First report and morphological , molecular characterization of Xiphinema chambersi Thorne , 1939 ( Nematoda , Longidoridae ) in Canada

A Xiphinema species, new to Canada was recovered from rhizosphere of oak trees in Ontario, Canada. Th e identity was confi rmed with morphological and molecular methods as X. chambersi Th orne, 1939. Female bodies are 2.1–2.4 mm long; odontostyle lengths are 110–118 μm; tail 110–177 μm long, arcuate, elongate-conoid, with hyline region 22 43 μm long. Vagina directed about 30 degrees posteriorly. Reproductive system is monodelphic with ovary refl exed anteriorly, vulva opening at 23–26% of the body. Males were not found. Th e 18S and ITS1 sequences of this population had 3–4 bp diff erences (99% identity) and 30 bp diff erences (97% identity) from two Arkansas populations respectively. Th e nematode population had three juvenile stages. Some variations of the morphometrics were observed comparing with the other populations. Th is is the fi rst report of X. chambersi in Canada.


Introduction
Xiphinema chambersi Th orne, 1939 was described from specimens from Virginia, USA.A more complete description of this species was added by Cohn and Sher (1972) based on the lectotypes.In USA, X. chambersi has been reported in 16 states and is widely distributed (Cohn and Sher 1972, Robbins and Brown 1991, Ye 2002, Lamberti et al. 2002).It was also reported in Japan (Shishida 1983) and Korea (Cho et al. 1992).Males were only reported in Arkansas (Ye 2002).Th e Japan (Shishida 1983) and Florida (Lamberti et al. 2002) populations have been reported having 3 juvenile stages, the other reported populations have not been specifi ed on the number of juvenile stages.Although Th orne (1939) described and illustrated a male, no males were seen on the syntype slides.Only 2 males were found in Arkansas (Ye 2002).Th is species has been reported to cause damage on sweet gum (Liquidambar styracifl ua) in Georgia (Ruehle 1971) and strawberry (Perry 1958).During a survey of nematodes of grasslands in Ontario, a population of Xiphinema was discovered.It was identifi ed as X. chambersi.Th e objective of this paper is to report and characterize X. chambersi in Ontario, Canada with morphological and molecular methods.

Materials and methods
Nematodes.Soil samples were collected with a soil probe from the rhizosphere of oak trees (Quercus rubra L.) at the Turkey Point Provincial Park of Ontario (42°42.460'N,80°20.375'W),Canada in 2009.Nematodes were extracted with the modifi ed pan method (Townshend 1963), fi xed in TAF, processed to glycerine and mounted on slides (Hooper 1970) for compound microscopic studies.A portion of the nematodes were frozen at -20°C in water for molecular studies.
Microscopic observation.Specimens were examined using Leica DM5500 B compound microscope using diff erential interference contrast and pictures were taken with Leica DFC 420 digital camera.Th e observed characters of the adults were compared with those of the specimens described by Th orne (1939) and the description by Cohn and Sher (1972).Measurements were made using a Leica micro application system on the images, and dimensions are expressed in a formula suggested by de Man (1880).Drawings were aided using a drawing tube.
SEM. Nematodes were fi rst fi xed in TAF (7% formalin, 2% triethanolamine, 91% distilled water), and then transferred to Seinhorst solution 1 (1% glycerol; 4% formalin: 95% distilled water) in a foam capsule.Th e nematodes then were processed through a serial alcohol dehydration by placing in the foam capsule in succession into 40, 60, 70, 90, and 100% ethanol each for 24 h. the foam capsule with the nematodes was placed into a Polarum E3100 Jumbo II CPD, where critical point drying of the nematodes was accomplished utilizing carbon dioxide.Th e nematodes were then glued on pins using the wood glue as adhesive.Th e pins with nematodes were then placed in an Emitech K550X Sputter Coater and coated for 1 min with gold-palladium.Nematodes were observed using a Philips XL 30 Scanning Electron Microscope.
Molecular study.Two populations of X. chambersi from Arkansas were included for the molecular comparisons.Both ITS and 18S genes of the Ontario population and Arkansas populations were PCR amplifi ed and sequenced.Primers were the same as described in Ye et al. (2004) and Neilson et al. (2004).Th e 25 μl PCR contained 12.5 μl 2X GoTaq DNA polymerase mix (Promega Corporation, Madison, WI 53711, USA), 1 μl each of 0.4-μM forward and reverse primers and 1 μl of DNA template.Th e thermal cycling program was as follows: denaturation at 95°C for 6 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 1 min.A fi nal extension was performed at 72°C for 10 min.PCR products were cleaned by Montage™ PCR Centrifugal Filter Devices (Billerica, MA, USA).PCR primers were used for direct sequencing by dideoxynucleotide chain termination using an ABI PRISM BigDye terminator cycle sequencing ready reaction kit (Applied Biosystems) in an Applied Biosystems 377 automated sequencer (Applied Biosystems) by Eurofi ns MWG Operon (Huntsville, AL35805, USA).Th e sequence was deposited in Genbank.Th e sequence was compared with other nematode species stored at the GenBank using the BLAST homology search program.Th e closest sequences were selected in the phylogenetic analysis.DNA sequences were aligned by Clustal W (http://workbench.sdsc.edu,Bioinformatics and Computational Biology group, Dept.Bioengineering, UC San Diego, CA).Maximal-parsmony (MP) analysis (Saitou and Nei 1987) was conducted on both ITS and 18S.Sites with missing data or gaps were treated as missing characters for the analysis.Th e robustness of the MP tree was tested using the bootstrap method (Felsenstein 1985) based on 2,000 replicates.

Results and discussion
Th e specimens are deposited in the Canadian National Collection of Nematode.Morphometrics data of the females and 3 stages of juveniles are in Table 1.

Morphological characterization
Th e main characters of X. chambersi from Ontario, Canada match well with the lectotype described by Cohn and Sher (1972) and Arkansas populations (Ye 2002) regarding to the body shape, size, the tail shape, hyline tail length, the odontostyle length, and the female gonad.It has only 3 juvenile stages, and no male was found.
Female.Body is C-to L-shaped when relaxed, tapering off gradually at both ends, more so posterior.Lip region is slightly setoff from the rest of body by a faint depression.Amphid aperture is about four-fi fth of the width of lip region.Reproductive system is monodelphic, opistodelphic with ovary refl exed.Vagina directed about 30 degrees angle posteriorly, lips thick.Intestine is transparent, pre-rectum is visible, and rectum is 45 μm long.Tail is 132 μm long, arcuate, elongate-conoid with 3 pairs of caudal pores, terminating in a cylindroid nonprotoplasmic tip with hyline region of 37.4 μm long.
Most of the morphometirc measurements of the females agree with those described by Cohn and Sher (1972) and Ye (2002) in the USA and Japan, some morphometrics variations have been observed: Th e averaged body length of the female of the population in Ontario, Canada is 2.2 ± 0.1 (2.1-2.4)mm is smaller than that of the populations  from Iowa, USA, the Florida population averaged 2.5±0.15(2.4-2.7)μm (Lamberti et al. 2002) the Arkansas population averaged 2.5 (2.1-2.8)μm (Ye 2002) and the Iowa population 2.4 (2.2-2.5)μm (Cohn and Sher 1972); but is larger than that of the population in Japan averaged 1.9 (1.8-2.0)μm (Shishida 1983).Th e odontostyle lengths of females from these populations are similar.Th e hyline tail length of the Ontario population averaged 37.4 ± 6.1 (22.0-43.4)μm is much longer than that of the population   Juvenile.Th e presence of three juvenile stages was determined.Th e body of the fi rst stage juvenile ventrally curved slightly when heat relaxed, the anterior end of the replacement odontostyle almost adjacent to the posterior end of the functional odontostyle, the tail has one pair of caudal pores.Th e body of the second stage juvenile is curved slightly more than the fi rst stage juvenile, the anterior end of the replacement odontostyle is behind the posterior end of the functional odontostyle.Th e tail has 2 pairs of caudal pores.Th e body of the third stage juvenile is ventrally curved slightly more than the second stage juvenile, especially in the tail region, the anterior end of the replacement odontostyle is more posterior to the end of the functional odontostyle.Th e tail has three pairs of caudal pores.Fig. 6 revealed X. chambersi from Canada only has 3 juvenile stages.
Male: not found

Molecular characterization
DNA Sequence: 2610 bp ribosome DNA segment consisting of the near-full-length 18S ribosomal RNA gene, the internal transcribed spacer 1 and partial 5.8S ribosomal RNA gene was PCR amplifi ed and sequenced for Ontario population of X. chambersi (genBank accession number HM138503).Th e 18S sequence of this population had only 3-4 bp diff erences (99% identity) from two Arkansas populations sequenced (AY283174 for population Xiph-41 and HM191718 for population Xiph-61).ITS1 region between Ontario population and Arkansas population Xiph-61 had 30 bp differences (97% identity, 1029 total characters) and 4 gaps.Phylogenetic trees based on the 18S and ITS of rDNA agreed with the results from previous studies (Ye et al. 2004, Neilson et al., 2004) (trees not shown).Both trees revealed the closest relationships being in the same highly supported monophyletic clade for the Ontario population and the Arkansas populations of X. chambersi.Due to the short branch length diff erence in phylogenetic trees and lack of suffi cient morphological diff erences among X.chambersi populations studied, the DNA sequence diff erences on 18S and ITS were considered as intraspecies variation when many Xiphinema species and populations were examined in the phylogenetic trees.

Figure 1 .
Figure 1.Drawings of female of Xiphinema chambersi from Ontario, Canada, A entire body B tail region C gonad D pharynx.

Figure 2 .
Figure 2. Juveniles and female of Xiphinema chambersi from Ontario, Canada, A fi rst stage juvenile B second stage juvenile C third stage juvenile D female.

Figure 3 .
Figure 3.Comparison of tails of Xiphinema chambersi from Ontario, Canada, A fi rst stage juvenile B second stage juvenile C third stage juvenile D female.

Figure 4 .Figure 6 .
Figure 4. Comparison of pharyngeal regions of diff erent stage of juveniles and female of Xiphinema chambersi from Ontario, Canada, A fi rst stage juvenile B second stage juvenile C third stage juvenile D female.

Figure 5 .
Figure 5. Micrographs of Xiphinema chambersi from Ontario, Canada, A SEM image of lip region of female B anus region C head D vulva E tail.