In March 2020, a cyst nematode was discovered during a Pale Potato Cyst Nematode survey conducted by Minnesota Department of Agriculture as part of the Animal and Plant Health Inspection Service (APHIS) efforts to survey states for the presence of PCN. The soil samples were collected from a potato field, located in Karlstad, Kittson County, Minnesota, USA. The cyst samples were sent to the Mycology and Nematology Genetic Diversity and Biology Laboratory (MNGDBL) for identification purposes. Based on the results of morphological and molecular studies this nematode was identified as
Cysts, second stage juveniles (J2), and eggs were obtained from two Minnesota soil samples collected from a location with the GPS coordinates: 48o57.248ʹN, 96o74.729ʹW. 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
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
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
The newly obtained sequences of the D2-D3 of 28S rRNA, ITS rRNA,
Measurements of second-stage juveniles from Minnesota (
Based upon this collective morphological and molecular data, we identify this nematode as