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1 US Department of Agriculture, Agricultural Research Service, Urbana, IL 61801, USA
2 Department of Crop Sciences, Turner Hall, University of Illinois, Urbana, IL 61801, USA
Correspondence
Gregory R. Noel
g-noel1{at}uiuc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and gyrB sequences of ‘Candidatus Paenicardinium endonii’ are DQ314214 and DQ314215.
Maximum-likelihood trees based on 16S rRNA gene and gyrB sequences showing the position of ‘Candidatus Paenicardinium endonii’ within the Bacteroidetes are available as supplementary material in IJSEM Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Wolbachia species are associated with the animal-parasitic nematodes Brugia malayi and Wucheria bancrofti (both Spiruida: Filariidae), which cause lymphatic filariasis and are vectored by mosquitoes, and Onchocerca volvulus (Spiruida: Onchocercidae), which causes onchocerciasis or river blindness and is vectored by black flies (Taylor, 2003; Saint André et al., 2002). Wolbachia species are believed to be the primary cause of acute inflammatory filarial disease and the inflammatory response of the cornea. Members of the nematode genera Steinernema and Heterorhabditis have a mutualistic association with endosymbiotic bacteria of the genus Xenorhabdus in which the nematode host vectors the bacterium into the haemocoel of the insect host, where the bacterium is deposited in the haemolymph, multiplies and kills the insect by inducing septicaemia (Kaya, 1993). Another group of nematode endosymbionts includes three candidate verrucomicrobial species of ‘Candidatus Xiphinematobacter’ associated with three species of plant-parasitic nematodes of the genus Xiphinema (Dorylaimida: Longidoridae) (Vandekerckhove et al., 2000). These nematodes reproduce by thelytokous parthenogenesis and the ‘Candidatus Xiphinematobacter’ organisms are inherited maternally. Another group of bacterial endosymbionts, which have characteristic microfilament-like structures (MLSs) similar to those of ‘Candidatus Cardinium hertigii’, infect the cyst-forming, plant-parasitic nematodes Globodera rostochiensis (potato cyst nematode), Heterodera glycines (soybean cyst nematode) and Heterodera goettingiana (pea cyst nematode) (all Tylenchida: Heteroderidae). These endosymbionts were first observed in females and males of two populations of G. rostochiensis and one population of H. goettingiana during transmission electron microscopy (TEM) studies (Shepherd et al., 1973) and subsequently in H. glycines juveniles (Endo, 1979). The endosymbionts have not been encountered in other populations of these species or in other genera of nematodes. Unlike their allies that infect arthropods (Zchori-Fein et al., 2004), the cyst-nematode-infecting endosymbionts are not known to affect host preference or reproduction or to cause other pathological manifestations (Shepherd et al., 1973). The objectives of our study were twofold. The first was to confirm observations of infection of second-stage juveniles (J2) of H. glycines and to identify sites of infection of the endosymbiont in females and males. The second objective was to characterize the endosymbiont and to determine its phylogenetic position within the bacterial domain by using 16S rRNA and gyrB gene sequences.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. Cysts (dead bodies of females containing eggs) were extracted from soil and added to pots containing steam-sterilized sand in which soybean seeds were planted. The cultures have been maintained in a greenhouse for many years by repeating the extraction and replanting process as needed.
Electron microscopy.
Male nematodes were obtained from infected soybean roots placed in a mist chamber. Vigorous individuals were selected with the aid of a dissecting microscope and placed in distilled water in a BPI dish. The water was drawn off with a micropipette and replaced with cold 2 % glutaraldehyde in 0.08 M sodium phosphate buffer, pH 7.2. Nematodes were cut in half, fixed for 1 week and rinsed with buffer. Heads and tails were placed in separate vials and post-fixed overnight in 1 % osmium tetroxide. Sections were selected individually, placed in triangular slits of excised agar approximately the size of the tip of a beam capsule and then covered with warm agar. Agar blocks containing nematode pieces were excised, dehydrated in an ethanol series (10, 25, 50, 75, 95 and 100 %) and placed in a 50 % solution of ethanol and n-butyl glycidyl ether (BGE) for 1 h. The 50 % solution was replaced with 100 % BGE and held overnight under vacuum. The sections were infiltrated under vacuum for several hours with 50, 75 and 100 % Quetol 654 (Electron Microscopy Sciences). Specimens were placed in fresh 100 % Quetol 654 and infiltrated overnight under vacuum and then embedded in Quetol 654 for 72 h at 70 °C. Females and J2 were prepared as for males except that nematodes were not cut. Ultrathin sections (90–100 nm) were cut with an ultramicrotome (Reichert) and mounted on uncoated grids. Sections were stained with aqueous saturated uranyl acetate for 40 min and with Sato's triple lead stain (Hanaichi et al., 1986) for 1 min. Sections were observed and photographed with a Hitachi H-600 transmission electron microscope.
PCR amplification, cloning and sequencing of the 16S rRNA and gyrB genes.
J2 presumably infected with the bacterial endosymbiont were hatched from eggs and rinsed by repeated centrifugation in sterile water. Nematodes were pipetted onto a column of sterile, moist silica and allowed to migrate through the 5-cm-long column into a reservoir of sterile water. Nematodes were concentrated by centrifugation under sterile conditions and used for extraction of DNA as described by Atibalentja et al. (2004). Briefly, several thousand nematodes in 200 µl sterile water were added to an equal volume of acid-washed 0.150–0.212 mm diameter glass beads (Sigma-Aldrich) in a screw-capped vial and subjected to vibration in a Mini-BeadBeater (BiosPec Products) at 5000 r.p.m. for 2 min. The nematode plus silica slurry was centrifuged at 16 000 g for 5 min and the supernatant was transferred into a 1.6 ml microfuge tube and stored at –20 °C. Combinations of universal primers 27f/1492r (Atibalentja et al., 2004; Lane, 1991) and UP-1/UP-2r (Yamamoto & Harayama, 1995) were used for PCR amplification of the 16S rRNA and gyrB genes, respectively, following procedures described previously (Atibalentja et al., 2004), except that the annealing temperature for the gyrB gene was set at 56 °C. Cloning and sequencing of PCR products were also conducted as reported previously (Atibalentja et al., 2004). Both DNA strands were sequenced from each of three and four clones, respectively, for the 16S rRNA and gyrB genes, and the resulting DNA fragments were assembled with the computer program SEQUENCHER (Gene Codes).
Phylogenetic analysis.
The consensus DNA sequences for the 16S rRNA and gyrB genes were subjected to sequence-similarity searches, using the BLAST algorithm (Altschul et al., 1997). The ORF finder utility of GenBank also was used to identify the putative open reading frame of the gyrB gene, and the deduced amino acid sequence was also submitted to BLAST. Homologous sequences from a selection of bacteria suggested by the BLAST results were retrieved from public databases and aligned with the corresponding sequence from the endosymbiont of H. glycines, using CLUSTAL X (Thompson et al., 1997). The alignments were edited manually to remove terminal and internal gap-rich regions. Phylogenetic analyses were conducted with PAUP* 4.0b10 (Swofford, 2003) under the maximum-parsimony (MP), maximum-likelihood (ML) and distance optimality criteria, following the procedures described by Zchori-Fein et al. (2004) unless indicated otherwise. Specifically, the HKY85 distance and the weighted least-squares objective function were used to construct the neighbour-joining (NJ) tree, which was used to estimate the parameters for ML reconstruction under a general time reversible (GTR) model of nucleotide substitution, with rate heterogeneity among sites (
) and a given proportion of invariable sites (I). The robustness of the inferred clades was assessed through bootstrap analysis, which, for MP and NJ trees, involved 10 000 replicates (of 100 random-addition sequence replicates each for MP). For ML trees, bootstrap analysis involved 1000 replicates of 10 random-addition sequence replicates each for the 16S RNA gene and 100 replicates of one random-addition sequence replicate each for the gyrB gene.
We also sought to identify signature sequences for the 16S rRNA gene of the H. glycines endosymbiont, using a slight modification of the method described by Zchori-Fein et al. (2004). Briefly, candidate oligonucleotides suggested by MacClade (Maddison & Maddison, 2001) were submitted to probe match in the Ribosomal Database Project (RDP II Release 9, http://rdp.cme.msu.edu/html) (Cole et al., 2003) and to BLAST search for short, nearly exact matches in GenBank only after they were selected by PRIMROSE (Ashelford et al., 2002) as oligonucleotide probes that exclusively targeted the 16S rRNA gene sequence of the H. glycines endosymbiont.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). For the most part, our observations confirmed those of Shepherd et al. (1973), who reported that the bacterium was most common in reproductive tissues of females including the ovary, oviduct wall and oocytes. We did not observe bacteria in the hypodermis of females, but found them in the hypodermal chords of males. Our study of J2 was done to confirm the presence of the bacterium in the second-stage infective juveniles. Bacteria were observed in the hypodermal chords, intestinal tissue and pseudocoelom. The research of Endo (1979) was an exhaustive study of the oesophageal region of infective juveniles. Bacteria observed in that study were most abundant in the sarcoplasm of stylet protractor muscles and muscles of the median bulb and the oesophageal glands. In our study, the bacterium was polymorphic in appearance due to the random orientation of individuals within nematode tissues and cells and sectioning of bacteria that often was tangential, especially along the length of the organisms. We found that the diameter of the bacteria ranged from 0.4 to 0.8 µm and the length ranged from 2.5 to 4.5 µm. These values are similar to those reported by Endo (1979), but greater than the values reported by Shepherd et al. (1973) for the endosymbionts in the nematodes G. rostochiensis and H. goettingiana and for ‘Candidatus C. hertigii’ (Zchori-Fein et al., 2004). MLSs were not observed in all bacteria (Fig. 2). It is unclear whether they were absent or in a plane or section that was not studied (Zchori-Fein et al., 2004). In our study and previous studies, some MLSs extended across the width of the cell, whereas some extended part way. The width of MLSs that we determined (17 nm) was similar to that of Endo (1979), but wider than the 14–15 nm reported by Shepherd et al. (1973). These differences might be due to artefacts of fixation and embedding or calibration of instruments. We observed what appeared to be bundles of MLSs in cross section (Fig. 2). However, cell walls were not apparent with these structures. Endo (1979) observed a cross section of MLSs inside a cell with a clearly defined cell wall.
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and Supplementary Fig. S1 available in IJSEM Online). Using the NJ tree under the ML criterion, the nucleotide substitution parameters were estimated as follows: A–C, 1.96; A–G, 7.26; A–T, 1.33; C–G, 0.08; C–T, 10.25; and G–T, 1.00. The proportion of invariant sites (I) was estimated at 0.56 and the shape parameter (
) of the gamma distribution was equal to 0.71.
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), only one, 5'-GGTCTTTAACTGACGCT-3', is present in the 16S rRNA gene sequence of the H. glycines endosymbiont (position 728). This oligonucleotide did not meet the PRIMROSE selection criteria for a probe (Ashelford et al., 2002). In contrast, we have identified two oligonucleotide sequences, 5'-CGCGATACAATCGTGTGTGTCC-3' (position 822) and 5'-CATCCCTAGAAATAGGGAGTTCCGAAA-3' (position 991), that are unique to the H. glycines endosymbiont.
gyrB gene.
A single band of the expected size also was obtained when DNA extracts from H. glycines J2 carrying the unidentified bacterium were amplified by PCR using the primers UP-1 and UP-2r. Cloning and sequencing of the resulting DNA fragment produced a 1237 bp sequence (G+C content 41.7 mol%) that contained a 397 aa partial ORF. BLAST searches using the DNA sequence or the deduced amino acid sequence of the partial ORF retrieved the gyrB gene of the ‘Candidatus Cardinium’ endosymbiont of Encarsia pergandiella (GenBank accession nos. AY332002 and AAQ98790) as the best match with the query sequence, sharing 83 and 81 % nucleotide and amino acid identity, respectively. Phylogenetic analyses using either the nucleotide or the amino acid sequence of the gyrB gene produced similar results for the position of the H. glycines endosymbiont. Therefore, the following discussion focuses only on the DNA-based analyses. The alignment of gyrB gene sequences used comprised 17 taxa and 861 characters, of which 263 were constant and 524 were parsimony-informative. MP analysis yielded two most-parsimonious trees, each 2742 steps long (consistency index 0.43). As was the case with the 16S rRNA gene, the two MP trees not only agreed between themselves but they also agreed with the NJ and the ML trees by placing the H. glycines endosymbiont in a lineage that had diverged earlier from the branch leading to the bacteria in the recently proposed ‘Candidatus Cardinium’ (Fig. 4 and Supplementary Fig. S2). Apparently, the speciation event was followed by a more rapid evolution in the branch containing the H. glycines endosymbiont than in the one leading to the cardinia. Estimates of nucleotide substitution parameters under the ML criterion, based on the NJ tree, were as follows: A–C, 2.36; A–G, 3.75; A–T, 1.20; C–G, 2.17; C–T, 7.66; and G–T, 1.00. The proportion of invariant sites (I) was estimated at 0.26 and the shape parameter () of the gamma distribution was equal to 1.27.
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The status of Candidatus is required by the International Committee on Systematics of Prokaryotes for prokaryotic organisms that are not culturable and thus cannot be characterized according to the International Code of Nomenclature of Bacteria (Murray & Stackebrandt, 1995). ‘Candidatus Paenicardinium endonii’ is Gram-negative and rod-shaped and is classified in the phylum Bacteroidetes, class Sphingobacteria, order Sphingobacteriales and family ‘Flexibacteraceae’ (Garrity & Holt, 2001).
Two 16S rRNA gene oligonucleotide sequences were reported as unique to ‘Candidatus Cardinium’ (Zchori-Fein et al., 2004), but only the sequence 5'-GGTCTTTAACTGACGCT-3' occurs in the H. glycines endosymbiont (position 728). Two additional oligonucleotide sequences, 5'-CGCGATACAATCGTGTGTGTCC-3' (position 822) and 5'-CATCCCTAGAAATAGGGAGTTCCGAAA-3' (position 991), are unique to the bacterium that infects H. glycines and, therefore, are diagnostic of ‘Candidatus Paenicardinium endonii’. The G+C content of the 16S rRNA gene of the bacterium is 52 mol%, compared with 48 mol% for ‘Candidatus C. hertigii’ (Zchori-Fein et al., 2004). ‘Candidatus Paenicardinium endonii’ shares 94 % 16S rRNA gene nucleotide sequence identity with the endosymbiont of Metaseiulus occidentalis and 93 % identity with ‘Candidatus C. hertigii’. The gyrB nucleotide sequence shares 83 % identity with the ‘Candidatus Cardinium’ endosymbiont of Encarsia pergandiella and 81 % identity with ‘Candidatus C. hertigii’. These levels of identity for the 16S rRNA and gyrB genes are indicative of a distinct taxon (Ludwig et al., 1998). The name ‘Candidatus Paenicardinium endonii’ is proposed on the basis of the 16S rRNA gene sequence (GenBank accession no. DQ314214), the gyrB gene sequence (accession no. DQ314215), the signature sequences listed above and the presence of MLSs. The bacterium found in the population of H. glycines from the Ames Plantation near Grand Junction, TN, USA, is the proposed type strain.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Zchori-Fein, E., Perlman, S. J., Kelly, S. E., Katzir, N. & Hunter, M. S. (2004). Characterization of a ‘Bacteroidetes’ symbiont in Encarsia wasps (Hymenoptera: Aphelinidae): proposal of ‘Candidatus Cardinium hertigii’. Int J Syst Evol Microbiol 54, 961–968.
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