DNA barcoding evidence for the North American presence of alfalfa cyst nematode, Heterodera medicaginis

Abstract Specimens of Heterodera have been collected from alfalfa fields in Kearny County, Kansas & Carbon County, Montana. DNA barcoding with the COI mitochondrial gene indicate that the species is not Heterodera glycines, soybean cyst nematode, H. schachtii, sugar beet cyst nematode, or H. trifolii, clover cyst nematode. Maximum likelihood phylogenetic trees show that the alfalfa specimens form a sister clade most closely related to H. glycines, with a 4.7% mean pairwise sequence divergence across the 862 nucleotides of the COI marker. Morphological analyses of juveniles and cysts conform to the measurements of H. medicaginis, the alfalfa cyst nematode originally described from the USSR in 1971. Initial host testing demonstrated that the nematode reproduced on alfalfa, but not on soybeans, tomato, or corn. Collectively, the evidence suggests that this finding represents the first record of H. medicaginis in North America. Definitive confirmation of this diagnosis would require COI sequence of eastern European isolates of this species.

Alfalfa cyst nematode, Heterodera medicaginis Kirjanova & Krall, 1971 was originally described from the USSR in 1971 and redescribed by Gerber and Maas (1982). The redescription added information missing in the original description regarding juvenile and male stages (Gerber and Maas, 1982). Host testing was conducted for the redescription that included 20 plant species from 7 plant families. These tests included Glycine max, Medicago lupulina, M. sativa, Phaseolus vulgaris, Pisum sativum, Trifolium pratense, T. repens, T. subterraneum, Vicia faba, V. sativa, Vigna sinensis, Galeopsis tetrahit, Dianthus plumarius, Spergula arvensis, Stellaria media, Beta vulgaris, Spinacia oleracea, Brassica oleracea, B. rapa, Rumex crispus, and Hebe andersonii. These host trials concluded that Heterodera medicaginis was only able to complete reproduction on alfalfa, Medicago sativa. The redescription included a narrative that provided morphological traits that could help discriminate the species from other members of the H. schachtii group. To discriminate the J2 stages of H. medicaginis and H. glycines Ichinohe 1952, it was determined that H. medicaginis possessed a longer stylet (25 µm vs 23 µm in H. glycines). Similarly, adult males could be identified by a longer stylet (29 µm vs 27 µm in H. glycines). The cyst stage of H. medicaginis was also notable because of its 'weakly developed, unbranched underbridge' (Gerber and Maas, 1982). Following the redescription, a DNA sequence of the internal transcribed spacer region was submitted to GenBank (Subbotin et al., 2001).
During the last five years, there have been unpublished reports of a cyst species reproducing on alfalfa (https://nematode.unl.edu/hetemedic.htm) in the Great Plains state of Kansas in the US. A follow-up of these reports, utilizing morphology, host trials, and DNA barcoding using the mitochondrial gene COI along with ITS1, the transcribed spacer region between 18S and 5.8S of the nuclear ribosomal repeat region, and the heat shock protein gene Hsp90 provide supporting evidence that Heterodera medicaginis is present in the US.

Nematode collections
The original North American collections of suspect alfalfa cyst specimens were made in western Kansas from alfalfa fields that bordered the Arkansas River near the city of Lakin, Kearny County, KS (Table 1). Some soil was sent to the Kansas State University Diagnostic Laboratory, the University of Idaho Nematology Laboratory, and cysts were sent to the University of Nebraska and the USDA Mycology and Nematology Genetic Diversity and Biology Laboratory (MNGDBL) at Beltsville, Maryland. Additionally, several cysts containing eggs and juveniles were isolated from an alfalfa planting in Carbon County, Montana during a Cooperative Agricultural Pest Survey in 2006 (Table 1).

Host testing
Preliminary host testing was conducted at Kansas State University using infested field soil containing an estimated 365 Heterodera sp. eggs and infective second-stage juveniles (J2), as well as 325 Meloidogyne hapla Chitwood, 1949 J2/100 cm 3 . The soil was placed into 450-cm 3 D40 Deepots (Stuewe and Sons Inc., Tangent, OR) and planted to either Kansas common alfalfa, an undetermined hybrid of corn, Flyer soybean, or Rutgers tomato. Nematode reproduction was determined after one and two months under greenhouse conditions. Heterodera females and cysts were dislodged from roots with water spray and collected on a 250-μ m-pore sieve and counted. Vermiform males and J2 of Heterodera and M. hapla were collected on a 25-μ m-pore sieve from one-and two-week incubations of roots in aerated water and counted.

Morphological and microscopic analysis
Cysts and infective juvenile stages were examined at the USDA MNGDBL and at the University of Nebraska Nematology Laboratory. Select juvenile measurements are presented in Table 2 alongside measurements from Gerber and Maas's (1982) redescription. Images of juveniles and adult males were taken with a Leica DMLB light microscope with differential interference contrast optics and a Leica DC300 video camera. All juveniles examined at the University of Nebraska were provided an identification number which links specimen images, measurements, and placement on phylogenetic trees. Cysts were prepared for scanning electron microscopy by fixation in a 4% formalin solution followed by a graded series of alcohol to 100% ethyl alcohol prior to critical point drying and coating with gold. Images were obtained on a Hitachi S-3000N scanning electron microscope located in the Morrison Microscopy Core Research Facility at the University of Nebraska.

Molecular analyses
The primers used for amplification of the COI gene region were: The sequencing primers were COI-F4a-Het and for the corresponding strand, an internal primer COI-R8-Het-5′-GAAAATGAGCTAC-CACATAATAAGTATCATGSARAACMACATC-CAAACTAGC-3′.
After removal of the primer sequences, amplification products from the Heterodera specimens were 862 base pairs. GenBank sequences used in this study generally were 100 to 300 nucleotides shorter than sequences generated with the new primer set. The ITS1 primer set used in the University of Nebraska Laboratory was reported in the study of Cherry et al. (1997).

Amplification conditions
Nematodes amplified at the UNL Nematology Laboratory were individually smashed in 18 µL of sterile H 2 O with a transparent microfuge micropipette tip on a coverslip, added to a 0.5 mL microfuge tube and stored at −20°C until needed. Amplification conditions were as follows: denaturation at 94°C for 5 min, followed by 45 cycles of denaturation at 94°C for 30 s, annealing at 48.0°C or 50.0°C for 30 s, and extension at 72°C for 90 s with a 0.5°C per second ramp rate to 72°C. A final extension was performed at 72°C for 5 min as described by Powers et al. (2014) and Olson et al. (2017). PCR   products were separated and visualized on 1% agarose using 0.5× TBE and stained with ethidium bromide. PCR products of sufficiently high quality were cleaned and sent for sequencing of both strands by the University of California, Davis DNA Sequencing Facility.
Nematodes analyzed in the Beltsville lab were smashed in worm extraction buffer and extracts prepared as described by Skantar et al. (2012). The ITS and 28S rDNA regions were amplified using primers TW81 and AB28, and D2A and D3B, respectively (Skantar et al., 2012). COI was amplified with primers  Het-CoxIF and HetCox-1R according to Subbotin et al. (2017). Partial Hsp90 fragments were amplified with primers U288 and L1110 (Skantar et al., 2012). PCR products were cleaned with the Monarch DNA Gel Extraction Kit (NEB, Ipswich, MA). ITS, COI, and Hsp90 amplicons were cloned using the Strataclone PCR Cloning Kit (Agilent, Santa Clara, CA) according to manufacturer's instructions. Plasmid clones of DNA were prepared with the Monarch Plasmid Miniprep Kit (NEB) and sequenced by Macrogen, Inc.

Data storage
Nucleotide sequences have been submitted to Gen-Bank and the Barcode of Life Database (BOLD).

Phylogenetic analysis
Hsp90 sequences obtained for the Kansas population were aligned with partial Hsp90 genomic DNA sequences from other cyst nematode species (new or from GenBank) using the MAFFT algorithm within Geneious 10.2.6. (https://www.geneious.com). The sequence data set was analyzed with Bayesian interference (BI) using the MrBayes module within Geneious under the model GTR with rate variation set to invgamma, 6 gamma categories, and outgroup set to Globodera pallida (Stone, 1973) Behrens, 1975. The Markov-Chain Monte Carlo (MCMC) values were set to 1×10 6 chain length, subsampling frequency 1,000, four heated chains, and a burn-in length of 10,000. Two runs were performed for each analysis. Topologies were used to generate a 50% majority rule consensus tree.
ITS1 and COI phylogenetic trees were constructed under maximum likelihood (ML) criteria in MEGA version 6. Sequences were edited using CodonCode Aligner version 8.0.1 (http://www.codoncode.com/) and aligned using MUSCLE within MEGA version 6 (Tamura et al., 2013). The gap opening penalty was set at −400 with a gap extension penalty of −200. For the COI tree, the General Time Reversible Model with Gamma distributed rates with Invariant sites (GTR + G + I) was determined to be the best substitution model by Bayesian information criterion using the best fit substitution model tool in MEGA 6.0., while the ITS1 tree used HKY. Both ML trees used a 'use all sites' option for gaps and 200 bootstrap replications to assess clade support.

Results
Host trial results indicated that among the crop species tested, only alfalfa was a suitable host for the Kansas alfalfa Heterodera population (Table 3). Mature females and cysts were recovered from alfalfa roots at two months after planting, but Heterodera J2 were recovered from alfalfa root incubations at both trial periods. In contrast, Meloidogyne J2 were recovered in large numbers from tomato roots, with lower numbers recovered from alfalfa and soybean roots. Figure 1 presents a maximum likelihood tree based on 862 base pairs of the COI mitochondrial gene from 154 specimens of heteroderid species. In total, 13 sequences from isolates collected from soil underneath alfalfa plantings form a well-supported homogeneous group that is a sister group to Heterodera glycines. The specimens from alfalfa are distinct from H. glycines at 42 nucleotide sites, with a mean pairwise P-distance (raw distance) of 4.7%. In total, 34 of the 42 nucleotide substitutions are at third-base pair positions in the COI gene. Three substitutions result in amino   Figure 3: Phylogenetic relationships of Heterodera species as inferred from analysis of a partial Hsp90 gene using Bayesian inference. Posterior probabilities more than 50% are given next to nodes. Newly obtained sequences are shown in bold. acid changes. The alfalfa specimens plus H. glycines form a group that is paired with H. schachtii Schmidt, 1871, with the three species constituting a well-supported clade (91 bootstrap value) that is joined to a second well-supported clade (bootstrap support, 98%) of other members of the H. schachtii group that include H. cicero Vovlas, Greco, and Di Vito, 1985, H. daverti Wouts & Sturhan, 1979, and H. trifolii Goffart, 1932. All six species within the H. schachtii group form a clade with a bootstrap support value of 100.
The ITS1 tree (Fig. 2) provides less clarity on the distinction between Heterodera medicaginis and H. glycines due to the sequence heterogeneity within both species. Depending on alignment parameters associated with gap creation and extension for ITS1, the diagnostic signal for this marker may be obscured. Most H. glycines sequences retrieved from GenBank cluster together apart, albeit with weak bootstrap support, from the suspected H. medicaginis sequences, including the single reference sequence from Russia (AF274391.1). Several GenBank H. glycines ITS se-quences of questionable identity fall outside the groupings of either species (LC208694.1, LC208695.1).
Four partial Hsp90 sequences were obtained from the Kansas population (Table 1). Although no Hsp90 sequence was available from a reference population of H. medicaginis, Bayesian analysis of partial Hsp90 genomic DNA showed that the Kansas population formed a distinct clade from H. glycines and other species from the Schachtii group (Fig. 3), giving further support for identification of this population as H. medicaginis.
The 28S sequence from the Kansas population was identical to H. glycines GenBank accession numbers (LC208677, KY795945, KY795944, KY795943, KX790324, GU475087, and DQ328692) and therefore did not discriminate H. medicaginis. The 28S sequence was submitted to GenBank (Accession No. MH793872) for future phylogenetic studies.
Measurements of J2 specimens are reported in Table 2 and illustrated ( Fig. 4A-F). The J2 has four lines in the lateral field and a tail with a finely rounded terminus (Fig. 4E-G). The male anterior end, posterior end, and entire body length showing spicules are also depicted ( Fig. 4H-J). Morphologically both cysts and juveniles conformed to Heterodera medicaginis except for one cone mount, which displayed molar-shaped bullae typical of H. schachtii. That cone, however, was slightly folded. Contrary to the redescription of H. medicaginis, the underbridge was moderately well-developed, about 100 mm long, with branches and heavily scattered bullae (Fig. 5B-D). Cysts were mostly oval, wide to lemon-shaped, brown in color, and with a cyst wall displaying a zig-zag pattern with few punctations (Fig. 6C-E). Cysts were ambifenestrate (Fig. 5A), the entire fenestra length ranging from 40 to 47 µm, fenestra width 30 to 32.5 µm or more, vulva  slit long with length 38 to 52 µm. The vulva to anus distance was 50 µm (Fig. 6E).

Discussion
A cyst nematode reproducing on alfalfa has been observed along the Arkansas River in western Kansas. A DNA record from a molecular survey from a Montana alfalfa field suggests the distribution may be wider than a single river valley in Kansas. DNA barcoding data by COI sequence rules out the identity of these cyst nematodes on alfalfa as being Heterodera glycines, H. schachtii, H. trifolii, or either of the two other members of the H. 'schachtii group.' Additionally, the morphology and measurements of juvenile and cyst stages are consistent with those of H. medicaginis. Cyst cone structure is more elaborate than was described in Gerber and Maas's (1982) redescription. Initial greenhouse host-reproduction trials using infested field soil are also consistent with the limited host-range of H. medicaginis. The presence of Meloidogyne hapla in these soils suggests that damage estimates should take both species into consideration. Collectively, these data support the identity of these North American cyst specimens as Heterodera medicaginis, the alfalfa cyst nematode. DNA sequence of the COI gene from Russian specimens will be necessary to definitively make the connection between US and confirmed eastern European isolates.