Molecular and morphological characterization of the alfalfa cyst nematode, Heterodera medicaginis, from Utah

Various stages: cysts, second-stage juveniles (J2), and eggs were sent to MNGDBL, Beltsville, MD from the University of Idaho. Juveniles used for morphological observations were separated from soil by sieving and Baermann funnel extraction. Juveniles were fixed in 3% formaldehyde and processed to glycerin by the formalin glycerin method (Hooper, 1970; Golden, 1990). 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 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.

Living nematode juveniles (J2) recovered from cysts were examined morphologically and molecularly for species identification.

The ITS 1&2 rDNA region was amplified with primers TW81 and AB28 (Skantar et al., 2012), producing a PCR amplicon of 985 bp. The PCR product was cleaned with the Monarch DNA Gel Extraction Kit (NEB, Ipswitch, MA) and then cloned using the Strataclone PCR Cloning Kit (Agilent, Santa Clara, CA). Cloned plasmid DNA was prepared with the Monarch Plasmid Miniprep Kit (NEB) and sequenced by Genewiz, Inc. The ITS rDNA sequence was assigned GenBank accession number MN308440. Hsp90 primers U831 [5′-AA(T/C)AA(A/G)AC(A/C) AAGCC(A/C/G/T)T(T/C)TGGAC-3′] and L1110 [5′-TC(A/G)CA(A/G)TT(G/A/C) TCCATGAT(A/G)AA(G/A/C)AC-3′] (Skantar and Carta 2004) were used to amplify a PCR product of 437 bp, which was cloned and sequenced as described above. Six clones containing the Hsp90 fragment were sequenced and submitted to GenBank under accession numbers MN311173-MN311178. Mitochondrial cytochrome oxidase I (COI) was amplified with primers Het-Cox1F and Het-Cox1R as described in the study of Subbotin et al. (2017). PCR amplicons of 430 bp were cleaned and sequenced directly with the same primers. The three identical sequences were submitted to GenBank under the accession number MN311179. DNA sequences were analyzed by BlastN. Evaluations of intraspecific and interspecific variation were conducted using sequence alignment algorithms within Geneious.