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J. Microbiol. Biotechnol. (2012), 22(12), 1605–1612 http://dx.doi.org/10.4014/jmb.1203.03068 First published online October 4, 2012 pISSN 1017-7825 eISSN 1738-8872 Physiological and Molecular Characterization of a Newly Identified Entomopathogenic Bacteria, Photorhabdus temperata M1021 Jang, Eun-Kyung1,2, Ihsan Ullah1, Jong-Hui Lim1, In-Jung Lee1, Jong-Guk Kim3, and Jae-Ho Shin1* 1 School of Applied Biosciences, Kyungpook National University, Daegu 702-701, Korea Food and Biological Resources Examination Division, Korean Intellectual Property Office, Daejeon 302-701, Korea 3 Department of Life Sciences and Biotechnology, College of National Sciences, Kyungpook National University, Daegu 702-701, Korea 2 Received: March 29, 2012 / Revised: July 4, 2012 / Accepted: July 26, 2012 The present study concerned the identification and characterization of a novel bacterial strain isolated from entomopathogenic nematodes collected from different regions in Korea. The bacterial isolate M1021 was Gramnegative, bioluminescent, and produced red colonies on MacConkey agar medium. A rod-shaped structure was confirmed by the electron micrograph. Fatty acid composition was analyzed by using the Sherlock MIDI system. The identification was further supported by 16S rDNA sequence analysis, which revealed 96-99% sequence homology with strains of Photorhabdus temperata. The location of the isolated strain of P. temperata in the phylogenetic tree was confirmed and it was named P. temperata M1021. P. temperata M1021 exhibited catalase, protease, and lipase activities when grown on appropriate media supplemented with respective substrates. The culture of P. temperata M1021 exhibited insecticidal activity against the larvae of Galleria mellonella and the activity was the highest after 3-4 days of cultivation with agitating at 28oC under 220 rpm. Antibacterial activity was also observed against Salmonella Typhimurium KCTC 1926 and Micrococcus luteus KACC 10488. Keywords: Antibacterial activity, entomopathogenic, Galleria mellonella, insecticidal toxicity, Photorhabdus temperata Photorhabdus spp. are symbiotically associated with entomopathogenic nematodes (EPNs) of the family Heterorhabditidae [7]. The Photorhabdus-Heterorhabditidae symbiotic complex passes through three stages during a complete life cycle. In the first symbiotic life cycle stage, *Corresponding author Phone: +82 53 9505716; Fax: +82 53 9537233; E-mail: [email protected] the infective form of the EPNs, called infective juveniles (IJs) [17], take Photorhabdus bacteria into their guts and then actively hunt for insect hosts. In the second stage (pathogenic stage), the nematodes enter into the hemocoel of an insect host through its respiratory spiracles or digestive tract [33], and the combined action of the nematodes and bacteria kills susceptible insect hosts within 48 h of infection [26, 33]. In the third stage (replicative stage), the bacterial cells multiply and convert the tissues of the insect cadaver into bacterial biomass using hydrolytic enzymes [8]. The insect dies of septicemia and the IJs feed on the multiplying symbiotic bacteria, and in this way complete 1-3 generations in the insect host cadaver [19]. Upon depletion of food resources, a large number of IJs have been produced, which contain colonies of the symbiotic bacteria in their guts, and this symbiotic association is then dispersed in search of new hosts [22]. During infection, the symbiotic bacteria show pathogenicity against the insects [8, 21]. They inhibit the insect immune response by suppressing phospholipase A2, which catalyzes phospholipids at the sn-2 position to release arachidonic acid [20]. The Photorhabdus bacteria also inhibit humoral immunity by down-regulating gene expression of antibacterial peptides including cecropin. They can directly induce apoptosis of insect immunocytes and hemocytes, resulting in complete immune suppression and septicemia [32]. Various antibiotics are synthesized from cultures of symbiotic bacteria whereby xenorhabdins and xenocoumacins are commonly produced by Xenorhabdus spp., whereas hydroxystilbenes and anthraquinones are produced by Photorhabdus spp. [29]. Currently, three species of Photorhabdus have been described to be associated with Heterorhabditis nematodes: P. asymbiotica, P. luminescens, and P. temperata. Based on their 16S rDNA sequences and metabolic properties, P. luminescens has been divided into 1606 Jang et al. five subspecies [10]. Four out of five subspecies including P. luminescens subsp. luminescens, P. luminescens subsp. laumondii, P. luminescens subsp. kayaii, and P. luminescens subsp. thracensis have been isolated from H. bacteriophora, and one P. luminescens subsp. akhurstii has been isolated from H. indica. Nematode association with P. temperata appears to be more diverse. It has been found to be associated with H. megidis, H. zealandica, H. marelatus, and H. downesi [12]. P. temperata has also been observed to be harbored by H. bacteriophora [4]. According to Boemare et al. [7], these reports do not modify the wellestablished concept of a one-to-one association between EPNs and their symbiotic bacteria, but it does represent a problem for bacterial classification. In fact, DNA-DNA hybridization and 16S rDNA sequence information suggest that Photorhabdus is a heterogeneous genus [3], and thus, subspecies definition is necessary [12]. Thus far, two P. temperata subspecies have been described: P. temperata subsp. temperata associated with H. megidis [12] and P. temperata subsp. cinerea associated with H. downesi [30]. Indigenous EPNs are perhaps more suitable for inundatory release against local insect pests because they have already adapted to the local climate and other population regulators [24] [5]. In addition, many countries are concerned about the introduction of exotic EPNs because they may have a negative impact on non-target organisms [5]. The main objective of the present study was to identify a novel bacterial strain associated with an endogenous EPN of Korea and investigate its pathogenicity against harmful insects. In addition, we aim to introduce our newly discovered strain into an agriculture field for use as a biocontrol agent. MATERIALS AND METHODS Collection of the Nematodes Approximately 200 soil samples were collected from different locations in South Korea. Each sample location consisted of a 50 m2 area. Approximately 250 g of soil from each sample location was placed in a pre-sterilized plastic bag and transported to the laboratory. EPNs were isolated from the soil samples through the insect baiting method [7]. The insect bait (G. mellonella larvae) was placed in 250 ml plastic containers along with a moist soil sample. The plastic containers containing the bait and soil sample were covered with a lid and maintained at room temperature (20 ± 2oC). Samples were moistened with water if they appeared to be dry at any point during baiting. Larvae bait was checked daily and any dead larvae were removed from the container. After 7-9 days of baiting, the dead larvae (brick red color) were carefully washed with distilled water and put in White traps until emergence of third-stage IJs [16]. Emerging nematodes were collected and then used to infect fresh G. mellonella larvae in order to produce nematodes for identification and establishment of bacterial cultures. Isolation and Identification of Symbiotic Bacteria Symbiotic bacteria were secluded by streaking insect larvae hemolymph on MacConkey agar medium. The culture plates were incubated at 28 ± 2oC for 48 h and then observed for colony growth. Bright-pink colonies grown on MacConkey medium (Becton, Dickinson and Company, USA) were transferred to NBTA plates (nutrient agar with 0.004% triphenyltetrazolium chloride and 0.025% bromothymol blue) and presumed Photorhabdus spp. were identified by their characteristic adsorption of blue dye [2]. The bacterial culture in Luria-Bertani (LB) medium was incubated at a temperature range of 28-36oC for 48 h in a shaking incubator at 220 rpm. Bacterial growth was assessed using a spectrophotometer to measure absorbance at OD600. The optimal incubation time was determined by incubating the culture broth at 28 ± 2oC for 1 to 8 days in a shaking incubator at 220 rpm. Gram staining was performed by application of the standard Gram stain procedure. The culture broth was deposited on formvar-coated grids for 15 s and followed by staining with 2% uranyl acetate for 25 min. The stained grids were evaluated using a transmission electron microscope. Genomic Analysis of the Isolated Bacteria Chromosomal DNA was extracted from the symbiotic bacteria using a QIAprep Spin Miniprep kit (Qiagen, Valencia, CA, USA). The extracted DNA segment containing the 16S rDNA gene (approximately 1.5 kb) was amplified by polymerase chain reaction (PCR) using primers 27F (5'-AGA GTT TGA TCC TGG CTC AG-3') and 1492R (5'-GGT TAC CTT GTT ACG ACT T-3'). The PCR mixture (50 µl) consisted of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 0.2 mM dNTPs, 0.2 pmol of each primer, and Taq DNA polymerase (Takara, Japan). Amplification was performed using a protocol consisting of 30 cycles that included 1 min of denaturation at 95oC, 30 s of annealing at 55oC, 1 min of elongation at 72oC, and a final extension of 5 min at 72oC. The PCR product was then purified (PCR purification kit; Solgent, Daejeon, Korea) and sequencing was conducted by a commercial laboratory (Solgent, Daejeon, Korea). The 16S rDNA gene sequences obtained from the isolated bacterial strain was edited using a multiple sequence editor (DNASIS, Hitachi, America, Ltd) and nucleotide comparison was investigated using the BLASTN program from the National Center for Biotechnology Information (NCBI). The nucleotide sequences were aligned using ClustalW2 from EBI (http://www.ebi.ac.uk/ Tools/clustalw2). Pair-wise evolutionary distances were computed using Poisson correction for multiple substitutions. The phylogenetic tree of the isolated strain was constructed using the maximun parsimony method [28] in the Mega 4.0 program. Lipase and Protease Activities The lipase activity produced by the bacterial isolate was determined using LB agar plates. Two sets of LB media were prepared such that one included Tween 20 and the other included Tween 80. Using autoclaved toothpicks, colonies of bacterial isolate were inoculated on the media. Escherichia coli DH5α (negative control) was inoculated on the same media and incubated at 28 ± 2oC for 48 h. LB agar supplemented with 10% (w/v) skim milk was used to determine the protease activity of the bacterial isolate. Using autoclaved toothpicks, bacterial colonies (test sample) were inoculated on culture plates and incubated at 28oC for 48 h. Bacillus subtilis 2232 was used as a positive control and E. coli DH5α was used as a negative control. ENTOMOPATHOGENIC BACTERIA, PHOTORHABDUS TEMPERATA M1021 Antibiotic and Catalase Activities An experiment was conducted to investigate the antibiotic activity of the bacterial isolate. Thirty microliter cultures of S. typhimurium KCTC 1926 (Korean Collection for Type Cultures) and M. luteus KACC 10488 (Korean Agricultural Culture Collection) were spread over the surface of two separate plates containing Tryptone soy agar (TSA) media [tryptone (Bacon) 17.0 g/l, soytone (Bacon) 3.0 g/l, glucose 2.5 g/l, sodium chloride 5.0 g/l, dipotassium hydrogen phosphate 2.5 g/l, and agar 15 g/l]. The bacterial isolate (test sample) and E. coli DH5α (negative control) were inoculated on the S. typhimurium and M. luteus culture plates. The culture plates were then incubated at 28oC for 48 h. The catalase activity was determined by growing the bacterial isolate on tryptone soy agar media. The culture plates were incubated at 28 ± 2oC for 48 h, and then 30 µl of 3% (v/v) H2O2 was added to the fully grown colonies of the bacterial isolate. E. coli DH5α was used as the positive control and Streptococcus agalactiae ATCC13813 (American Type Culture Collection) was used as the catalase negative control. Analysis of Fatty Acid Composition The fatty acids were analyzed by gas chromatography (Model 6890; Hewlett Packard, Canada) using the Microbial Identification software package [25]. Four different types of reagents were used for fatty acid analysis: Reagent I (45 g sodium hydroxide, 150 ml methanol, and 150 ml distilled water), Reagent II (325 ml certified 6.0 N hydrochloric acid and 275 ml methyl alcohol), Reagent III (200 ml hexane and 200 ml methyl tert-butyl ether), and Reagent IV (10.8 g sodium hydroxide dissolved in 900 ml distilled water). The sample was prepared according to the requirements of the Sherlock microbial identification system (MIDI). (1) Harvesting: A 4 mm loop was used to harvest about 40 mg of bacterial cells from the third quadrant (second or first quadrant if slow growing) of the quadrant streaked plate. The cells were placed in a clean 13×100 culture tube. (2) Saponification: 1.0 ml of Reagent I was added to each tube containing harvested cells. The tubes were securely sealed with Teflon-lined caps, vortexed briefly, and then heated in a boiling water bath for 5 min. The tubes were vigorously vortexed for 510 s and then returned to the water bath in order to complete the 30 min heating. (3) Methylation: The cooled tubes were uncapped and 2 ml of Reagent II was added. The tubes were then capped and briefly vortexed. After vortexing, the tubes were heated for 10 ± 1 min at 80 ± 1oC. Note that this step was critical in terms of time and temperature. (4) Extraction: Addition of 1.25 ml of Reagent III to the cooled tubes was followed by recapping and gentle tumbling on a clinical rotator for approximately 10 min. The tubes were then uncapped and the aqueous (lower) phase was pipetted out and discarded. (5) Base Wash: Approximately 3 ml of Reagent IV was added to the organic phase remaining in the tubes. The tubes were recapped and tumbled for 5 min. Following uncapping, approximately 2/3 of the organic phase was pipetted into a GC vial, which was then capped and ready for analysis. Fatty acids produced by the bacterial isolate were measured by GC (Model 6890; Hewlett Packard, Canada). Insect Breeding G. mellonella larvae were bred from eggs of wax moth (collected from Daegu, Korea) using artificial media. The media ingredients consisted of wheat bran (600 g), rice bran (600 g), yeast extract (4.5 mg), CaCO3 (2 mg), glycerol (250 ml), water (175 ml), honey 1607 (600 ml), and vitamin B-complex (600 mg). All the ingredients, except the honey and vitamins, were mixed thoroughly. Then the mixture and honey were autoclaved in separate containers. After sterilization, the honey, vitamins, and other ingredients were all mixed together. Eggs laid by wax moth on butter paper were added into 150 g of the media and then incubated at 27 ± 1oC and relative humidity 50 ± 5%, whereby the eggs hatched. The small larvae were then transferred to a larger container containing a large amount of medium. Toxicity Test of the Bacterial Culture Supernatants An experiment was designed to examine the pathogenicity of the isolated bacteria. The bacteria were cultured in LB medium for 48 h at 28 ± 2oC, and then the culture broth was centrifuged at 12,000 rpm in order to harvest the supernatant. Three microliters of the culture supernatant was injected into the hemocoel of individual G. mellonella larvae using a 10 µl Hamilton syringe. Each injected larva was transferred to a 90 mm Petri dish and then incubated at 25 ± 2oC and 50% relative humidity. The larva mortality rate was evaluated after five days. Ten larvae were tested using each bacterial sample and the experiment was repeated three times. RESULTS Morphological Characteristics of the Isolate The bacterial strain isolated from the EPNs was grown on NBTA at 28 ± 2oC, producing colonies of up to 1.0 mm in diameter after 48 h. The bacterial isolate grew well on all Fig. 1. Biochemical analysis of P. temperata M1021. (A) Colony morphology of P. temperata M1021. (B) Catalase activity of P. temperata M1021, forming bubbles in response to the addition of H2O2. (C) Antibiotic activity of P. temperata M1021 against S. typhimurium. (D) Antibiotic activity of P. temperata M1021 against M. luteus. (E) Lipase activity of P. temperata M1021 in medium supplemented with Tween. (F) Protease activity of P. temperata M1021 in medium supplemented with skim milk. Clear zones around the colonies of P. temperata M1021 in Figures C, D, E, and F confirmed the positive results. 1608 Jang et al. Fig. 2. Morphological characterization of P. temperata M1021. (A) Rod-shaped P. temperata strain M1021 as viewed on an electron micrograph. (B) Gram-negative nature of the P. temperata strain M1021 isolated from EPNs. the media tested in this study. The colony morphology was granulated, convex, and opaque (Fig. 1A). The bacterial isolate was streaked on NBTA plates and the potential Photorhabdus sp. was identified based on phenotypic criteria associated with bioluminescence and colony morphology. The primary colonies appeared as mucoid, convex, and greenish or dark blue with a clear zone due to dye absorption and TTC reduction. Electron micrograph (Fig. 2A) results revealed the rod shape of the bacterial isolate and Fig. 2B illustrates their Gram-negative characteristic. Genotypic Characteristics of the Isolate The PCR product was purified and sequenced, revealing a length of 1,498 bp. The 16S rDNA sequence from the present study was aligned against a database available at the NCBI. The identification of bacterial isolate was carried out by the maximum parsimony (MP) method. Nine sequences (8 references and 1 clone) were selected for the construction of a phylogenetic tree with 1,000 bootstrap replications. These strains were selected from BLAST search showing maximum sequence homology and query coverage, as well as lowest E values. Xenorhabdus was used as the out-group. BLAST search showed that bacterial isolate M1021 has 99.8% sequence homology against Fig. 3. Phylogenetic tree of the selected Photorhabdus was constructed by maximum parsimony with bootstrap analysis of 1,000 replicates. The tree was constructed using 9 taxa (8 references and 1 clone). Numbers at branch points indicate different clones having the same, or nearly same, sequence. M1021 formed a subclade with P. temperata with a bootstrap support of 100%; therefore, M1021 was identified as a new strain of P. temperata. P. temperata strain XINachT, 98% against P. luminescens subsp. luminescens strain HbT, 97% against P. asymbiotica strain ATCC 43950T, and 96% against P. luminescens subsp. luminescens strain ATCC 29999T. In the dendrogram, bacterial isolate M1021 formed a clade (99% bootstrap support) with P. temperata (Fig. 3). On the basis of sequence homology and phylogenetic analysis, isolate M1021 was thus identified as a new strain of P. temperata. It was named Photorhabdus temperata M1021. The 16S rDNA sequence was deposited into GenBank under the accession number HQ647119. Catalase Activity The bacterial catalase activity was determined by growing the P. temperata M1021 on a TSA plate at 28 ± 2oC for 48 h, and then 30 µl of 3% H2O2 (v/v) was poured over the fully grown colonies (Fig. 1B). Bubble formations over the surface of colonies indicated the presence of catalase. S. agalactiae was used as a catalase negative control and E. coli DH5α was used as a catalase positive control. Antibiotic Synthesis Antibiotic synthesis by P. temperata M1021 was confirmed using a simple experiment. Thirty microliter cultures of S. typhimurium and M. luteus were spread over the surfaces of two different media plates containing tryptone soy agar, and the P. temperata M1021 and E. coli DH5α colonies were streaked over the plates. The results (Fig. 1C and 1D) indicated that antibiotics were produced by P. temperata M1021, which killed both S. Typhimurium and M. luteus in their respective culture plates. The secretion of antibiotics functioned to form clear zones around the P. temperata M1021 colonies. No clear zones formed around the E. coli DH5α colonies, which were included as a negative control. Lipase and Protease Activities Lipase and protease activities were found in the P. temperata strain M1021. LB media containing either Tween 20 or Tween 80 were used to determine the lipase activity. Results (Fig. 1E) indicated that P. temperata M1021 was capable of producing the lipase, which degraded both the Tween 20 and Tween 80 present in the LB media, forming digestion zones around the colonies. The protease activity was determined by incubating the P. temperata M1021 on LB agar supplemented with 10% (w/v) skim milk. The formation of clear zones around the P. temperata M1021 colonies confirmed the secretion of proteases (Fig. 1F). Fatty Acid Composition The fatty acid composition of P. temperata M1021was analyzed using a Sherlock MIDI system. The results (Fig. 4) revealed 14 different fatty acids (even and odd numbered, normal, iso, and anteiso; fatty acids are represented by a ENTOMOPATHOGENIC BACTERIA, PHOTORHABDUS TEMPERATA M1021 1609 Fig. 4. Fatty acid composition of P. temperata strain M1021. The fatty acids were analyzed with a Sherlock MIDI system following the manufacturer instructions. binumeric system in which the first number refers to the chain length and the second number refers to the number of double bonds) emerging between 12:0 and 18:0. Growth Conditions Based on our results, the optimum growth conditions for P. temperata M1021 (Fig. 5A) indicated that P. temperata M1021 showed best growth activity at 28 ± 2oC and 220 rpm. The effect of the incubation time was also evaluated, with the highest growth rate recorded at the fourth and fifth days of incubation (Fig. 5B). After a long exponential phase, the growth curve for P. temperata M1021 reached a stationary phase on the fifth day of incubation. A declination in growth was observed beyond 5 days of incubation. Pathogenicity of P. temperata M1021 Approximately 3 µl of the filter extract was injected into fifth instar larvae of G. mellonella. The results (Fig. 5C) indicated that P. temperata M1021 was highly virulent and caused 100% mortality within 48 h. The LB medium used as a negative control showed no evidence of pathogenicity at any stage of larval incubation. The majority of the larvae injected with the filtered extract died 24-36 h after injection and turned a red-brown color typical of insects infected with P. temperata. After a long exponential phase, the growth curve of the P. temperata M1021 culture reached a stationary phase on the sixth day of incubation (Fig. 5C). The insecticidal activity contained in the supernatant gradually increased as the incubation time increased, and maximum pathogenicity was noticed in the mid and late stages of the exponential phase (Fig. 5C). Fig. 5. Optimal conditions for the growth of P. temperata M1021. (A) P. temperata M1021 was incubated in LB medium at different temperatures (25-36oC) for 48 h. (B) The growth curve indicates that P. temperata M1021 reached an exponential phase on the fourth day of its life cycle. The culture was incubated at 28oC in a shaking incubator at 220 rpm. (C) Pathogenicity patterns of the P. temperata M1021 against the fifth instar larvae of G. mellonella. Three microliters of bacteria was injected into the hemocoel. Ten larvae were injected and their mortality was assessed after 24 h. The histogram shows the mortality rate of G. mellonella, and closed circles ( ● ) show the growth curve at 600 nm. DISCUSSION Photorhabdus spp. are Gram-negative, bioluminescent bacteria that live in symbiotic association with soil nematodes (Heterorhabditidae). In nature, the complex formed by the bacterium-nematode has evolved to be an obligate insect pathogen. EPNs require their own symbiotic 1610 Jang et al. bacteria for successful parasitism against their insect hosts [1]. Two genera of symbiotic bacteria, Photorhabdus and Xenorhabdus, are found in EPNs [7]. This study was designed to identify and characterize the bacteria isolated from soil nematodes (Heterorhabditis) collected from different locations across Korea. Our morphological identification results coincided with positive key characteristics associated with P. temperata, including motility, Gram negativity, rod shape, highly bioluminescent, and adsorbing dye on NBTA and MacConkey plates [11, 18, 27]. Optimal bacterial growth for Photorhabdus spp. was observed at 28 ± 2oC. This temperature had been previously optimized by Akhurst and Boemare [2]. Morphological characteristics of the bacteria provide some information regarding the identification, but nowadays molecular and phylogenetic approaches have absolutely changed the dynamics of the identification techniques. Genomic DNA sequencing is an objective, reproducible, and rapid technique for identification of bacterial strains. Sequencing of 16S rDNA has gained an immense importance in bacterial identification in recent times, due to the presence of highly conserved sequence of nucleotides in this part of the DNA. Genotypic characteristics strongly supported the hypothesis that the bacterial strain isolated from the EPN belonged to P. temperata. Phylogenetic analysis of our discovery confirmed its location in the phylogenetic tree and it was named P. temperata M1021. The identification was further confirmed by the fatty acid composition as analyzed by the Sherlock microbial identification system. The fatty acid profile of P. temperata M1021 was comparable with the previous analysis of fatty acid composition of P. temperata subsp. temperata [20, 31]. P. temperata releases secondary metabolites such as antibiotics, bacteriocins, lipases, and proteinases, and it is tempting to speculate that the more rigid membranous structures associated with P. temperata may facilitate the release of these secondary metabolites [34] as compared with other bacteria. This hypothesis may also explain the increase, instead of the expected decrease, of the structural order of the fatty acids released from P. temperata cultured at reduced temperatures. The surface area that a given quantity of phospholipids occupy in their respective membranes decreases at lower temperatures because of the reduced thermal motion in their component acyl chains [23]. This reduction in surface area may induce the opening of additional channels on the cell surface through which different secondary metabolic products may pass. In this manner, the bacteria may maintain proper levels of these compounds in the insect host, thus protecting the cadaver and the bacteria from colonization by other bacterial species as well as providing digestible food for the nematode host at reduced temperatures [13]. The P. temperata M1021 discovered during the present study secreted insecticidal toxin into its growth medium. The toxin was extremely lethal when injected into the fifth instar larvae of G. mellonella. The hemocoelic injection of the bacterial extract resulted in complete mortality of the insect larvae within 48 h. In typical insect infections, upon entry into the host hemocoel, the nematodes release the symbiotic bacteria in their guts [15, 29]. The presence of the bacterial symbiont is required in order to kill the insect host and to digest the host’s tissues, thereby providing nutrients for nematode growth and development [9]. It was observed during the incubation of P. temperata M1021 that after a long exponential phase, the bacteria entered into a stationary phase at the fifth day of the incubation period. The insecticidal activity of P. temperata M1021 gradually increased with culture time and reached a peak at the mid and late stages of the exponential phase. The insecticidal activity of entomopathogenic bacteria could be due to the secondary metabolites they produce, such as toxins and enzymes [6, 33]. It is notable that there was a conflict between the results of the present study and previous reports, which observed that culture extracts after 24 h of incubation exhibited the highest insecticidal activity [14]. Considering the combined results, we inferred that the toxins were extracellular in nature and secreted in the mid exponential phase by the strain used in the present study, and secreted in the early exponential phase in the strains used previously [6, 14]. The proposed strain of the present study was named as P. temperata M1021. Acknowledgment This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0009969) and Korea Ministry of Environment as the Eco-Innovation project. REFERENCES 1. Akhurst, R. J. 1982. Antibiotic activity of Xenorhabdus spp., bacteria symbiotically associated with insect pathogenic nematodes of the families Heterorhabditidae and Steinernematidae. J. Gen. Microbiol. 128: 3061-3065. 2. Akhurst, R. J. and N. E. Boemare. 1988. A numerical taxonomic study of the genus Xenorhabdus (Enterobacteriaceae) and proposed elevation of the subspecies of X. nematophilus to species. J. Gen. Microbiol. 134: 1835-1845. 3. Akhurst, R. J., R. G. Mourant, L. Baud, and N. E. Boemare. 1996. Phenotypic and DNA relatedness between nematode symbionts and clinical strains of the genus Photorhabdus (Enterobacteriaceae). 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