pink core and outer layer pink to bluish\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 4px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003eIrregular\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003eUndulated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003eFlat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eTransparent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003eClear zone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eRough and Glistening\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003ePink layer turn to red color\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 4px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003eIrregular\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003eUndulated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003eFlat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eTransparent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003eClear zone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eRough and Glistening\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003eRed color\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. \u0026nbsp; Morphological characteristics of primary and secondary forms of \u003cem\u003eP. luminescens\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTest\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 28px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUASR22PL1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 28px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUASR22PL2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 28px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUASR22PL3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePrimary form\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSecondary form\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePrimary form\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSecondary form\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePrimary form\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSecondary form\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003eSimple staining\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e+ ve\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e+ ve\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e+ ve\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e+ ve\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e+ ve\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e+ ve\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003eGram staining\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e- \u0026nbsp;ve\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e- ve\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e- \u0026nbsp;ve\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e- ve\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e- \u0026nbsp;ve\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e- ve\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003eMotility\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e+ ve\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e+ ve\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e+ ve\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e+ ve\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e+ ve\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e+ ve\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003eBioluminescence\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e+ ve\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e+ ve\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e+ ve\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e+ ve\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e+ ve\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e+ ve\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003eShape\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003eMedium to long rod\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003eMedium to long rod\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003eMedium to long rod\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003eMedium to long rod\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003eMedium to long rod\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003eMedium to long rod\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. Identification of \u003cem\u003eP. luminescens\u003c/em\u003e based on sequence similarities from NCBI Genebank data and their accession numbers\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 5px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSl. No.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIsolates\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAccession number\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNCBI Identity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSimilarity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAccession no. of NCBI Identity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 5px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cem\u003eP. luminescens\u003c/em\u003e UASR22PL-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003eOQ472600\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34px;\"\u003e\n \u003cp\u003e\u003cem\u003ePhotorhabdus luminescens\u0026nbsp;\u003c/em\u003eKZ2R1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e99%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003eKP224438\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 5px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cem\u003eP. luminescens\u003c/em\u003e UASR22PL-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003eOQ473650\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34px;\"\u003e\n \u003cp\u003e\u003cem\u003ePhotorhabdus luminescens\u003c/em\u003e strain KZ2R1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e99%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003eKP224438\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 5px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cem\u003eP. luminescens\u003c/em\u003e UASR22PL-3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003eOQ472608\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34px;\"\u003e\n \u003cp\u003e\u003cem\u003ePhotorhabdus luminescens\u003c/em\u003e strain CAU9727\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e95.79%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003eMF429509.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\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":"international-journal-of-tropical-insect-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtis","sideBox":"Learn more about [International Journal of Tropical Insect Science](http://link.springer.com/journal/42690)","snPcode":"42690","submissionUrl":"https://www.editorialmanager.com/jtis/default2.aspx","title":"International Journal of Tropical Insect Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Photorhabdus luminescens, Heterorhabditis indica, Symbiotic Bacteria, Biological Control","lastPublishedDoi":"10.21203/rs.3.rs-6982668/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6982668/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study aimed to identify and characterize \u003cem\u003ePhotorhabdus luminescens\u003c/em\u003e, a bacterial endosymbiont of the entomopathogenic nematode \u003cem\u003eHeterorhabditis\u003c/em\u003e spp., collected from various crop ecosystems in Raichur, Karnataka. Both phenotypic and genotypic analyses were employed to confirm bacterial identity. Three distinct bacterial strains named UASR22PL1, UASR22PL2, and UASR22PL3 were isolated by streaking haemolymph obtained from \u003cem\u003eGalleria mellonella\u003c/em\u003e larvae infected with \u003cem\u003eHeterorhabditis\u003c/em\u003e spp. All strains exhibited Gram-negative bacillus morphology characteristic of \u003cem\u003eP. luminescens\u003c/em\u003e. After 72 hours of incubation, bacterial colonies appeared smooth, small, round, glistening, and convex with entire margins. They developed a red pigmentation with a bluish hue, and over time, transitioned from primary to secondary form due to prolonged static incubation of the culture. The phylogenetic tree constructed further confirmed that the isolated strains belonged to the species \u003cem\u003eP. luminescens\u003c/em\u003e, establishing a close group of \u003cem\u003eP. luminescens\u003c/em\u003e strain KZ2R1.. Known for their potent insecticidal activity, these three native isolates have potential applications in biocontrol strategies for managing significant agricultural pests.\u003c/p\u003e","manuscriptTitle":"Description and Characterization of Indigenous Isolates of the Symbiotic Bacterium Photorhabdus luminescens (Thomas and Poinar)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-04 16:56:14","doi":"10.21203/rs.3.rs-6982668/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-05T08:45:33+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-25T22:48:18+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-15T07:52:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"15668018441730209906103360129783175972","date":"2025-07-31T22:33:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"123878930055068439907235993631292479437","date":"2025-07-30T16:00:25+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-30T07:05:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-02T03:01:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-02T03:00:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"International Journal of Tropical Insect Science","date":"2025-06-26T10:43:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-tropical-insect-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtis","sideBox":"Learn more about [International Journal of Tropical Insect Science](http://link.springer.com/journal/42690)","snPcode":"42690","submissionUrl":"https://www.editorialmanager.com/jtis/default2.aspx","title":"International Journal of Tropical Insect Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"83fb602e-4aea-4c5c-9afd-123a8bce55f1","owner":[],"postedDate":"August 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-28T11:23:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-04 16:56:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6982668","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6982668","identity":"rs-6982668","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}