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

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 cm³. The soil was placed into 450-cm³ 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-μm-pore sieve and counted. Vermiform males and J2 of Heterodera and M. hapla were collected on a 25-μm-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:

• COI-F4a-Het-5′-CAGTTATATAATTCTTTTATTACTAGTCATGCATTAATTATRATTTTTTTTYTRGTTATACC-3′.

• COI-R10b-Het 5′-CCAAAAAAAAAAAAAATCACTATAATCYAAATATTTACGDGG-3′

• The sequencing primers were COI-F4a-Het and for the corresponding strand, an internal primer COI-R8-Het-5′-GAAAATGAGCTACCACATAATAAGTATCATGSARAACMACATCCAAACTAGC-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 H₂O 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 Exraction 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 GenBank 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 × 10⁶ 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.

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.