JOURNAL OF NEMATOLOGY Article | DOI: 10.21307/jofnem-2019-016 e2019-16 | Vol. 51 DNA barcoding evidence for the North American presence of alfalfa cyst nematode, Heterodera medicaginis Thomas Powers1,*, Andrea Skantar2, Tim Harris1, Rebecca Higgins1, Peter Mullin1, Saad Hafez3, Abstract 2 4 Zafar Handoo , Tim Todd & Specimens of Heterodera have been collected from alfalfa fields 1 Kirsten Powers in Kearny County, Kansas and Carbon County, Montana. DNA 1University of Nebraska-Lincoln, barcoding with the COI mitochondrial gene indicate that the species is Lincoln NE 68583-0722. not Heterodera glycines, soybean cyst nematode, H. schachtii, sugar beet cyst nematode, or H. trifolii, clover cyst nematode. Maximum 2 Mycology and Nematology Genetic likelihood phylogenetic trees show that the alfalfa specimens form a Diversity and Biology Laboratory sister clade most closely related to H. glycines, with a 4.7% mean (MNGDBL), USDA, Beltsville, MD. pairwise sequence divergence across the 862 nucleotides of the COI 3Department of Plant Pathology, marker. Morphological analyses of juveniles and cysts conform to the University of Idaho, Boise, ID. measurements of H. medicaginis, the alfalfa cyst nematode originally described from the USSR in 1971. Initial host testing demonstrated 4Kansas State University, that the nematode reproduced on alfalfa, but not on soybeans, Manhattan Kansas. tomato, or corn. Collectively, the evidence suggests that this finding *Email: [email protected] represents the first record of H. medicaginis in North America. Definitive confirmation of this diagnosis would require COI sequence This paper was edited by of eastern European isolates of this species. Erik J. Ragsdale. Received for publication Key words September 4, 2018. Alfalfa cyst nematode, COI DNA barcode, Detection, Diagnosis, Heterodera medicaginis, Taxonomy. Alfalfa cyst nematode, Heterodera medicaginis J2 stages of H. medicaginis and H. glycines Ichinohe Kirjanova & Krall, 1971 was originally described from 1952, it was determined that H. medicaginis pos- the USSR in 1971 and redescribed by Gerber and sessed a longer stylet (25 µm vs 23 µm in H. glycines). Maas (1982). The redescription added information Similarly, adult males could be identified by a longer missing in the original description regarding juvenile stylet (29 µm vs 27 µm in H. glycines). The cyst stage of and male stages (Gerber and Maas, 1982). Host test- H. medicaginis was also notable because of its ‘weakly ing was conducted for the redescription that included developed, unbranched underbridge’ (Gerber and 20 plant species from 7 plant families. These tests Maas, 1982). Following the redescription, a DNA se- included Glycine max, Medicago lupulina, M. sativa, quence of the internal transcribed spacer region was Phaseolus vulgaris, Pisum sativum, Trifolium pratense, submitted to GenBank (Subbotin et al., 2001). T. repens, T. subterraneum, Vicia faba, V. sativa, Vigna According to Subbotin et al. (2010), H. medicaginis sinensis, Galeopsis tetrahit, Dianthus plumarius, Sper- is known from the Russian regions of Rostov, Stav- gula arvensis, Stellaria media, Beta vulgaris, Spinacia ropol Territory, Krasnodar Territory, and Kabardino oleracea, Brassica oleracea, B. rapa, Rumex crispus, Balkaria, as well as from Ukraine, Kazakhstan, and and Hebe andersonii. These host trials concluded that Uzbekistan (Baidulova, 1981). Furthermore, unidenti- Heterodera medicaginis was only able to complete re- fied cyst nematodes were observed on lucerne roots production on alfalfa, Medicago sativa. The redescrip- in Poland (Brzeski, 1998). tion included a narrative that provided morphological During the last five years, there have been unpub- traits that could help discriminate the species from other lished reports of a cyst species reproducing on alfalfa members of the H. schachtii group. To discriminate the (https://nematode.unl.edu/hetemedic.htm) in the Great 1 © The Society of Nematologists 2019. DNA barcoding evidence for the North American presence of alfalfa cyst nematode, Heterodera medicaginis Plains state of Kansas in the US. A follow-up of these redescription. Images of juveniles and adult males reports, utilizing morphology, host trials, and DNA were taken with a Leica DMLB light microscope barcoding using the mitochondrial gene COI along with differential interference contrast optics and a with ITS1, the transcribed spacer region between Leica DC300 video camera. All juveniles examined 18S and 5.8S of the nuclear ribosomal repeat re- at the University of Nebraska were provided an gion, and the heat shock protein gene Hsp90 provide identification number which links specimen imag- supporting evidence that Heterodera medicaginis is es, measurements, and placement on phylogenetic present in the US. trees. Cysts were prepared for scanning electron microscopy by fixation in a 4% formalin solution Materials and Methods followed by a graded series of alcohol to 100% ethyl alcohol prior to critical point drying and coat- Nematode collections ing with gold. Images were obtained on a Hitachi S-3000N scanning electron microscope located in The original North American collections of suspect the Morrison Microscopy Core Research Facility at alfalfa cyst specimens were made in western Kansas the University of Nebraska. from alfalfa fields that bordered the Arkansas River near the city of Lakin, Kearny County, KS (Table 1). Molecular analyses Some soil was sent to the Kansas State University Di- agnostic Laboratory, the University of Idaho Nematol- The primers used for amplification of the COI gene ogy Laboratory, and cysts were sent to the University region were: of Nebraska and the USDA Mycology and Nematology Genetic Diversity and Biology Laboratory (MNGDBL) • COI-F4a-Het-5′-CAGTTATATAATTCT- at Beltsville, Maryland. Additionally, several cysts con- TTTATTACTAGTCATGCATTAATTATRAT- taining eggs and juveniles were isolated from an alfalfa TTTTTTTYTRGTTATACC-3′. planting in Carbon County, Montana during a Cooper- • COI-R10b-Het 5′-CCAAAAAAAAAAAAAAT- ative Agricultural Pest Survey in 2006 (Table 1). CACTATAATCYAAATATTTACGDGG-3′ • The sequencing primers were COI-F4a-Het Host testing and for the corresponding strand, an internal primer COI-R8-Het-5′-GAAAATGAGCTAC- Preliminary host testing was conducted at Kansas CACATAATAAGTATCATGSARAACMACATC- State University using infested field soil contain- CAAACTAGC-3′. ing an estimated 365 Heterodera sp. eggs and in- fective second-stage juveniles (J2), as well as 325 After removal of the primer sequences, amplifica- Meloidogyne hapla Chitwood, 1949 J2/100 cm3. The tion products from the Heterodera specimens were soil was placed into 450-cm3 D40 Deepots (Stuewe 862 base pairs. GenBank sequences used in this study and Sons Inc., Tangent, OR) and planted to either generally were 100 to 300 nucleotides shorter than se- Kansas common alfalfa, an undetermined hybrid of quences generated with the new primer set. The ITS1 corn, Flyer soybean, or Rutgers tomato. Nematode primer set used in the University of Nebraska Labora- reproduction was determined after one and two tory was reported in the study of Cherry et al. (1997). months under greenhouse conditions. Heterodera fe- males and cysts were dislodged from roots with wa- Amplification conditions ter spray and collected on a 250-μ m-pore sieve and counted. Vermiform males and J2 of Heterodera and Nematodes amplified at the UNL Nematology Lab- M. hapla were collected on a 25-μ m-pore sieve oratory were individually smashed in 18 µL of sterile from one- and two-week incubations of roots in H2O with a transparent microfuge micropipette tip aerated water and counted. on a coverslip, added to a 0.5 mL microfuge tube and stored at −20°C until needed. Amplification Morphological and microscopic analysis conditions were as follows: denaturation at 94°C for 5 min, followed by 45 cycles of denaturation at Cysts and infective juvenile stages were examined at 94°C for 30 s, annealing at 48.0°C or 50.0°C for the USDA MNGDBL and at the University of Ne- 30 s, and extension at 72°C for 90 s with a 0.5°C braska Nematology Laboratory. Select juvenile per second ramp rate to 72°C. A final extension measurements are presented in Table 2 alongside was performed at 72°C for 5 min as described by measurements from Gerber and Maas’s (1982) Powers et al. (2014) and Olson et al. (2017). PCR 2 JOURNAL OF NEMATOLOGY Table 1. Collection data for specimens used in this study. Specimens 1 to 15 were examined as fixed specimens. Specimen ID Species Locality Host Marker GenBank Accession No. 1 H. medicaginis Kearny County, Kansas Alfalfa N/A 2 H. medicaginis Kearny County, Kansas Alfalfa N/A 3 H. medicaginis Kearny County, Kansas Alfalfa N/A 4 H. medicaginis Kearny County, Kansas Alfalfa N/A 5 H. medicaginis Kearny County, Kansas Alfalfa N/A 6 H. medicaginis Kearny County, Kansas Alfalfa N/A 7 H. medicaginis Kearny County, Kansas Alfalfa N/A 8 H. medicaginis Kearny County, Kansas Alfalfa N/A 9 H. medicaginis Kearny County, Kansas Alfalfa N/A 10 H. medicaginis Kearny County, Kansas Alfalfa N/A 11 H. medicaginis Kearny County, Kansas Alfalfa N/A 12 H. medicaginis Kearny County, Kansas Alfalfa N/A 13 H. medicaginis Kearny County, Kansas Alfalfa N/A 14 H. medicaginis Kearny County, Kansas Alfalfa N/A 15 H. medicaginis Kearny County, Kansas Alfalfa N/A P169028 H. medicaginis Kearny County, Kansas Alfalfa 18S AY912048 N838 H. schachtii Goshen County, Wyoming Soybean COI MK093062 N839 H. schachtii Goshen County, Wyoming Soybean COI MK093063 N864 H. schachtii Goshen