Diversity of Xenorhabdus and Photorhabdus spp. and Their Symbiotic Entomopathogenic Nematodes from Thailand

Xenorhabdus and Photorhabdus spp. are bacterial symbionts of entomopathogenic nematodes (EPNs). In this study, we isolated and characterized Xenorhabdus and Photorhabdus spp. from across Thailand together with their associated nematode symbionts, and characterized their phylogenetic diversity. EPNs were isolated from soil samples using a Galleria-baiting technique. Bacteria from EPNs were cultured and genotyped based on recA sequence. The nematodes were identified based on sequences of 28S rDNA and internal transcribed spacer regions. A total of 795 soil samples were collected from 159 sites in 13 provinces across Thailand. A total of 126 EPNs isolated from samples taken from 10 provinces were positive for Xenorhabdus (n = 69) or Photorhabdus spp. (n = 57). Phylogenetic analysis separated the 69 Xenorhabdus isolates into 4 groups. Groups 1, 2 and 3 consisting of 52, 13 and 1 isolates related to X. stockiae, and group 4 consisting of 3 isolates related to X. miraniensis. The EPN host for isolates related to X. stockiae was S. websteri, and for X. miraniensis was S. khoisanae. The Photorhabdus species were identified as P. luminescens (n = 56) and P. asymbiotica (n = 1). Phylogenenic analysis divided P. luminescens into five groups. Groups 1 and 2 consisted of 45 and 8 isolates defined as subspecies hainanensis and akhurstii, respectively. One isolate was related to hainanensis and akhurstii, two isolates were related to laumondii, and one isolate was the pathogenic species P. asymbiotica subsp. australis. H. indica was the major EPN host for Photorhabdus. This study reveals the genetic diversity of Xenorhabdus and Photorhabdus spp. and describes new associations between EPNs and their bacterial symbionts in Thailand.


Introduction
Xenorhabdus and Photorhabdus spp. are bacterial symbionts of entomopathogenic nematodes (EPNs) belonging to the genera Steinernema and Heterorhabditis, respectively. These Gram-negative bacilli inhabit the intestine of infective juvenile stage EPNs present in soil. EPNs infect the larval stage of a diverse range of insects and release their bacterial cargo, which multiply and secrete proteins and secondary metabolites that are lethal to the insect larva and suppress the growth of other competing environmental bacteria, fungi, nematodes and protists. The dead insect cadaver provides a nutrient source for completion of the nematode lifecycle. When nutrients are depleted, the bacteria re-associate with the infective juvenile stage EPNs, which then disperse into the environment to search for a new insect host [1].
Sixty-one species of Steinernema and 24 species of Heterorhabditis nematodes have been identified to date [2,3,4]. A total of 21 Xenorhabdus spp. have been described in association with P. luminescens [8]. The objectives of this study were to isolate and identify EPNs and their associated Xenorhabdus and Photorhabdus spp. from across Thailand and describe their phylogeography. We also defined the relationship between the bacteria and EPN species and defined the association of EPNs recovery and various soil parameters.

Collection of soil samples
A total of 795 soil samples from 159 sites were collected from 13 provinces in Thailand between 4 July 2009 and 22 October 2009 (the rainy season). Samples were taken from random areas of natural grassland, roadside verges, woodland, and the banks of ponds and rivers. No specific permits were required for the described field studies. For each site, 5 soil samples were randomly taken in an area of approximately 100 m 2 at a depth of 10-20 cm using a hand shovel. Approximately 500 g of each soil sample was placed into a plastic bag. The longitude, latitude and altitude of each sampling site were recorded using GPSMAP 60CSx (Garmin, Taiwan). The temperature, pH and moisture of each sample were recorded using a Soil PH & Moisture Tester (Model: DM-15, Takemura electric works, Ltd, Japan). Soil samples were maintained at 25-32uC (ambient temperature) during transportation to our laboratory in Bangkok.

Isolation of entomopathogenic nematodes (EPN)
EPNs were recovered from soil samples using an established insect (Galleria mellonella) baiting technique, as described previously [9]. In brief, each soil sample was placed into a plastic box and 5 last instar G. mellonella larvae were placed on the soil surface. The box lid was put in place, the box inverted and stored at 25uC for 5 days. Dead G. mellonella were collected and each G. mellonella cadaver placed into a White trap [10]. This was maintained in the dark at 20-25uC to allow for the emergence of infective juvenile nematodes (IJs). Soil samples associated with death of G. mellonella were re-baited three times using fresh larvae. Emergent nematodes were collected and pooled for a given soil sample and used to infect new G. mellonella larvae to confirm entomopathogenicity and to amplify the number of nematodes. This was performed by adding 500 ml containing approximately 100 EPNs onto a sterile petri dish. Two insect larvae were placed in the dish, which was sealed with parafilm and incubated in the dark at 25uC. The insects were observed daily, dead G. mellonella collected and IJs harvested as above. Nematodes were maintained at 13uC in distilled water prior to molecular characterisation.
Isolation and identification of Xenorhabdus and Photorhabdus spp. from nematodes Dead insects were cleaned by immersing in 75% ethanol prior to dissecting on a sterile petri dish. Xenorhabdus and Photorhabdus spp. were obtained by streaking 1 ml of haemolymph onto nutrient bromothymol blue agar (NBTA) [11]. Plates were sealed with parafilm and incubated at ambient temperature (25uC) for 4 days. In an initial pilot study of the first 35 soil samples, colonies of presumptive Xenorhabdus and Photorhabdus spp. on NBTA were selected based on colony morphology and the catalase test [11]. Twelve different colony types were identified which were further characterized using PCR and sequencing of a region of the 16S rRNA gene, as described previously [12,13]. This demonstrated that 3 specific colony types were Xenorhabdus spp. and 4 specific colony types were Photorhabdus spp. Sequence analysis of a region of the 16S rRNA gene confirmed the presence of a region at position 208-211 (E. coli numbering) that distinguished between the two species (Xenorhabdus for TTCG and TGAAAG in Photorhabdus), as described previously [13]. Colony morphology was used thereafter to select colonies for further analysis. Xenorhabdus was characterized based on a dark blue or dark red colony colour with a convex or umbonated surface and swarming on NTBA after 3 to 4 days at room temperature (25uC), and catalase test negative. Photorhabdus was characterized based on a light or dark green colony colour with a convex or umbonated surface on NTBA after 3 to 4 days at room temperature (25uC), and catalase test positive.
Species identification and phylogenetic analysis of all bacterial isolates were subsequently performed on the basis of recA sequence. Total genomic DNA was extracted from a 3 ml LB overnight culture of Xenorhabdus or Photorhabdus spp. using a Genomic DNA Mini Kit (Geneaid Biotech Ltd., Taiwan). A 890 bp region of the recA gene was amplified by PCR using the following primers: recA_F (59-GCTATTGATGAAAATAAACA-39) and recA_R (59-RATTTTRTCWCCRTTRTAGCT-39) [6]. PCR was performed in a total volume of 50 ìl containing 3.5 mM MgCl 2 , 1 mM of each dNTP, 0.5 ìM of each primer, 0.05 unit of Taq DNA polymerase (Promega, USA), and 2.5 ìl of DNA in 16 reaction buffer using a PTC-200 Peltier Thermal Cycler (MJ research INC., Watertown, Massachusetts, USA). PCR cycling parameters were an initial step of 94uC for 5 min, followed by 30 cycles of 94uC for 1 min, 50uC for 1 min and 72uC for 2 min and a final extension of 72uC for 7 min. PCR products were visualized on ethidium bromide stained agarose-gel electrophoresis and purified using Gel/PCR DNA Fragment Extraction Kit (Geneaid Biotech Ltd., Taiwan). Purified PCR products were sequenced by Macrogen Inc. (Korea). Sequences have been deposited in Genbank under accession numbers JQ973956 to JQ974024 for Xenorhabdus spp., and JQ973899 to JQ973955 for Photorhabdus spp. Species identification was performed using a BLASTN search of recA against the NCBI nucleotide database (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi), and the match with the highest similarity score selected. Multiple nucleotide sequences representing all of the known species and subspecies of Photorhabdus and Xenorhabdus spp. were downloaded from the NCBI database, aligned with sequences from the study isolates, and trimmed to a 646 bp region using Clustal W [14] using MEGA software version 5.05 [15]. Maximum likelihood trees were reconstructed using Nearest-Neighbor-Interchange (NNI) and Tamura-Nei model [16] using MEGA software version 5.05 [15]. Bootstrap analysis was carried out with 1,000 datasets.

Molecular characterisation of entomopathogenic nematodes
Genomic DNA was extracted from approximately 50 infective juveniles for each sample, as described previously [17]. Molecular identification of Steinernema and Heterorhabditis spp. was performed by PCR amplification and sequencing of a region of the 28S rDNA gene and two ITS regions of the rDNA gene, respectively. The primers and methods used were as described previously [17,18], with the exception that cycling conditions were modified to eradicate non-specific bands. These were as follows: 28S rDNA gene; 95uC for 5 min, 35 cycles of 94uC for 1 min, 55uC for 30 sec and 72uC for 45 sec, and a final extension at 72uC for 7 min; ITS region of the rDNA; 95uC for 5 min, 35 cycles of 94uC for 1 min, 50uC for 30 sec and 72uC for 1 min, and final extension at 72uC for 7 min. Amplified products were visualized on ethidium bromide stained agarose-gel electrophoresis and purified using a Gel/PCR DNA Fragments Extraction Kit (Geneaid Biotech Ltd., Taiwan). Sequencing was performed by Macrogen Inc. (Korea) and edited using SeqManII software (DNASTAR Inc., Wisconsin, USA). Species identification was performed using a BLASTN search against a nucleotide database (http://www. ncbi.nlm.nih.gov/blast/Blast.cgi).

Data analysis
Statistical analysis was performed using STATA version 11 (Stata Corp, College Station, Tx, USA). The value of soil pH, temperature and moisture were compared between EPN positive and negative groups. These parameters were not normally distributed and were analysed using the Mann-Whitney test. Results were expressed as the median and interquartile range (IQR).

Identification and phylogenetic analysis of Xenorhabdus isolates
The 69 Xenorhabdus isolates were identified as X. stockiae (n = 52), closely related to X. stockiae (n = 14), and closely related to X. miraniensis (n = 3). These findings were replicated in a phylogenetic analysis. A maximum likelihood tree reconstructed using the 69 Xenorhabdus sequences together with sequences downloaded from GenBank is shown in Figure 2. The Thai isolates fell into four distinct groups. Group 1 included 52 study isolates and a sequence from the NCBI database derived from X. stockiae. Group 2 included 13 study isolates but no reference sequence, and group 3 included one study isolate that was most closely related to group 2. The fourth cluster contained the remaining 3 Thai isolates, which were most closely related to X. miraniensis. Geographical mapping of the largest phylogenetic group containing the 52 X. stockiae isolates demonstrated that these were recovered from diverse geographical locations including 7 provinces (Table 2), as were the 13 isolates belonging to group 2.

Identification and phylogenetic analysis of Photorhabdus isolates
The 57 Photorhabdus isolates were identified as P. luminescens (n = 56) and the human pathogenic species P. asymbiotica (n = 1). The P. luminescens isolates were sub-speciated as subsp. hainanensis (n = 45), P. luminescens subsp. akhurstii (n = 8), a sub-species most closely related to subsp. laumondii (n = 2), or a subspecies related to hainanensis and akhurstii but no reference identified in the database (n = 1). The isolate of P. asymbiotica was identified as P. asymbiotica subsp. australis. These findings were replicated in a phylogenetic analysis. A maximum likelihood tree reconstructed using the 57 Photorhabdus sequences together with sequences downloaded from GenBank are shown in Figure 3. A total of 56 sequences clustered with sequences from known P. luminescens isolates, and one Thai isolate clustered with P. asymbiotica and was most closely related to P. asymbiotica subsp. australis. The sequences from the 56 Thai P. luminescens isolates were distributed as groups or single isolates on 4 branches of the tree. Group 1 contained 45 Thai isolates and included sequences belonging to P. luminescens subsp. hainanensis, group 2 contained 8 Thai isolates and a reference sequence for P. luminescens subsp. akhurstii, group 3 contained 1 Thai isolate and no reference sequence, and group 4 contained two Thai isolates that were most closely related to P. luminescens subsp. laumondii. The P. asymbiotica isolate was on a distinct branch of the tree. Geographical mapping of the largest phylogenetic group containing the 45 isolates residing in group 1 demonstrated that these were recovered from numerous provinces ( Table 2).

Molecular characterization of EPNs
Nighty-five EPNs were identified using BLASTN searches of partial sequences of 28S rDNA and ITS, and the relationship described between EPNs and their bacterial symbionts. The remaining 31 EPNs were lost through fungal contamination. The EPNs associated with 62/69 Xenorhabdus isolates are shown in Figure 2. X. stockiae were associated with S. websteri, and X. miraniensis were associated with S. khoisanae. The EPNs associated with 33/57 Photorhabdus isolates are shown in Figure 3. The single isolate of P. asymbiotica was associated with H. indica. The remaining 32 isolates of P. luminescens were associated with H. indica (n = 26), H. sp. SGmg3 (n = 3), H. sp. SGgi (n = 1), H. baujardi (n = 1) and H. bacteriophora (n = 1).

Discussion
The primary objective of this study was to isolate Xenorhabdus and Photorhabdus spp. from a geographical region that has been under-explored, and to phylogenetically characterize these isolates. Additional objectives were to determine the characteristics of the soil from which EPNs were isolated, characterize the EPN phylogeny, and describe the bacterial/EPN partners.
Phylogenetic analysis of 69 Xenorhabdus isolates demonstrated that X. stockiae predominated, with a small number of X. miraniensis (n = 3). Isolation of X. stockiae is consistent with the previous  isolation from the environment in Thailand of X. stockiae strain TH01, the recA sequence of which fell within the largest phylogenetic cluster of 52 X. stockiae isolates identified here. A further 14 isolates were most closely related to X. stockiae, but showed evidence of evolutionary divergence. The Steinernema host species associated previously with X. stockiae isolated in Thailand was reported to be S. siamkayai [6,7], and the relationship found is this study between X. stockiae and the host nematode S. websteri is a new observation. Elsewhere, S. websteri has been reported to be associated with X. nematophila [19]. X. miraniensis has been isolated previously from Australia but not from Thailand [7]. The three X. miraniensis isolates described in this study were from the same subdistrict of Chaiyaphum and were associated with S. khoisanae. This nematode host has been reported previously in association with an unknown species of Xenorhabdus in South Africa [19]. Phylogenetic analysis of 57 Photorhabdus isolates demonstrated that 56 of these were P. luminescens, which could be sub-divided into several subspecies including subsp. hainanensis and subsp. akhurstii, together with isolates that were closely related to subsp. laumondii. P. luminescens subsp. hainanensis and subsp. laumondii have not been isolated previously from Thailand, but P. luminescens subsp. akhurstii was isolated previously from H. indica MP17 in Khon Kaen, from H. indica MP111 in Krabi and from a potentially new Heterorhabditis sp. MP68 in Kanchanaburi, Thailand [8]. P. luminescens subsp. akhurstii was restricted in our study to H. indica, while P. luminescens subsp. hainanensis was isolated from H. indica, H. sp. SGgi and H. sp. SGmg3. Elsewhere, subsp. akhurstii has been found in association with H. bacteriophora (in Iran, Hungary, Argentina and the USA) and H. indica (China) [8], and P. luminescens subsp. hainanensis has been isolated from an unknown Heterorhabditis sp. in China [6]. Isolates of P. luminescens of unknown subspecies were isolated in our study from H. baujardi and H. bacteriophora. H. bacteriophora has been associated previously with P. luminescens subsp. akhurstii, caribbeanensis, kayaii, kleinii, laumondii and luminescens, and P. temperata subsp. cinerea, khanii, stackebrandtii [8]. H. baujardi has been described in Vietnam [20], Brazil [21] and Cameroon [22], and in Brazil was found to carry P. luminescens [23].
A single isolate of P. asymbiotica was also cultured, which was in association with the nematode H. indica. It is likely that a more detailed characterization might assign this nematode to the gerradi subspecies that was shown to vector the Australian isolate, P. asymbiotica Kingscliff [24]. P. asymbiotica is an emerging pathogen that has been reported to cause locally invasive soft tissue infection and disseminated bacteremia. Clinical cases have been identified in both Australia and the USA [5,24]. Our study represents the first reported isolation of P. asymbiotica from the Asian continent, and is an important indicator for the potential for clinical infection with this pathogen.
Our study demonstrated that EPNs could be isolated from diverse soil types in Thailand with a wide temperature, moisture and pH range. Steinernema or Heterorhabditis nematodes positive for Xenorhabdus and Photorhabdus spp. were isolated from 28% and 27% of the 159 sampling sites, respectively. Isolation of Steinernema has been reported from numerous countries across Europe, with rates of isolation varying from 2.2% to 36.8% [25]. Heterorhabditis is distributed throughout North and South America, Australia, Europe, Asia and Africa [6,7]. In Thailand, the host nematode species isolated to date in association with Xenorhabdus and Photorhabdus have been limited to S. siamkayai (associated with X. stockiae [6,7]) and H. indica (associated with P. luminescens [8]). Our     The whole genome sequences of Xenorhabdus and Photorhabdus contain numerous genes encoding proteins and secondary metabolite synthetic enzymes that presumably make compounds that have a role in killing the insect and protection against other invading microorganisms in the infected insect cadaver [26]. The secondary metabolites have diverse chemical structures and a wide range of bioactive properties including antibiotic, antimycotic, insecticidal and nematicidal activity. For example, X. nematophilia produces xenocoumacins [26], and Photorhabdus all produce stilbene derivatives [27]. These genera also produce a range of bioactive protein molecules including the Photorhabdus proteins which have activity against other bacteria such as lumicins [28] or against the insect host, such as Mcf, Tc toxins and the PirAB toxins which show larvicidal activity against the vectors of dengue [29,30]. We propose that the findings from this study could form a starting point for the rational choice of isolates for future studies focused on the discovery of novel antimicrobial and insecticidal compounds.