Nematode samples and DNA extraction: The species used in this study are listed in , along with the geographic origin and host plant of the populations. Three populations of H. stephanus, including one from Dillsburg, Pennsylvania (on Kentucky bluegrass) and two from the campus of Clemson University, Clemson, South Carolina (one on dogwood and one on river birch) were used for the morphological and molecular characterizations. Two populations of H. columbus from South Carolina (one from a cotton field and one from a soybean field), and two populations of H. galeatus from South Carolina (one on St. Augustine grass and one on bermudagrass) were also included in the analyses. Forty-seven original sequences generated in this study were added to GenBank, but publicly available sequences from other authors for several Hoplolaimus species and two outgroup species were included for comparison. The accession numbers for all sequences included in this study are listed in .
Species, site of initial recovery, plant host, and GenBank Accession number for populations used in this study.
DNA was extracted from individual nematodes hand-picked from each population, using Sigma Extract-N-Amp kit (XNAT2) (Sigma, St. Louis, MO). The manufacturer's protocol was modified by reducing all volumes to one eighth of the recommended amounts. One nematode was placed into a 0.2 ml centrifuge tube containing 12.5 μl of the kit's Extraction Solution. The nematode was then crushed using the tip of a <10 μl pipette tip, followed by adding 3.5 μl of the kit's Tissue Prep solution to the tube. The tube was then vortexed, followed by a brief centrifugation to collect contents. Tubes were then incubated at 55 °C for 10 minutes, followed by incubation at 95 °C for 3 minutes. Next, 12.5 μl of the kit's Neutralization Solution was added to each tube. The extracted DNA was used for PCR or stored at -20°C.
Morphological characterizations: At least 8 females and 4 males from each population were used for the morphological diagnosis. Measurements and observation of morphological diagnostic characters were made using temporary mounts in water and with an Olympus BX60 microscope equipped with the software iSolution Lite (Image and Microscope Technology i-Solution, Inc.).
Scanning electron microscopy (SEM) observations were conducted on a Hitachi Analytical Tabletop Microscope TM3000. Male and female specimens of H. stephanus and H. galeatus were fixed in 2.5% glutaraldehyde for at least 2 hours, then passed through a graded series of ethanol dehydration (25%, 50%, 75%, 90%, 95% , 100% ethanol, 15 min each), followed by critical point drying with hexamethyldisilazane (HMDS) and platinum coating prior to examination.
Molecular characterization of populations was conducted using species-specific primers and methodology as described by Bae et al. (2009)
for discrimination of H. galeatus, H. columbus,
and H. magnistylus
H. stephanus species-specific primer design:
The species-specific primers for H. stephanus
were designed using comparative ITS1 region sequence alignments () of Hoplolaimus columbus, H. galeatus, H. magnistylus, H. seinhorsti, H. concaudajuvencus
and H. stephanus
(gi226431011, gi186920200, gi186920202, gi186920203, gi186920204, H. stephanus
SCST01). Five putative sets of primers were designed with the Primer-Blast application on NCBI (http://www.ncbi.nlm.nih.gov/tools/primer-blast
). After testing under a range of annealing temperatures (54-61°C), species-specific primer pair Hs-1f (5’- CCTGCCTTGGGGGTCGCTTG-3’) and Hs-1r (5’- GCCAGTGTGTTCCGCTCGCA-3’) were chosen and optimal PCR reactions with these primers were performed with annealing temperature of 60°C.
Partial ITS1 region sequence alignment of five Hoplolaimus species. The highlighted portions indicate variation among the five species, used to design Hoplolaimus stephanus species-specific primer pair.
PCR Amplification of actin, ITS1 and LSUD genes:
The actin region was amplified by the primers Act1-f (5’-CCAAATCATGTTCGAGACGTT-3’) and Act1-r (5’-GAACATAGCCTCTGGGCAAC-3’). These primers were designed using comparative sequence alignments of Heterodera cynodontis, Heterodera avenae, Heterodera schachtii, Heterodera latipons, Heterodera glycines
and Caenorhabditis elegans
from GenBank (Accession numbers gi167472950, gi41387727, gi41387723, gi41387721, gi26422171, gi18314322, gi133952034). The best putative primers were selected using the Primer-Blast application of NCBI (http://www.ncbi.nlm.nih.gov/tools/primer-blast
). Amplifications were performed in 20μl reactions, each containing: 5 μl PCR-grade water, 10 μl of ReadyMix TAq PCR Reaction Mix with MgCl2
(Sigma, St. Louis, MO) (20 mM Tris-HCl pH 8.3, 100 mM KCl, 3 mM MgCl2
, 0.002% gelatin, 0.4 mM dNTP mixture (dATP, dCTP, dGTP, and dTTP), and 0.06 units of Taq DNA Polymerase/μl), 1.0 μl of each primer (20 μM), and 2 μl of DNA template. The PCR reactions were performed on a PTC-100 Peltier thermal cycler (MJ Research, Inc. Watertown, Massachusetts) with the following run parameters: one initial denaturation cycle at 95°C for 3 min, followed by 36 cycles at 95°C for 45s, 46°C for 1.5 min, 72°C for 2 min and final extension at 72°C for 10 min.
The ITS1 and LSU-D region were amplified using the primers Hoc-1f (5’-AACCTGCTGCTGGATCATTA-3’), Hoc-2r (5’-CCGAGTGATCCACCGATAA-3’), LSUD-f (5’-ACCCGCTGAACTTAAGCATAT-3’) and LSUD-r (5’-TTTCGCCCCTATACCCAAGTC-3’), respectively, designed by Bae et al. (2008)
. PCR reactions were performed as described above, using the following parameters: an initial denaturation cycle at 95°C for 3 min, 36 cycles at 95°C for 45s, 59°C for 1.5 min, 72°C for 2 min, and a final extension cycle at 72°C for 10 min. Each reaction included a negative control without DNA template. After amplification, 5 μ
l of each reaction were loaded onto a 1.5% agarose gel (120 V, 50 min) and photographed under UV light. At least three replicates were performed for each population-primer set combination. PCR products were purified and concentrated with Bio-Rad PCR Kleen Spin Columns (Bio-Rad, Hercules, CA). Purified DNA was sent to the Clemson University Genomics Institute (Clemson, SC) for direct sequencing in both directions. Amplification primers were used as sequencing primers. The sequences were edited and aligned using BioEdit 7.0 (Hall, 1999
Sequences of ingroup and outgroup taxa were aligned using ClustalW (Thompson et al., 1994
). Sequences of Globodera rostochiensis
and Heterodera glycines
were used as the outgroup taxon for actin, ITS1 and 28S region (). The files were converted from FASTA to NEXUS format using DNA Sequence Polymorphism Version 5.10.01 (DnaSPv5) (Librado and Rozas, 2009
). A best fit model of nucleotide substitution was selected using the GTR+I+G model with the Akaike Information Criterion (AIC) among 56 different models using ModelTest v 3.7 (Posada and Crandall, 1998
). The Akaike-supported model, the base frequencies, the proportion of invariable sites, and the gamma distribution shape parameters and substitution rates were used in phylogenetic analyses. Bayesian inference was implemented for each gene separately using MrBayes 3.1.2 program (Huelsenbeck and Ronquist, 2001
) running the chain for 100,000 generations with the Markov Chain Monte Carlo (MCMC) method, a sample frequency of 10 and burn-in value of 250. We estimated the posterior probabilities of the phylogenetic trees (Larget and Simon, 1999
) using the 50% majority rule. The phylogenentic trees were viewed with TreeView1.6.6 (Page, 1996