Turfgrasses are widely used as groundcovers in a variety of sporting, residential, and recreational contexts, and maintaining high quality and aesthetics is an essential feature of turfgrass management. Control of newly emerging diseases and nematodes presents an ongoing challenge to the health, quality, production, and maintenance of turfgrass. The barley root-knot nematode,
A similar study by Ye et al. (2015) analyzed data from 51 RKN populations collected from turfgrasses within the eastern United States. In contrast with the western U.S. golf courses surveyed, the most prevalent RKN species on the East Coast were
Molecular markers continue to be a useful tool for distinguishing
The Banbury Golf Course in Bandon, Oregon reported a poorly performing section that had been planted entirely from seed, including a majority percentage of
Two soil samples were sent to the USDA Mycology and Nematology Genetic Diversity and Biology Laboratory (Beltsville, MD) in November of 2019. The origin of the soil samples was Banbury Golf Course in Ada County, Idaho. Nematodes were recovered from soil using nested 20, 60, and 500 μm sieves, with juveniles (J2s) recovered from the 500 μm sieves using the sieving decanting method, followed by sugar centrifugal flotation (Jenkins, 1964). Nematodes were fixed in 3% formaldehyde and processed to glycerin by the formalin glycerin method (Golden, 1990; Hooper, 1970). Females recovered from the soil samples were fixed in 3% formaldehyde solution. Photomicrographs of the specimens were made with a Nikon Eclipse Ni compound microscope, using a Nikon DS-Ri2 camera (Nikon USA, Melville, NY). Measurements were made with an ocular micrometer on a Leica WILD MPS48 Leitz DMRB compound microscope (Leica, Wetzlar, Germany). All measurements are in micrometers unless otherwise stated.
The molecular identification was performed using DNA extracted from three separate juveniles (J2) as templates in PCR reactions. Individual J2 were mechanically disrupted in 20 μl of extraction buffer (125 mM KCl, 25 mM Tris-Cl, pH 8.3, 3.75 mM MgCl2, 2.5 mM DTT, 1.125% Tween 20), then stored in PCR tubes at −80 °C until needed. To prepare DNA extracts, frozen nematodes were thawed, 1 μl proteinase K (from 2 mg/ml stock solution) was added, and tubes were incubated at 60 °C for 60 min, followed by 95 °C for 15 min to deactivate the proteinase K and centrifuged briefly prior to use in PCR. All PCR reactions contained 0.75 μl of each primer and 0.125 μl of Dream Taq (0.625 Units) per 25 μl reaction. The 28S reaction included 2 μl of DNA template; the rest contained 3 μl. Ribosomal PCR products were amplified from the 28S D2–D3 expansion segment as described by De Ley et al. (2005) with primers D2A (5′- 1334–1340) and D3B (5′-TCGGAAGGAACCAGCTACTA-3′), and from the intergenic spacer (IGS-2) according to Blok et al. (2002) with primers 5S (5′-TTAACTTGCCAGATCGGACG-3′) and 18S (5′-TCTAATGAGCCGTACGC-3′). Mitochondrial DNA products were amplified from the variable-marker region between the mitochondrial COII and the large (16S) rRNA gene, as described by Powers and Harris (1993), with primers 1RNAF (5′-TACCTTTGACCAATCACGCT-3′) and COIIR (5′-GGTCAATGTTCAGAAATTTGTGG-3′), and partial COI according to Derycke et al. (2010), with primers JB3 (5′-TTTTTTGGGCATCCTGAGGTTTAT-3′) and JB5 (5′-AGCACCTAAACTTAAAAC ATAATGAAAATG-3′). Hsp90 sequences were amplified according to Nischwitz et al. (2013) with primers RKN-d1F (5′- GCYGATCTTGTYAACAACCYTGGAAC-3′) and RKN-5R (5′-TCGAACATGTCAAAAGGAGC-3′). PCR products were cleaned with the Monarch DNA Gel Extraction Kit (NEB, Ipswitch, MA), and Hsp90 products were cloned using the Strataclone PCR Cloning Kit (Agilent, Santa Clara, CA), prepared with the Monarch Plasmid Miniprep Kit (NEB), and sequenced with vector M13F and M13R primers.
Other PCR amplicons were sequenced directly with their corresponding PCR primers, except the 28S which was sequenced with the primers D3A and D3B. All PCR and cloned products were sequenced by Genewiz, Inc. (South Plainfield, NJ, USA). GenBank accession numbers were assigned as follows: 28S rDNA (MT406252); IGS-2 rDNA (OQ72994 to OQ72997); mitochondrial COII to 16S DNA (MT408951 to MT408953); COI (MT408951 to MT408953); and Hsp90 (MT408947 to MT4089450). Newly obtained sequences were compared with publicly available sequences in GenBank using BlastN. Multiple sequence alignments of each marker were constructed with ClustalW or MAFFT within Geneious, with outgroups chosen based on previous studies. Phylogenetic trees were constructed by Bayesian Inference (BI) under the GTR + I + G model implemented within the MrBayes plugin in Geneious. BI analysis for each gene included a random starting tree and was run with four chains for 2 × 106 generations. Two runs were performed for each analysis. Trees were sampled every 1000th generation, with 25% of results discarded as burn-in. Remaining samples were used to generate a 50% majority-rule consensus tree. Posterior probabilities (PP) are shown on appropriate clades.
Morphological measurements of
Morphometrics of infective second-stage juveniles of
Linear (μm) | ||||
Body length | 427.9 ±16.73 (390 – 460) | 435 (418 – 465) | 421 ± 8.1 (410 – 429) | 429 ± 16.1 (397 – 467) |
Maximum body width | 16.2 (15.0 – 17.5)* | 15 ± 0.95 (14 – 17.5) | 14.1 ± 0.6 (13.3 – 14.5) | 15.9 ± 0.7 (14.4 – 17.3) |
Stylet length | 12.09 ± 0.56 (11.0 – 13.0) | 14 (13 – 15) | 13.3 ± 0.5 (12.6 – 13.9) | 11.7 ± 0.6 (10.8 –1 2.4) |
Body width at anus | 10.3 ± 0.9 (9.0 – 12.0)* | 11 (9 –13) | - | - |
Tail length | 70.3 ± 5.04 (62.0 – 80.0) | 70 (52 – 78) | 66.0 ± 3.9 (61.0 –70.0) | 68.2 ± 8.0 (55–78) |
Hyaline tail terminus length | 21.4 ± 2.16 (17.0 – 26.0) | n.d. | 17.9 ± 1.8 (15.8 – 19.6) | 24.7 ± 2.6 (19.6 – 29.8) |
Lines in lateral field | 4 | 4 | - | - |
Ratios | ||||
a = body length/greatest body diameter | 35.61 ± 2.21 (32.24 – 40.91) | 28 (25 – 32) | 30 ± 1.5 (28.3 – 31.8) | 27.1 ± 1.9 (24.2 – 30.7) |
c = body length/tail length | 6.11 ± 0.43 (4.94 – 6.79) | 6.2 (n.d.) | 6.4 ± 0.3 (6.0 – 6.7) | 6.4 ± 0.8 (5.6 – 8.0) |
Values were calculated based on 10 specimens.
Amplification of the IGS-2 ribosomal DNA region and the D2–D3 expansion segment of region 28S yielded sequences of 1334 to 1340 bp and 775 bp, respectively. Amplification of the mitochondrial interval between COII and the large (16S) rRNA gene yielded sequences of 416 to 441 bp, and from the COI gene, 752 to 796 bp. Hsp90 PCR generated sequences ranging from 1031 to 1043 bp.
Amplification of the D2–D3 expansion region of 28S rDNA yielded identical sequences from three individual J2. Similarity matches from BlastN against the GenBank database included other
Amplification of the IGS-2 rDNA from three J2 gave two identical sequences and a third that was 99.6% identical (7 bp differences). These IGS-2 sequences from
The phylogenetic tree (Fig. 4) inferred from alignment of newly obtained
PCR of the mitochondrial interval COII-16S from three J2 gave two identical sequences, and a third that varied at 3 bp from the others (99.6 to 100% identity). BlastN yielded top hits to both
In a previous study of RKN populations from golf course turfgrass (McClure et al., 2012), mtCOII-16S interval could not distinguish populations
The presence of mixed
Amplification of mitochondrial COI gave two identical sequences from separate J2. BlastN comparison to GenBank resulted in matches to
In the Bayesian tree resulting from the multiple sequence alignment generated with selected RKN species (Fig. 6), all
Hsp90 amplification from three juveniles resulted in four cloned PCR products varying in length from 1031 to 1042 bp, with 98.8 to 99.4% identity (6 to 12 bp differences) among clones. BlastN comparison to sequences in GenBank indicated that the best matches to
Based upon the unambiguous similarity of these DNA markers with those previously reported for the species (McClure et al., 2012, Nischwitz et al., 2013, Hodgetts et al., 2016; Powers et al., 2018) and the morphological data (Franklin, 1965; Karssen et al., 2002; Zhao et al., 2017, Suresh et al., 2017; Santos et al., 2020), we identify this isolate as
The greens at Banbury Golf Course were originally planted as creeping bent grass (
This study represents the first report of
In the United Kingdom, mixed populations of