Characterization of Vittatidera zeaphila (Nematoda: Heteroderidae) from Indiana with molecular phylogenetic analysis of the genus

Cysts, white females, and J2 were obtained from soil and roots associated with corn plants from Spencer County, Indiana. Juveniles were separated from soil by sieving and Baermann funnel extraction or were collected from cysts removed from fresh roots and kept in water in watch glasses. Juveniles were fixed in 3% formaldehyde and processed to glycerin with a formalin glycerin method (Hooper, 1970; Golden, 1990). Females and some cysts were removed from roots after fixation for 12 hr in 3% formaldehyde solution.

Photomicrographs of cyst vulval cones, and J2 were made with an automatic 35-mm camera attached to a compound microscope having an interference contrast system. Roots and whole cysts were photographed under a dissecting microscope Nikon SMZ18, and light microscopic images of fixed nematodes were taken on a Nikon Eclipse Ni compound microscope using a Nikon DS-Ri2 camera. Measurements were made with an ocular micrometer on a Leica WILD MPS48 Leitz DMRB compound microscope. All measurements are in micrometers unless otherwise stated.

The molecular identification was performed using DNA extracted from single nematodes as template in PCR reactions. Single juveniles were mechanically disrupted with sharp forceps tips in 20 µl nematode extraction buffer (500 mM KCl, 100 mM Tris-Cl (pH8.3), 15 mM MgCl₂, 10 mM dithiothreitol (DTT), 4.5% Tween 20 and 0.1% gelatin) (Thomas et al., 1997) and stored at −80°C until needed. To prepare DNA extracts, frozen nematodes were thawed, 1 µl proteinase K (from 2 mg/ml stock solution) was added, and the tubes were incubated at 60°C for 60 min, followed by 95°C for 15 min to deactivate the proteinase K. Two or five microliters of extract were used for each PCR reaction.

ITS: Amplification of the internal transcribed spacer region ITS1&2 rDNA contained 0.2 µM each primer, TW81 (Joyce et al., 1994) and AB28 (Howlett et al., 1992), 1.5 mM MgCl₂, 0.2 mM dNTPs, 1U Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA), 3 µl nematode DNA extract, and supplied enzyme reaction buffer in a total volume of 25 µl. Cycling included one step of 95°C for 2 min, followed by 35 cycles of 95°C for 30 sec, 55°C for 30 sec, and 72°C for 90 sec, finished with one cycle at 72°C for 5 min (Skantar et al., 2007).

28S: Amplification of the 28S large ribosomal subunit (LSU) D2-D3 expansion segment included the primers D2A [5′-ACAAGTACCGTGAGGGAAAGTT-3′] and D3B [5′-TCGGAAGGAACCAGCTACTA-3′] and were amplified as previously described (De Ley et al., 2005; Ye et al., 2007).

18S: The 18S (small subunit: SSU) sequence was amplified with primers in one fragment with forward primer 18S-CL-F3: [5′-CTTGTCTCAAAGATTAAGCCATGCAT-3′] (Carta and Li, 2018) and reverse primer 1912R according to Holterman et al. (2006). Reactions contained 2 µl nematode DNA extract, 0.5 μl 10 μM primers, 0.5 μl 10 mM dNTP, 1U Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA), 0.75 μl 50 mM MgCl₂, and 2.5 µl PCR buffer in a total volume of 25 µl. PCR cycling conditions were 95°C for 3 min, 35X (94°C for 30 sec, 50°C for 40 sec, 72°C for 70 sec), 72°C for 5 min, 4°C until finish.

COI: Mitochondrial cytochrome oxidase I (COI) was amplified with primers Het-coxiF [5′-TAGTTGATCGTAATTTTAATGG-3′] and Het-coxiR [5′-CCTAAAACATAATGAAAATGWGC-3′]. Amplifications were performed in 25 µl reactions with 1x PicoMaxx (Agilent) buffer, 0.2 mM dNTPs, 0.3 µM each primer, 0.125 µl Dream Taq DNA Polymerase (Thermo Fisher), 0.5 µl PicoMaxx Taq, and 3 µl DNA extract. Cycling conditions were as described previously (Subbotin, 2015).

Hsp90: Heat shock protein 90 (Hsp90) fragments were amplified with degenerate primers U288 [5′-GAYACVGGVATYGGNATGACYAA-3′] and L1110 [5′-TCRCARTTVTCCATGATRAAVAC-3′] (Skantar and Carta, 2004). Cycling was performed with 1× PicoMaxx reaction buffer, 0.2 mM dNTPs, 1.5 mM MgCl₂, 0.3 µM each primer, 1.25 U PicoMaxx Taq, 1 U Platinum Taq, and 3 µl nematode DNA extract. PCR cycling conditions were 94°C 2 min, followed by 45 cycles of [94°C 20 sec, 65°C 5 sec, 60°C 5 sec, 55°C 5 sec, 45°C 5 sec, 68°C 3 min], ending with 1 cycle of 68°C for 15 min.

PCR products were analyzed by electrophoresis on 2% agarose with 1X SB (sodium borate-EDTA) buffer. Gels were stained with ethidium bromide and visualized using the U:Genius gel documentation system (Syngene, Frederick, MD). Hsp90 fragments were cloned using the Strataclone PCR Cloning Kit (Agilent, Santa Clara, CA) according to manufacturer’s instructions. Plasmid clone DNA was prepared with the QiaPrep Spin Miniprep Kit (Qiagen, Valencia, CA) and digested with Eco RI to verify the presence of insert. Sequencing was performed by Genewiz, Inc. Direct sequencing of PCR amplicons was used to obtain the 28S sequences (assigned GenBank accession numbers MK121965-MK121968), the ITS 1&2 rDNA sequence (MK121952), the 18S rDNA sequence (MK182465), and COI sequences (MK253554-MK253558). Accession numbers were assigned for new sequences from cloned Hsp90 of V. zeaphila (MK580824-MK580829), Heterodera avenae (MH484608, MH484609, and MH484611), and the outgroup Helicotylenchus digonicus (MK580830).

Raw sequence reads were processed in Sequencher 5.4.6 (Genecodes, Inc., Ann Arbor, MI). Multiple DNA sequence alignments were created using Geneious Prime 2019.0.3 (www.geneious.com) with built-in parameters or the MAFFT plug-in, with auto-selection of best algorithm depending on data. Alignments of Hsp90 intron regions were adjusted further using Geneious Alignment with free end gaps and identity (1.0/0.0) cost matrix and inspected visually to ensure preservation of exon boundaries and reading frames. Phylogenetic analysis using Bayesian inference (Huelsenbeck and Ronquist, 2001) was performed via the CIPRES Gateway (Miller et al., 2010), except where noted otherwise. The model of nucleotide evolution was determined by jModelTest 2.1.3 (Darriba et al., 2012), with the best-fit model GTR+I+G selected for Hsp90. The parameters for base frequency, proportion of invariable sites, and gamma distribution shape, and substitution rates according to Akaike Information Criteria (AIC) generated in a custom command block implemented in MB. Bayesian analysis was run with four chains for 2 × 10⁶ generations, with Markov chains sampled at intervals of 500 generations. Two runs were performed for each analysis. After burn-in samples were discarded and convergence evaluated, the remaining samples were retained for further analysis. Topologies were used to generate 50% majority rule consensus trees with posterior probabilities given on appropriate clades. Trees were visualized in Geneious. Alternative alignments of Hsp90 genomic sequences or coding regions were made using Clustal Omega (Sievers et al., 2011) and MUSCLE (Edgar, 2004) plug-ins for Geneious, and corresponding trees were made in MB or IQ-TREE (Nguyen et al., 2015) to investigate the effect of removing introns or partitioning the data on tree topology and support values.

COI alignments were likewise made within Geneious, using Atalodera carolynae as the outgroup. Phylogenetic reconstruction based on COI alignments were performed in IQ-TREE, within which ModelFinder (Kalyaanamoorthy et al., 2017) determined the best-fit model to be K3Pu + F + I + G4. The consensus tree was constructed from 1,000 bootstrap trees using UFBoot2 (Hoang et al., 2018) with bootstrap values indicated on appropriate branches. Alternative MB trees were made for comparison, including runs where rate parameters were independently specified for codon position.