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Applied and Environmental Microbiology, November 1998, p. 4246-4254, Vol. 64, No. 11
Institut National de la Recherche Agronomique,
Received 24 February 1998/Accepted 24 August 1998
The genetic diversity of symbiotic Xenorhabdus and
Photorhabdus bacteria associated with
entomopathogenic nematodes was examined by a restriction fragment
length polymorphism analysis of PCR-amplified 16S rRNA genes (rDNAs). A
total of 117 strains were studied, most of which were isolated from the
Caribbean basin after an exhaustive soil sampling. The collection
consisted of 77 isolates recovered from entomopathogenic nematodes in
14 Caribbean islands and of 40 reference strains belonging to
Xenorhabdus and Photorhabdus spp.
collected at various localities worldwide. Thirty distinctive 16S rDNA
genotypes were identified, and cluster analysis was used to distinguish
the genus Xenorhabdus from the genus
Photorhabdus. The genus Xenorhabdus
appears more diverse than the genus
Photorhabdus, and for both genera the bacterial
genotype diversity is in congruence with the host-nematode taxonomy.
The occurrence of symbiotic bacterial genotypes was related to the
ecological distribution of host nematodes.
Xenorhabdus and
Photorhabdus spp.
(Enterobacteriaceae) are symbiotically associated with
the entomopathogenic nematodes (EPNs). Both partners of each
bacterial-helminthic complex act together to kill insect prey by
producing toxins and septicemia. Nematodes reproduce in the insect
cadaver, feeding on the produced bacterial biomass and the insect
tissues metabolized by the bacteria (4). When nematodes are
harvested freshly from soil samples, all of the bacterial
isolations from the intestinal contents of infective juveniles showed
the presence of Xenorhabdus spp. in
Steinernema spp. and Photorhabdus
spp. in Heterorhabditis spp. It has been postulated that
this high specificity is mainly due to the effect of a series of
antimicrobial end products excreted by the symbiont itself during the
multiplication of the nematodes in the parasitized insects
(12). When infective juveniles escape the insect cadaver, they harvest symbiont cells in their intestine, securing among the
generations the perenniality of the symbiotic association between both partners.
Axenic nematodes and symbionts are generally
entomopathogenic by themselves, but the bacterial partner
requires assistance from the nematode to achieve inoculation (the 50%
lethal dose is usually less than 50 cells, depending on the test
insects) (16). In natural conditions, symbionts are
inoculated into the insect hemolymph by their host nematode, which acts
as a living syringe on the target insects. In some symbioses, both
partners must participate after inoculation to achieve pathogenesis, as with, for instance, the Steinernema glaseri-Xenorhabdus
poinarii symbiosis (3).
Xenorhabdus and Photorhabdus
spp. appear to display a high and monophyletic diversity: five
Xenorhabdus species have been described
(X. nematophilus, X. poinarii,
X. bovienii, X. beddingii, and
X. japonicus [6, 23]), and only one
Photorhabdus species has been described
(P. luminescens [11]). However,
within the P. luminescens species several genomic
groups have been recognized by DNA-DNA hybridization (7) and
suggested by 16S rDNA sequencing (20, 31). Thus, the number
of species could be underestimated.
In both genera, identification of new bacterial isolates or species is
difficult because most strains are phenotypically very similar and fail
to give positive results in many classical tests for identification
(10) and because of a lack of sufficient members per
taxon. Consequently, only a few species have been described, and some
of these are represented by only a few isolates (6).
Ecological data relating bacterial symbionts with their nematode host
or their environment remain weak. Thus, studies on the taxonomic
diversity and distribution of members of Xenorhabdus and Photorhabdus spp. are needed. As a first
step, we recently used a PCR-restriction fragment length polymorphism
(RFLP) method applied to the 16S rRNA gene to rapidly identify new
isolates of both genera of symbionts (13). This approach
distinguished Xenorhabdus and
Photorhabdus species and identified groups as effectively as did DNA-DNA hybridization (7, 13). The
method was applied on a limited number of bacterial strains
obtained after isolation from nematode collections. In the current
study, a larger and more comprehensive sampling of symbionts was
undertaken in order to learn more about their ecological distribution
relative to host taxonomy and environmental factors.
An exhaustive sampling was performed among 14 islands in the Caribbean
basin. Caribbean islands are interesting because they are assumed to
present limited soil imports and are subject to the same climate and
because previous studies on native EPNs are available (8, 21,
27). The sampling is based on two surveys: (i) one is an
exhaustive soil collection based on a grid map covering all the seven
Guadeloupe islands (Grande Terre and Basse Terre, Marie-Galante, La
Désirade, Petite Terre, Les Saintes, Saint-Martin, and
Saint-Barthélemy) from which nematodes were trapped to study their distribution (14), and (ii) the other is a collection of nematodes recovered at random from seven other neighboring Caribbean
islands (Martinique, Saint-Vincent, Cuba, Jamaica, Puerto Rico, the
Dominican Republic, and Trinidad and Tobago) (19). The
purpose of this study was to examine the diversity of EPN symbionts by
PCR ribotyping (13) and to correlate the results to nematode
taxonomy throughout the Caribbean islands and to the sampling
environment (soil type, rainfall, elevation, and vegetal covering or
crops) in the Guadeloupe islands. Finally, the isolates were compared
to additional Xenorhabdus and
Photorhabdus strains isolated from nematodes
widely distributed throughout the rest of the world and from human
clinical samples in order to determine whether Caribbean bacterial
symbionts represent distinct genotypes of
Xenorhabdus and Photorhabdus.
Exhaustive nematode sampling in the Guadeloupe islands.
Nematodes were collected from the seven Guadeloupe islands. Exhaustive
soil sampling was conducted between February and December of 1996. To
harvest each sample, three portions of soil were chosen randomly in a
100-m2 area. Each portion consisted of a core 5.5 cm in
diameter at a 25-cm depth (ca. 0.6 dm3 of soil). The three
portions were mixed, giving 1.8 dm3 from which 0.6 dm3 was used for recovering the nematodes as previously
described (14). In all, 538 soil samples were collected
according to a square grid with points spaced at 2-km intervals and
covering the whole surface of the Guadeloupe islands. EPNs were
isolated by using the Galleria trap technique (9)
and identified by using morphological criteria, isozyme analysis, and
satellite DNA probes (17, 19). The presence of nematodes was
related to site location, elevation, rainfall, soil type, and
vegetation (14).
Random sampling in other Caribbean islands.
Nematodes were
also collected from the seven other Caribbean islands listed above. In
contrast to the exhaustive Guadeloupe survey, these samples were
collected randomly from a variety of ecosystems, including croplands,
orchards, grasslands, salt marches, and forests. On Martinique,
Saint-Vincent, Trinidad and Tobago, and Jamaica, samples were collected
by the Institut National de la Recherche Agronomique (INRA) laboratory
in Guadeloupe. From the three other Caribbean islands, nematodes were
collected and identified by E. Arteaga and M. Montes (Ministerio de
Agricultura, Estación Nacional de Sanidad de los
Cítricos, La Habana, Cuba), W. Figueroa (University of Puerto
Rico, Río Pedras, Puerto Rico), and L. Garrido and A. Carro
(Universidad Autónoma de Santo Domingo, Engombe, Santo Domingo).
After delivery of the biological material, the taxonomic position of
this nematode collection was checked again (19) by using the
methods mentioned above.
Bacterial isolates and reference strains.
Individual
bacterial colonies were isolated from the infective stages by the
hanging-drop technique (25). We examined 77 isolates from
the Caribbean area, including 31 strains from the Guadeloupe islands
and 46 strains from the remaining Caribbean islands (Table
1). To identify the isolates, their
phenotypic properties and RFLP patterns of amplified 16S ribosomal DNA
(rDNA) were compared to those of 27 reference strains previously
studied (10, 13). Thirteen other reference strains,
including seven Xenorhabdus and six
Photorhabdus strains, were added (Table
2). The
seven new Xenorhabdus strains included strain USFL52
of X. bovienii, strain SK72 of X. poinarii, and five other Xenorhabdus spp. that
originated from different parts of the world and from new species of
host nematodes, i.e., Steinernema monticolum
(29), S. scapterisci, S. serratum, S. kushidai, and S. riobrave (Table 2). Five opportunistic
Photorhabdus spp. isolated from human patients
at the Centers for Diseases Control (Atlanta, Ga.) (15) and
strain Q614, which is the only known nonluminescent
Photorhabdus strain, were also examined
(5).
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
PCR-Ribotyping of Xenorhabdus and
Photorhabdus Isolates from the Caribbean Region
in Relation to the Taxonomy and Geographic Distribution of Their
Nematode Hosts
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
TABLE 1.
List of the 77 bacterial isolates used in this study
TABLE 2.
List of the 43 Xenorhabdus,
Photorhabdus, and other
Enterobacteriaceae strains used as references in RFLP
analysis of 16S rRNA genes
Phenotypic characterization. To verify in a preliminary step that the isolates belonged to the Xenorhabdus and Photorhabdus genera, we used conventional phenotypic criteria (10) and compared the results with reference strains. All of the tests were conducted at 28°C. Cellular morphology and motility were assessed by microscopic examination of 24-h-old nutrient broth cultures. Dye adsorption of bromothymol blue was tested on nutrient agar supplemented with 0.004% (wt/vol) triphenyltetrazolium chloride and 0.0025% (wt/vol) bromothymol blue (NBTA medium) for Xenorhabdus isolates (2), and dye adsorption of neutral red was tested on MacConkey agar for Photorhabdus isolates (10). Antimicrobial activity was determined by the method of Akhurst (1) with Micrococcus luteus as the indicator microorganism. Other tests included the use of API 20E and API 20NE strips (Biomerieux, Craponne, France); catalase; bioluminescence; phospholipase (lecithinase); lipolysis on Tween 20, 40, 60, 80, and 85; and pigmentation (10).
Nucleic acid extraction.
Cells were grown on nutrient agar
plates for 48 h at 28°C, scraped off in TE8 buffer (50 mM
Tris-HCl, 20 mM EDTA; pH 8), and centrifuged in a microcentrifuge tube
for 2 min at 10,000 × g. The cell pellets were washed
twice in TE8 buffer and stored at 
20°C. DNA extraction was
performed with the nucleic acid extraction kit Isoquick (ORCA Research,
Inc., Bothell, Wash.) according to the rapid DNA extraction protocol of
the manufacturer. The DNA pellets were dissolved in 100 µl of pure
water and diluted 20- to 100-fold to be used as templates for PCR.
16S rDNA restriction analysis. 16S rDNAs were amplified by using the primers and reaction conditions previously described (13). For each isolate, 6 to 17 µl of amplified 16S rDNA was digested overnight with 5 U of restriction endonuclease (GIBCO BRL, Cergy-Pontoise, France). PCR products of Xenorhabdus isolates and reference strains were digested separately with six tetrameric endonucleases previously found to produce polymorphic digests (13): CfoI, HinfI, DdeI, AluI, HaeIII, and MspI. Amplified DNAs from the Photorhabdus isolates were digested with three endonucleases (AluI, CfoI, and HaeIII) which were sufficient to generate all the genotypes of Photorhabdus strains previously studied (13). DNA digests were then analyzed by horizontal electrophoresis at 6 V/cm in 3% (wt/vol) Nusieve or Metaphor agarose (Tebu, Le Perray en Yvelines, France) gels in 0.5× TBE buffer (44.6 mM Tris-base; 44.6 mM boric acid; 1 mM EDTA; pH 8) containing 0.5 mg of ethidium bromide per liter. The gels were visualized under UV light with an imager (The Imager, software version 2.03; Appligene, Inc., Strasbourg, France). Genetic relationships between two amplified 16S rRNA genes were evaluated by determining the presence or absence of DNA restriction fragments of a given length. Dice's similarity coefficient, based on the proportion of shared restriction fragments, was calculated, and the distance matrix was determined by the Nei and Li method (22). Distance values were displayed as a dendrogram by using the unweighted-pair-group method with arithmetic means (UPGMA) (28).
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Isolation and phenotypic characterization of bacterial symbionts. All bacterial isolates from the Guadeloupe and other Caribbean island surveys shared the common phenotypic properties with all reference strains (data not shown) and therefore belonged to Xenorhabdus and Photorhabdus genera (10).
Of the 538 soil samples collected from the seven Guadeloupe islands, 31 (5.8%) contained Heterorhabditis nematodes (Fig. 1), from which 31 Photorhabdus strains were isolated (Table 1). No Steinernema spp. were found, and therefore no Xenorhabdus spp. were obtained from the Guadeloupe islands. Of the Heterorhabditis nematodes, 27 were H. indica (87%), three were H. bacteriophora (10%), and one was not identified (3%) (Table 1). H. indica was mainly located in coastal areas and rarely inland (Fig. 1): 25 isolates originated from soil marshes, sandy beaches, and the slopes of a limestone cliff (soil pH, 8.0 to 9.3; elevation, 0 to 75 m), and 2 isolates originated from pastures (vertisol at pH 6.5 to 7.5; elevation,
240 m). The three
H. bacteriophora were found in cropland soil (vertisol
at pH 6.5 to 7.5; elevation, 25 m), orchard soil (sand at pH 9;
elevation, 25 m), and rainforest soil (oxisol at pH 5.5;
elevation, 350 m). No other inland areas provided any EPNs (Fig. 1).
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, H. indica; 
,
H. bacteriophora; and 
, Heterorhabditis
sp. The symbiont strain and genotype number are shown beside each
symbol.
Amplified 16S rDNAs and RFLP data. In all, 72 Photorhabdus, 5 Xenorhabdus, and 43 reference strains (including 20 Xenorhabdus and 20 Photorhabdus spp. and 3 other genera of Enterobacteriaceae) were further investigated by PCR ribotyping. 16S rDNA genes of all 120 strains were amplified by using PCR primers representing regions of the 16S rDNA conserved in bacteria. All of the strains produced a single band of about 1,600 bp. Polymorphic restriction patterns were obtained with the six endonucleases used. Results of the Photorhabdus and Xenorhabdus patterns are presented in Fig. 2 except for the CfoI patterns that were the same as those shown previously (13). By combining all of the restriction patterns, the 120 strains could be grouped into 33 genotypes (Table 3).
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RFLP analysis of Photorhabdus isolates and reference strains. The 72 Caribbean Photorhabdus strains were divided into four genotypes (numbered 12, 13, 27, and 28 in Table 1) based on their RFLP patterns (Fig. 2).
A subsample of eight Photorhabdus isolates belonging to each of the four genotypes defined above was typed with three additional tetrameric enzymes (HinfI, MspI, and DdeI). No more polymorphism was observed. RFLP patterns from the Caribbean strains were compared to those of 20 Photorhabdus reference strains, of which 14 had been previously typed (13). The six new reference strains (1216-79, 2407-88, 2617-87, 3105-77, 3265-86, and Q614) were separated into two newly defined genotypes (Table 2): genotype 29 included five opportunistic Photorhabdus clinical strains, which were all identical, and genotype 30 included the unique strain Q614 from Australia. Thus, among all the P. luminescens strains tested, 12 genotypes were defined (genotypes 10 to 17 and 27 to 30 [Table 3]), 4 of which were novel. Three restriction enzymes (AluI, CfoI, and HaeIII) were sufficient to resolve them. Three of the four genotypes observed in Caribbean Photorhabdus isolates were also observed among Photorhabdus reference strains originating from other parts of the world: genotype 12 was represented by the reference strain IS5, a symbiont of H. indica from Israel; genotype 27 matched the genotype of strain D1, a symbiont of H. indica from Australia; and genotype 13 corresponded to those of the symbiotic strains HP88 and K80 from H. bacteriophora and Heterorhabditis sp., respectively. In contrast, genotype 28 was specific to two Photorhabdus isolates from the Guadeloupe islands and was not observed among the reference strains.RFLP analysis of Xenorhabdus isolates and reference strains. The amplified 16S rDNAs from the five Caribbean Xenorhabdus isolates (CU01, JM26, PR06-A, VC01, and FRM16) were analyzed with six endonucleases and were compared to the genotypes of 20 Xenorhabdus reference strains, of which 13 had been analyzed previously and shown to belong to nine 16S rDNA genotypes (13). By combining the different restriction patterns (Fig. 2; Table 3), each of the five Caribbean isolates belonged to a distinct 16S rDNA genotype, four of which were novel (Table 1). The seven newly investigated Xenorhabdus reference strains (SK72, USFL52, JP02, KR1, UY61, CN01, and USTX62) were grouped into seven different 16S rDNA genotypes (named genotypes 3, 6, 18, 22 to 24, and 26), five of which were novel (Table 2). In all, 18 Xenorhabdus genotypes were defined, half of which were novel (Table 3). The Caribbean isolate CU01, obtained from S. cubanum, and the reference strain SK72, a symbiont of S. glaseri, were identical to the type strain (G6T) of the species X. poinarii. The new reference strain USFL52 from Florida exhibited the same pattern as T228T, the type strain of X. bovienii.
Genetic relationships between amplified 16S rRNA genes. Comparison of the restriction profiles obtained with Photorhabdus and Xenorhabdus isolates revealed 30 distinctive genotypes. The three additional genotypes in Table 3 correspond to the other Enterobacteriaceae. To estimate the genetic relationships between PCR-amplified 16S rDNAs, we calculated a matrix of pairwise genetic distances for the 33 genotypes defined with the six restriction enzymes (Table 3). A mean of 33 restriction fragments per genotype was analyzed. The distance matrix was used to construct a dendrogram based on a UPGMA algorithm (Fig. 3). Twenty-three of the 16S rDNA genotypes were represented by only one to two strains, and the remaining seven were represented by multiple strains (Fig. 3). Two major groups (I and II) were delineated at a genetic distance of 0.038 and corresponded to Xenorhabdus and Photorhabdus genera, respectively. As expected, the three other Enterobacteriaceae genera branched apart from these two groups.
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Bacterial genotype distribution in relation to host nematodes and geography. We isolated 72 Photorhabdus and 5 Xenorhabdus spp. from the Caribbean basin. Among the Photorhabdus spp. four genotypes were identified. All of the 63 isolates of the genotypes 12 and 27 originated from H. indica. The two reference strains IS5 and D1, isolated from H. indica, also shared these genotypes. Genotype 12 was therefore the most prevalent (56 of 63 isolates) of the Photorhabdus genotypes, and it occurred throughout the Caribbean region. It was restricted to the coastal areas in the Guadeloupe islands (Fig. 1), but it also occurred inland in the Dominican Republic and Puerto Rico. Genotype 27 was rare and found only in Petite Terre, Jamaica, Martinique, and in the northern Guadeloupe islands (Saint-Martin and Saint-Barthélemy).
All seven isolates of genotype 13 originated from H. bacteriophora and matched the reference H. bacteriophora strain HP88. Three other genotypes (genotypes 10, 16, and 28) also were found in isolates from H. bacteriophora (strains Hb, C1, and FRG29). Genotype 28 was new, was restricted to Guadeloupe, and was not detected among the reference strains collected in the rest of the world. Steinernema spp. were very scarce in the Caribbean basin and therefore could not be related to geographical origins. The five Xenorhabdus isolates corresponded to five different genotypes, and each of them was isolated from a different species of Steinernema: S. cubanum, S. bicornutum, S. puertoricense, Steinernema sp.1, and Steinernema sp.2, the two latter being new species not yet described.| |
DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Based on RFLP analysis of 16S rDNA from bacterial symbionts, host nematode characterization, and the geographical distribution of genetic bacterial groups, we described here the genetic composition of EPN symbiotic bacteria in the Caribbean basin and compared them to symbiotic strains collected at various localities throughout the world.
The sampling intensity allowed us to estimate a higher degree of diversity compared with the Xenorhabdus and Photorhabdus strains studied previously. Thirteen new genotypes were detected and successfully added to the 17 previously defined ones (13); this was done without substantially altering the phylogenetic relationships established previously by clustering analysis. Thus, PCR-RFLP analysis applied to 16S rDNA proved to be a rapid and sensitive typing method for distinguishing strains of the Xenorhabdus and Photorhabdus genera. Because we found new genotypes and new restriction patterns among Xenorhabdus and Photorhabdus spp., the number of endonucleases required to generate all of the genotypes has to be reconsidered. HaeIII, CfoI, and AluI have to be used to differentiate all of the Photorhabdus genotypes, and five restriction enzymes (CfoI, HinfI, MspI, HaeIII, and DdeI) are necessary in order to distinguish all of the Xenorhabdus genotypes.
The addition of new genotypes in the 16S rDNA clustering analysis revealed an unusually high level of genetic diversity within the Xenorhabdus genus compared to previous descriptions (13, 20, 26, 30, 31). This development likely resulted from the large number of studied strains originating from 16 identified Steinernema species and from various localities worldwide. For instance, S. arenarium, S. bicornutum, S. scapterisci, and S. serratum, whose symbionts were not previously typed, proved to harbor divergent Xenorhabdus symbionts that were distantly related to described species. Most of the genotypes were so divergent that they may represent new species. However, due to a lack of similar bacterial isolates, new Xenorhabdus species could not be described. Compared to 16S ribosomal sequencing studies (20, 30, 31), the phylogenetic position of the symbiont of S. kushidai, which is closely related to X. nematophilus, was corroborated, whereas the phylogenetic position of the symbiont of S. riobrave was different. Because novel Xenorhabdus strains were detected, the complete 16S rRNA genes should be sequenced in order to refine the phylogenetic tree of the Xenorhabdus genus.
Clustering analysis of the Photorhabdus genotypes revealed two major subgroups corresponding to the host nematodes and their ecological data. The first subgroup, II-a, included symbionts of H. indica and H. bacteriophora, which were found in the Caribbean and other tropical regions, whereas the second subgroup, II-b, included symbionts of H. megidis and H. zealandica, which were limited to the temperate regions. Previous 16S rDNA sequencing analyses corroborate the delineation between symbionts of H. indica and H. bacteriophora and the symbionts of H. megidis (20, 31). Moreover, within subgroup II-a, the similarity between symbionts of H. indica and H. bacteriophora is also corroborated by ribosomal sequencing (20).
Because of a higher number of isolates and a precise identification of their symbiotic nematodes that were not available in our previous data (13), a clear relationship between 16S rDNA genotypes and Heterorhabditis species origins was detected. Thus, Photorhabdus genotypes 12 and 27 were exclusively associated with H. indica, whereas Photorhabdus genotypes 13 and 28 were only associated with H. bacteriophora. Yet in two cases (strains HbT and C1) an inconsistency was observed. HbT and C1 were isolated from nematodes initially named H. bacteriophora but which are now known to differ from the typical H. bacteriophora represented by HP88 (18). The native host-nematodes of HbT and C1 may belong to two distinct species or subspecies that are different from the species of H. bacteriophora associated with genotypes 13 and 28. Because the identification of Heterorhabditis spp. is difficult, the characterization of their bacterial symbionts may help resolve some difficult taxonomic questions regarding their hosts.
Geographical grouping of the Photorhabdus genotypes was linked to nematode distribution. Bacterial genotypes associated with H. indica are restricted to tropical areas as is their host H. indica (24), whereas genotypes associated with H. bacteriophora seem to be more homogeneously distributed, as is their host H. bacteriophora (18). Furthermore, in the Guadeloupe islands, most of the H. indica isolates were found in coastal sandy soils, and all of the H. bacteriophora were found in the vertisols of croplands and the oxisols of forests. These results agree with previous studies indicating that Heterorhabditis spp. mainly occur in coastal areas (18). However, some H. indica nematodes were isolated from inland soils in Puerto Rico and the Dominican Republic, possibly as a result of soil material transfer on these relatively developed islands. Both genotypes associated with H. indica (genotypes 12 and 27) were spread throughout the Caribbean basin, suggesting that the host species is the predominant determinant of geographic distribution. To evaluate more accurately the selective pressure applied by the host nematode versus those that might be applied by soil factors on symbiont populations, further studies on the possible occurrence of free-living Xenorhabdus and Photorhabdus isolates in soil are required.
The high degree of Xenorhabdus diversity is congruent with the wide diversity of associated Steinernema nematodes and, with only one exception, the genotypes reflect the nematode host species. This exception is represented by the genotype of X. poinarii, which is associated with two nematode species: S. glaseri and S. cubanum. These two nematode species are closely related because they share morphological and ITS-based similarities (18). A complementary study of the symbionts by phenotypic characterization and DNA-DNA hybridization is in progress to confirm this finding. If verified, this would be the second reported case of a Xenorhabdus species associated with different Steinernema species, X. bovienii associated with S. feltiae, S. kraussei, and S. affine that occur in the same region and environment (11).
Molecular tools, such as 16S rDNA PCR-RFLP analysis for bacterial typing, along with satellite DNA probes and isozyme analysis for EPN identification, are fast and accurate ways of comparing bacterium-nematode associations on a large geographical scale. A high level of taxonomic congruence has been detected by using this approach between symbiont pairs, a finding that supports an early coevolution of these symbioses. The perenniality of the association may have resulted from the vertical transmission of symbiotic bacteria during monoxenic sepsis produced during parasitism and from an early intestinal contamination of infective juveniles escaping insect cadavers. Each species of nematode seems to secure a very restricted microbial niche that is more or less specific to a particular Xenorhabdus or a Photorhabdus species.
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ACKNOWLEDGMENTS |
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The technical assistance of Eliane Bonifassi and Anne Lanois for the isolation and first characterization of the reference symbionts is gratefully acknowledged. We also appreciate being able to use the biological material provided by Eva Arteaga, Enrique Cabanillas, Agueda Carro, Wilfredo Figueroa, Luis Garrido, Nelson Simões, Grover Smart, and Patricia Stock. We thank Alan Kirk for revising the English of the manuscript.
This work was supported by the MENRT grant 95-5-10697.
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FOOTNOTES |
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* Corresponding author. Mailing address: Laboratoire de Pathologie Comparée, C.P. 101, Université Montpellier II, 34095 Montpellier Cedex 5, France. Phone: 33-4-67143740. Fax: 33-4-67144679. E-mail: boemare{at}ensam.inra.fr.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Applied and Environmental Microbiology, November 1998, p. 4246-4254, Vol. 64, No. 11
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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