The cactus cyst nematode,
Cysts, white females, second-stage juveniles (J2), and eggs were obtained from soil and roots associated with cactus plants from the cactus collection in Meridian, Idaho, USA. Juveniles for morphological observations were separated from soil by sieving and using Baermann funnel extraction, or were recovered from cysts removed from fresh roots and kept in water in watch glasses. Juveniles were fixed in 3% formaldehyde and processed to glycerin by the formalin glycerin method (Golden, 1990; Hooper, 1970). Females and some cysts were typically removed from roots after fixation for 12 hr in 3% formaldehyde solution. Photomicrographs of cyst vulval cones, females, 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, and light microscopic images of fixed nematodes were taken on a Leica WILD MPS48 Leitz DMRB compound microscope. Measurements were made with an ocular micrometer on a Leica WILD MPS48 Leitz DMRB compound microscope. All measurements are in micrometers, unless otherwise stated.
Living nematode juveniles (J2) recovered from the cysts were examined morphologically and molecularly for species identification at the MNGDBL. Observations of morphological characters critical for identification (Fig. 1A–E and Fig. 2A–C) indicated that the specimens were
The ITS 1&2 rDNA region was amplified with primers TW81 and AB28 (Joyce et al., 1994) and conditions as described previously (Skantar et al., 2012), producing PCR amplicons of 985 bp. The PCR products were cleaned with the Monarch DNA Gel Extraction Kit (NEB, Ipswitch, MA) and then cloned using the Strataclone PCR Cloning Kit (Agilent, Santa Clara, CA). Six ITS rDNA clones representing three J2 were prepared with the Monarch Plasmid Miniprep Kit (NEB) and sequenced by Genewiz, Inc. (MH477533-MH477538). The 28 S rDNA D2-D3 expansion segment was amplified using primers D2A and D3B (DeLey et al., 1999) and conditions as described previously (Skantar et al., 2012). Hsp90 sequences were amplified with primers U288 and L1110 and gave products of 1219 bp. These were cloned and sequenced as described above and submitted to GenBank under accession numbers MH484605-MH484607.
Separate alignments of ITS rDNA and Hsp90 genomic DNA sequences were constructed using the MAFFT algorithm within Geneious v. 10.2.6. For ITS, the best-fitting model of nucleotide substitution GTR + I + G was estimated using jModelTest based on the Akaike Information Criterion. Phylogenetic relationships were estimated with Bayesian interference (BI) on the CIPRES Science Gateway (
Measurements of second-stage juveniles (
The ITS rDNA sequences from this population varied from 0 to 7 bp among each other. Intraspecific variation among all available ITS sequences of
Three 28 S rDNA amplicons of 721 bp were obtained from three separate J2 and gave rise to identical sequences (MH478572-MH478574). MegablastN search of the NCBI NR database showed 99% identity to a single sequence of
Partial Hsp90 sequences were aligned with selected sequences from other
Based upon this collective morphological and molecular data, we identify this isolate as