Molecular Characterisation and Diagnosis of Root-Knot Nematodes (Meloidogyne spp.) from Turfgrasses in North Carolina, USA

Root-knot nematodes (Meloidogyne spp.) are the most common and destructive plant-parasitic nematode group worldwide and adversely influence both crop quality and yield. In this study, a total of 51 root-knot nematode populations from turfgrasses were tested, of which 44 were from North Carolina, 6 from South Carolina and 1 from Virginia. Molecular characterisation was performed on these samples by DNA sequencing on the ribosomal DNA 18S, ITS and 28S D2/D3. Species-specific primers were developed to identify turfgrass root-knot nematode through simplex or duplex PCR. Four species were identified, including M. marylandi Jepson & Golden in Jepson, 1987, M. graminis (Sledge & Golden, 1964) Whitehead, 1968, M. incognita (Kofoid & White, 1919) Chitwood, 1949 and M. naasi Franklin, 1965 through a combined analysis of DNA sequencing and PCR by species-specific primers. M. marylandi has been reported from North Carolina and South Carolina for the first time. Molecular diagnosis using PCR by species-specific primers provides a rapid and cheap species identification approach for turfgrass root-knot nematodes.


Introduction
Turfgrasses are used worldwide for lawns of home and office buildings, athletic fields, other recreational facilities, and roadsides. In the United States, there are more than 50,000,000 lawns and 16,000 golf courses and the turfgrass area was estimated to be 30 million acres in 2007 [1,2]. In North Carolina (NC), there are 664 golf courses [http://www.golflink.com/golfcourses/state.aspx?state=NC] and the turfgrass industry is a 2.3 billion dollar a year industry (http://www.golf2020.com/media/32940/nc_golf_full_rpt_sri_final_29apr2013.pdf). However, maintenance of turfgrass is very challenging due to damage by various pests, including nematodes. During a survey from 2010 to 2013, 29 species of plant-parasitic nematodes belonging to 22 genera in 15 families were found associated with bermudagrass (Cynodon dactylon), creeping bentgrass (Agrostis stolonifera), and zoysiagrass (Zoysia japonica) in NC and South 18S, ITS and 28S D2/D3, then to develop and validate turfgrass RKN species-specific primers for a reliable and rapid PCR assay to support our diagnostic services and to allow species identification of RKNs through a combined analysis of DNA sequencing and PCR by species-specific primers. The specificity and application of the assay were demonstrated.

Nematode samples
A total of 51 RKN populations from turfgrasses were tested in this study, which comprised of 44 from NC, 6 from SC and 1 from Virginia (Va) ( Table 1). These samples were submitted to the Nematode Assay Laboratory of the Agronomic Division, NCDA&CS voluntarily from golf courses, sod farms and homeowners' lawns. Some of the samples were collected during a plantparasitic nematode survey of 111 golf courses in 39 counties in NC and SC in the summer 2011 [3]. No specific permissions were required in sampling for plant-parasitic nematodes and no endangered or protected species were involved. In addition, nine non-turfgrass nematode populations belonging to M. arenaria  Table 1). The identification of these reference species had already confirmed by DNA sequencing and PCR by speciesspecific primers in other projects (data not shown herein). Nematodes were extracted from soil samples by a combination of elutriation [21] and centrifugation [22] methods. The nematode sample was poured into a counting dish (7.5 cm L × 3 cm W × 1.5 cm H) and the nematodes were identified and counted under a Nikon Diaphot 200 inverted microscope (Tokyo, Japan). Further species confirmation was performed with a Leica DM2500 compound microscope (Leica Microsystems Inc., Buffalo Grove, IL, USA) with interference contrast at up to 1,000× magnification.
DNA extraction. For molecular analysis, a single or up to 10 nematodes of the J2 from the same sample were hand-picked into 10-μl AE buffer (10 mM Tris-Cl, 0.5 mM EDTA; pH 9.0) on a glass microscope slide (7.5 cm x 2.5 cm). The nematodes were then macerated with a pipette tip into pieces and collected in 50-μl AE buffer and stored at -20°C.
DNA amplification, cleaning and sequencing. The primers used for PCR and DNA sequencing are given in Table 2. The primers SSUF07/SSUR26 [23], 18S965/18S1573R [24], and 18SnF/18SnR [25] were used to amplify the ribosomal DNA near-full-length 18S gene. The primers rDNA2/ rDNA1.58S [26,27] were used to amplify the ITS1 rDNA region. The primers D2a/D3b [28] were used to amplify the partial rDNA 28S gene D2/D3 domain. PCR for these genes was also conducted using various combinations of universal forward and reverse primers designed for Meloidogyne to ensure high success in PCR (Table 2). These primers were based on the conserved sites from a multiple alignment of many representative Meloidogyne species from the GenBank and their approximate positions are shown in Fig 1. The primer selection criteria were as follows: Tm (melting temperature) 55 to 60°C, primer length 18 to 22 bp, and absence of secondary structure when possible. These primers were synthesized by Integrated DNA Technologies, Inc. (Coralville, Iowa, USA). The 25-μl PCR was performed using 12.5-μl 2X Apex Taq red master mix DNA polymerase (Genesee Scientific Corporation, San Diego, CA, USA), 9.5-μl water, 1-μl each of 10-μM forward and reverse primers, and 1μl of DNA template according to the manufacturer's protocol in a Veriti 1 thermocycler (Life Technologies, Carlsbad, CA, USA). The thermal cycler program for PCR was as follows: denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 45 s, and extension at 72°C for 1 min. A final extension was performed at 72°C for 10 min. PCR products were cleaned using ExoSap-IT (Affymetrix, Inc., Santa Clara, CA, USA) . The sequences used in phylogenetic analysis were chosen from the highest match based on BlastN result in GenBank against the four RKN species recovered from this study. The model of base substitution in the DNA sequence data was evaluated using MOD-ELTEST version 3.06 [29]. The Akaike-supported model [30], the proportion of invariable sites, and the gamma distribution shape parameters and substitution rates were used in phylogenetic analyses using DNA sequence data. Bayesian analysis was performed to confirm the tree topology for each gene separately using MrBayes 3.1.0 [31], running the chain for 1,000,000 generations and setting the 'burnin' at 1,000. Markov Chain Monte Carlo (MCMC) methods were used within a Bayesian framework to estimate the posterior probabilities (pp) of the phylogenetic trees [32] using the 50% majority-rule. The λ2 test for homogeneity of base frequencies and phylogenetic trees was performed using PAUP Ã version 4.0 (Sinauer Associates, Inc. Publishers, Sunderland, MA, USA).

Simplex PCR by species-specific primers
The species identification of M. incognita was confirmed using PCR by species-specific SCAR primers Inc-K14-F/Inc-K14-R which produce a 399-bp DNA fragment [33]. Mn28SFs/ RK28SUR in 28S D2/D3 were designed specific for M. naasi producing a 272-bp DNA fragment based on JN019291. Primers Mg28SFs/RK28SUR and Mm28SFs/RK28SUR in 28S D2/  Table 1) to test the scenario if a mixed species was present. The PCR condition is the same as described above. Duplex PCR by ITS species-specific primers and 28S universal primers The 25-μl duplex PCR was performed using 12.5-μl 2X Apex Taq red master mix DNA polymerase, 7.5-μl water, 1-μl each of 10-μM forward and reverse primers specific for M. graminis and M. marylandi, plus 1-μl each of 10-μM primers RK28SF/MR as internal positive control, and 1-μl of DNA template. The PCR condition is the same as described above.

Root-knot nematode identification
The J2s of RKNs were recovered from the turfgrass soil samples. Species identification in this study was based on the combined analysis of DNA sequencing on the rDNA 18S, ITS and 28S D2/D3 (Table 1) and PCR by species-specific primers ( Table 3). Four species were recovered including M. marylandi, M. graminis, M. incognita and M. naasi; the results are given in Table 1.

DNA sequencing
The rDNA 18S, ITS and 28S D2/D3 were successfully sequenced; their accession numbers from the GenBank are presented in Table 1 [7,[35][36][37][38], ITS [7,39], 28S [7,39] and IGS [40]. Therefore, the conserved ribosomal DNA can't separate these tropical RKNs. The mitochondrial DNA has a faster rate of evolution than the corresponding nuclear genes, creating sufficient nucleotide variation for species-level analyses [15]. The region of the mitochondrial genome flanked by the COII gene and the large (16S) ribosomal gene were successfully applied in large-scale regional RKN survey through PCR and RFLP [14]. Unfortunately, numerous attempts using the same primers [14] or designing new primers for turfgrass RKNs in this project were not successful, with a low rate of success in PCR and insufficient DNA sequence data to generate any meaningful results. Thus, the use of mitochondrial genome on molecular identification for turfgrass RKNs needs further study.

Simplex PCR by species-specific primers
Results of simplex PCR by species-specific primers are given in Table 3   for M. marylandi. Results of 28S primers and ITS primers agree with each other. Primer set Mn28SFs/RK28SUR are positive only for M. naasi. However, M. naasi is rather rare in this study and only one population  was available for further PCR testing by specie-specific primers. Two other populations (11-30383 and 11-30385)  The simplex PCR results for testing four common Meloidogyne species from turfgrass using species-specific primers are presented in Fig 4. Fig 4A amplified a 198-bp DNA fragment in 28S D2/D3 using Mg28SFs/RK28SUR for M. graminis, but the other three species failed to get any PCR products. Fig 4B amplified a 198-bp DNA fragment in 28S D2/D3 using Mm28SFs/ RK28SUR for M. marylandi, but the other three species failed to get any PCR products. Fig 4C  amplified a 272-bp DNA fragment in 28S D2/D3 using Mn28SFs/RK28SUR for M. naasi, but the other three species failed to get any PCR products. Fig 4D amplified a 399-bp DNA fragment in SCAR using Inc-K14-F/Inc-K14-R for M. incognita, but the other three species failed to get any PCR products. All these four samples produced a 612-bp DNA fragment using RK28SF/MR. All results are positive if the DNA is from a mixture of four species. Water used as a negative control in all these assays was negative.

Duplex PCR by ITS species-specific primers and 28S universal primers
Results of duplex PCR by ITS species-specific primers and 28S universal primers are given in Table 3 and agree with simplex PCR results. The duplex PCR results for testing two most common Meloidogyne species (M. marylandi and M. graminis) from turfgrass using ITS speciesspecific primers and 28S universal primers are presented in Fig 5. Fig 5A amplified a 267-bp DNA fragment using MgmITSF/MgITSRs and a 612-bp DNA fragment using RK28SF/MR for M. graminis, but the other three species only amplified a 612-bp DNA fragment by RK28SF/ MR. Fig 5B amplified a 323-bp DNA fragment in ITS using MgmITSF/MmITSRs and a 612-bp DNA fragment using RK28SF/MR for M. marylandi, but the other three species only amplified a 612-bp DNA fragment by RK28SF/MR. Water used as a negative control in all these assays was negative. The duplex PCR provides any assay to detect the target species and any RKNs in a single reaction to prevent false negatives caused by failure of the PCR for any reason.
In conclusion, this study characterized DNA sequences on rDNA 18S, ITS and 28S D2/D3 on a wide range of RKN populations from turfgrasses mainly from NC. Universal primers were also developed for PCR on the genus Meloidogyne for these three gene fragments. Analysis of the sequences through BlastN search and phylogenetic analysis revealed four distinct species, namely M. marylandi, M. graminis, M. incognita and M. naasi, the first two being the predominant species in NC. This result is different from the western United States where M. naasi was determined to be the most common species [7,12]. In this same study [7,12],.M. minor was only detected from Washington and M. chitwoodi and M. fallax only from California, but none of these three species were detected in the current study.
Species-specific primers on rDNA 28S D2/D3 were developed to identify turfgrass RKN through simplex PCR by species-specific primers on M. marylandi, M. graminis and M. naasi. Species-specific primers on ITS were also developed to identify two most common species M. marylandi and M. graminis to allow species confirmation using an additional marker through simplex or duplex PCR. SCAR primers Inc-K14-F/Inc-K14-R [33] were employed to identify M. incognita which produces a 399-bp DNA fragment. In addition, the RKN-universal primers RK28SF/MR were designed and included to amplify a 612-bp DNA fragment as a RKN endogenous control to detect the presence of RKN rDNA 28S gene, so that a RKN-negative sample can still be evaluated to exclude false negatives due to instrument, pipetting, reagent, and/or reaction failure. Compared with other molecular diagnosis [7,14,16,17,19,20,49], this assay only requires routine PCR and electrophoresis and is simple, cheap and rapid (<4 h), without further restriction digestion, DNA sequencing or expensive real-time PCR equipment and reagents. This molecular diagnosis using PCR by species-specific primers provides a rapid species identification approach for turfgrass RKN independent of morphology.