Molecular and morphological characterization of a first report of Cactodera torreyanae Cid del Prado Vera & Subbotin, 2014 (Nematoda: Heteroderidae) from Minnesota, the United States of America

Cysts, second stage juveniles (J2), and eggs were obtained from two Minnesota soil samples collected from a location with the GPS coordinates: 48^o57.248ʹN, 96^o74.729ʹW. Juveniles were fixed in 3% formaldehyde and processed to glycerin by the formalin glycerin method (Golden, 1990; Hooper, 1970). Cysts contained viable eggs and second stage juveniles (J2) which were examined morphologically and molecularly for species identification. Observations of morphological characters critical for identification were cyst shape, color and nature of fenestration, cyst wall pattern, J2 stylet length, shape of stylet knobs, and shape and length of tail and hyaline tail terminus (Fig. 1). Photomicrographs of cyst vulval cones, females, and J2 were made with an automatic Nikon Eclipse Ni compound microscope using a Nikon DS-Ri2 camera. Measurements were made with an ocular micrometer on a Leitz DMRB compound microscope. All measurements are in micrometers. Mexican population of C. torreyanae was also used for molecular study.

Figure 1:

DNA was isolated from single juveniles disrupted in 20 µl Nematode Extraction Buffer. DNA extraction, amplification, purification of PCR products, cloning, and sequencing were performed as described in Skantar et al. (2012) and Subbotin (2021a). The following primer sets were used for PCR: the forward D2A (5′ – ACA AGT ACC GTG AGG GAA AGT TG – 3′) and the reverse D3B (5′ – TCG GAA GGA ACC AGC TAC TA – 3′) primers for amplification of the D2-D3 expansion segments of 28S rRNA gene; the forward TW81 (5′ – GTT TCC GTA GGT GAA CCT GC – 3′) and the reverse AB28 (5′ – ATA TGC TTA AGTT CAG CGG GT – 3′) primers for amplification of the ITS1-5.8S-ITS2 of rRNA gene, the forward JB3 (5′ – TTT TTT GGG CAT CCT GAG GTT TAT -3′) and the reverse JB4.5 (5′ – TAA AGA AAG AAC ATA ATG AAA ATG -3′) primers or the forward Het-coxiF (5′ – TAG TTG ATC GTA ATT TTA ATG G – 3′) and the reverse Het-coxiR (5′ – CCT AAA ACA TAA TGA AAA TGW GC – 3′) primers for amplification of the partial COI gene (Skantar et al., 2021; Subbotin, 2021a).

DNA sequencing was conducted by University of Maryland Center for Biosystems Research and Genewiz, Inc. ITS rRNA gene sequences were obtained from cloned amplicons; sequences of 28S and COI genes were obtained directly from PCR products. New sequences were submitted to GenBank under the following accession numbers: ITS rRNA gene (MZ753794-MZ753798); 28S rRNA gene (MZ753815-MZ753817, MZ773251) and COI gene (MZ753804, MZ753805, MZ773253, MZ773254).

The newly obtained sequences of the D2-D3 of 28S rRNA, ITS rRNA, COI genes were aligned using ClustalX 1.83 with corresponding published gene sequences (Cid del Prado Vera and Subbotin, 2014; Escobar-Avila et al., 2021; Li et al., 2021; Skantar et al., 2021). Sequence alignment was analyzed with Bayesian inference (BI) using MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003) under the GTR + G + I model and with PAUP* 4b10 (Swofford, 2002) as described by Subbotin (2021b).

Measurements of second-stage juveniles from Minnesota (n = 7) included lengths of body (range = 421–510 μm, mean = 464.6 μm), stylet well developed (22.5–23.0 μm, 22.6 μm) with rounded basal knobs, tail (35.0–52.0 μm, 41.0 μm), and hyaline tail terminus (18.0–20.0 μm, 18.9 μm). The lateral field had four distinct lines. Shapes of the tail, tail terminus, and stylet knobs were also consistent with those of Mexican C. torreyanae. The cysts (n = 2) were lemon shaped, dark brown in color and dark brown in color, circumfenestrate, cone top with denticles present. Cyst wall with a wavy zigzag pattern in the middle. The fenestra length = 25.0, 32.5 μm, fenestra width = 28.0, 35.0 μm and the vulva slit to anus distance = 35.0, 37.5 μm. Morphometrics and morphology of cysts were consistent with those of Mexican C. torreyanae (Cid del Prado Vera and Subbotin, 2014) except for presence of denticles in the Minnesota population. Morphometrically, this species is also similar to Cactodera weissi (Steiner, 1949) Krall & Krall, 1978 in having small rounded to lemon shaped cysts with a circumfenestrate distinct vulval cone and in the J2 body and tail lengths and rounded stylet knobs. No males were found.

The D2-D3 expansion segments of 28S rRNA gene. The alignment was 722 bp in length and contained 13 Cactodera sequences, including four new sequences of C. torreyanae and two sequences of the outgroup taxa. Phylogenetic relationships within the genus Cactodera are given in Figure 2. The Minnesota C. torreyanae sequences clustered together (PP = 100%) with the sequences of the Mexican population of this species. The difference between the Minnesota and Mexican sequences was 0.1% (1 bp).

Figure 2:

The ITS rRNA gene. The alignment was 1030 bp in length and contained 19 Cactodera sequences, including five new sequences of C. torreyanae and two sequences of the outgroup taxa. Phylogenetic relationships within the genus Cactodera are given in Figure 3. The Minnesota C. torreyanae sequences clustered together (PP = 100%) with the sequences of the Mexican population of this species. The difference between the Minnesota and Mexican sequences was 0.7 to 1.4% (6–13 bp).

Figure 3:

The COI gene. The alignment was 452 bp in length and contained 11 Cactodera sequences, including four new sequences of C. torreyanae and two sequences of the outgroup taxa. Phylogenetic relationships within the genus Cactodera are given in Figure 4. The Minnesota C. torreyanae sequences clustered together (PP = 100%) with the sequences of the Mexican population of this species. The difference between the Minnesota and Mexican sequences was to 1.5% (6 bp). The difference between C. milleri and C. solani sequences was 1.1% (4 bp).

Figure 4:

Based upon this collective morphological and molecular data, we identify this nematode as Cactodera torreyanae. The closest species morphologically and molecularly is C. weissi but was distinguishable based upon direct comparison of ITS rRNA, 28S rRNA and COI gene sequences. To our knowledge this is the first report of the Cactodera torreyanae from United States and first report of this cyst nematode species from a potato field. Definite host plant for this nematode remains unknown.