Meloidogyne aegracyperi n. sp. (Nematoda: Meloidogynidae), a root-knot nematode parasitizing yellow and purple nutsedge in New Mexico

Abstract Meloidogyne aegracyperi n. sp. is described from roots of purple nutsedge in southern New Mexico, USA. Mature females are small (310–460 µm), pearly white, with their egg masses completely contained inside root galls. The neck is often at a 90 to 130° angle to the protruding posterior end with the perineal pattern. The distance of the dorsal esophageal gland orifice (DGO) to the base of the stylet is relatively long (4.0–6.1 µm), and the excretory pore is level with the base of the stylet. The anterior portion of the rounded lumen lining of the metacorpus contains 3 to 10 small vesicles. The perineal pattern has a rounded dorsal arch with a tail terminal area that is smooth or marked with rope-like striae. Only two males were found. The body twists 90° throughout its length. The DGO to the base of the stylet is long (3.0–3.3) µm. The cephalic framework of the second-stage juvenile is weak, and the stylet is short (10.1–11.8 µm). The DGO to the base of the stylet is long (3–5 µm). The tail is very long (64–89 µm) and the hyaline portion of the tail is very narrow, making the tail finely pointed. Eggs are typical for the genus and vary in length (85.2–99.8 µm) and width (37.1–48.1 µm), having a L/W ratio of (2.1–2.6). Maximum likelihood phylogenetic analyses of the different molecular loci (partial 18S rRNA, D2-D3 of 28S rRNA, internal transcribed spacer (ITS) rRNA, cytochrome oxidase subunit II (COII)-16S rRNA of mitochondrial DNA gene fragments and partial Hsp90 gene) placed this nematode on an independent branch in between M. graminicola and M. naasi and a cluster of species containing M. chitwoodi. M. fallax, and M. minor. Greenhouse tests showed that yellow and purple nutsedge were the best hosts, but perennial ryegrass, wheat, bentgrass, and barley were also hosts.

Yellow nutsedge (Cyperus esculentus L.) and purple nutsedge (C. rotundus L.) are perennial weeds of global importance that can enhance survival and population densities of Meloidogyne incognita (Kofoid and White, 1919) Chitwood, 1949 and result in injury to crops (Schroeder et al., 1993(Schroeder et al., , 1994Thomas et al., 2004). Shoot growth of both nutsedges is not affected by M. incognita parasitism; numbers and size of reproductive tubers increase as nutrient resources are reallocated to roots, and galls do not develop on roots (Mauk et al., 1999;Schroeder et al., 1999). During routine bioassays of nutsedge cultures maintained in the greenhouse, galling was observed on roots of purple nutsedge, but was not present on any comingled roots of M. incognita-susceptible 'Rutgers' tomato (Solanum lycopersicum L.). Subsequent dissection of the galls revealed small, mature Meloidogyne females and egg masses that were primarily contained inside the root tissue. Eggs were recovered from roots of purple nutsedge, but not that of tomato following extraction with sodium hypochlorite. Additional pots of purple nutsedge and Rutgers tomato that were established in pasteurized soil using surface-sterilized nutsedge tubers and inoculated with eggs recovered from galled purple nutsedge roots showed similar results: root galling and egg recovery from nutsedge, but no galling or egg recovery from tomato.
Additional research on the morphology and host range conducted at Virginia Tech and New Mexico State University revealed several unusual morphological characters and a unique host range that indicated it was a new species. The perineal pattern, shape of the female stylet, and shape of the male head and stylet were unique and different from those of any other described species. Meloidogyne aegracyperi n. sp. is described herein, and the common name 'nutsedge root-knot nematode' is proposed. The specific epithet was derived from the Latin word 'aegra' and the host plant name, meaning 'diseased Cyperus.'

Materials and methods
Meloidogyne aegracyperi n. sp. was established from field collected soil and plants from the type locality and propagated on purple nutsedge to maintain stock cultures that were kept in a greenhouse at 22 to 28°C. All nematodes used in morphologic, morphometric, and host range studies were from these cultures.

Morphological studies
Males and second-stage juveniles (J2) were extracted from washed galled roots incubated in a moist chamber. Light microscopy (LM) observations were made from specimens mounted on 5% water agar pads and paralyzed with 0.1 m sodium azide (Eisenback, 2012). Females and J2 were prepared for scanning electron microscopy (SEM) according to Eisenback (1985). Perineal patterns were prepared for SEM according to Charchar and Eisenback (2001). They were observed and photographed by LM with negative contrast as reported by Eisenback (2012). Eggs were measured in fresh tap water mounted on agar pads. All LM observations except for perineal patterns were made with a bright field microscope, and at least 100 specimens were observed. However, only two males were recovered from hundreds of infected nutsedge plants. Measurements were made of females in 2% glutaraldehyde in 0.1 M cacodylic acid buffer, pH 7.2; perineal patterns were mounted in glycerin, and extracted stylets (Eisenback et al., 1980) were measured with the SEM. In total, 30 specimens of J2 and females were randomly selected for measurements along with the two males.

Sample isolation
In total, 24 individual females were isolated from nutsedge roots and transferred into separate polymerase chain reaction (PCR) tubes containing 20 μ l of lysis buffer (0.2 M Tris-Cl, pH 7.8) and stored at −20°C.

Lysis, PCR, and sequencing
Lysis was performed using a rapid single tube lysis procedure (Solano and Hanson, unpubl. data). Briefly, samples were removed from −20°C and incubated at 90°C for 10 min. After heating, 30 μ l of Proteinase K digestion mixture was added to each sample (5 μ l Proteinase K (Qiagen Inc., Valencia, CA), 3 μ l of 10 × Platinum Taq DNA Polymerase PCR Buffer (Invitrogen, Carlsbad CA), and 22 μ l water per sample).
Samples were then sonicated for 8 min in a Branson 2510 ultrasonic cleaner then incubated for 30 min at 60°C. After Proteinase K digestion samples were frozen at −80°C for 10 min then incubated at 90°C for 10 min. After heating, 50 μ l of water was added to each tube and samples were well mixed then stored at −20°C prior to PCR.
Sequence-based identification of individual nematodes was performed using 18S rRNA and heat shock protein 90 (Hsp90) markers with primers used for amplification and sequencing listed in Table 1. Amplification of the 18S rRNA was performed using two primer sets; 79 F deg plus 1629 R deg which amplifies the majority of the 18S rRNA gene and 983 F deg plus Nema 28S R AG which amplifies the 3′ ~1/2 of the 18S rRNA gene and the ITS region. The previously described primer set RKN-d1F plus RKN-5R was used for amplification and sequencing of the Hsp90 gene (Skantar and Carta, 2004). All amplification reactions were performed in 40 μ l PCR reactions using NEBNext Q5 Hot Start HiFi PCR Master Mix (New England BioLabs, Ipswich, MA). PCR reactions contained: 2 µl of nematode lysate, 4 µ l of each primer, 17.2 µ l of water, and 20 µ l of the 2 × PCR master mix. The cycling conditions for the 18S rRNA reactions were 94°C for 2 min, followed by 34 cycles of 94°C for 20 sec, 58°C for 30 sec, and 65°C for 80 sec, with a final extension at 68°C for 10 min. Cycling conditions used for the Hsp90 gene were 94°C for 2 min, followed by 40 cycles of 94°C for 30 sec, 55°C for 20 sec, and 68°C for 90 sec, with a final extension at 68°C for 5 min. PCR products were run on a 1% agarose gel in SB buffer (Brody and Kern, 2004) and stained with SYBR Gold according to manufacturer's instructions (Invitrogen Inc., Carlsbad, CA). Gels were visualized on a digital gel imager (UVP EC3 Imaging System, UVP Inc). PCR reactions that contained the expected size amplicons were treated with ExoSAP-IT (Affymatrix Inc., Santa Clara, CA) according to manufacturer's instructions. Amplicon concentrations in ExoSAP-IT-treated reactions were determined using a commercial SYBR green-I based DNA quantification kit (Invitrogen Inc., Carlsbad, CA) and read on a fluorescent plate reader (Synergy HTX Multi-Mode Microplate Reader). Automated dideoxy sequencing was performed by Genewiz Inc. (South Plainfield, NJ). Sequence editing, assembly, and analysis were performed using the integrated sequence analysis package, Genious 9.0.2 (Kearse et al., 2012) with the MAFFT aligner being used to generate multiple sequence alignments. Maximum likelihood phylogenetic trees were generated using Mega 6.06 with default parameters and 500 × bootstrapping (Tamura et al., 2013).

Description Female
Mature females with their egg masses are usually contained completely inside galled root tissues. They are very small (373 µm long) and pearly white.
Their body shape is unique from many other species because the neck is often at a 90 to 130° angle to the protruding posterior end that contains the perineal pattern. Lip region low, cephalic framework weakly developed, with one head annule. The cone of the stylet slightly curved dorsally, posterior edges of the knobs angular, and tapering onto the shaft. The distance of the dorsal esophageal gland orifice (DGO) to the base of the stylet relatively long (4-6 µm). Excretory pore level, with the base of the stylet, is present. The lining of the metacorpus triradiate, with the posterior and anterior portions rounded. Numerous (3-10) small vesicles present in the anterior metacorpus. Two, small, rounded esophago-intestinal cells at the base of the metacorpus, followed by a large nucleated dorsal esophageal gland lobe with two smaller nucleated subventral esophageal gland cells. The didelphic ovary is typical for the genus. Six, large rectal gland cells connect to the rectum and produce the gelatinous matrix forming the egg mass. The perineal pattern is raised on a protuberance at the posterior end of the body. It contains a rounded dorsal arch with a tail terminal area that is usually smooth, but may be marked with thick lines and many horizontal, rope-like striae. Phasmids are typical for the genus. The vulval lips are usually flattened, but they may be rounded and slightly protruding. Smooth, regular striae surround the vulva and tail terminal area and give the appearance of a dorso-ventrally elongated oval pattern.

Male
Two males were found. The characteristics are as follows: anterior end tapering, labial disc slightly concave around the stoma, one distinct head annule, cephalic framework slight, stylet shaft tapering posteriorly. Body twisting 90° throughout its length. Stylet knobs rounded and set-off from the shaft. The distance of the DGO to the base of the stylet is long (3-3.3 µm). Esophageal glands overlapping the intestine ventrally. Four lines in the lateral field. Paired spicules with gubernaculum are typical for the genus. Tail tip slightly set-off from the remainder of the body.

Second-stage juvenile
It has a body with a very long tail and tail terminus. Cephalic framework is weak, stylet is small, with a constriction near the junction of the shaft and knobs. In SEM, the head has a slit-like oral opening placed on the rounded labial disc and surrounded by six small pit-like openings of the inner labial sensilla. Small depressions in the cuticle on the dorsal and ventral lip pairs mark the outer labial sensilla. Rounded knobs,

DNA sequence-based identification
DNA sequences were manually edited to remove low quality sequence and PCR priming regions prior to making assemblies for both the 18S rRNA and Hsp90 loci. Ambiguity-free assemblies with an average length of 1,055 bp and covering ~2/3 of the 18S rRNA gene were generated for 12 of the 18S rRNA amplicons while assemblies with an average length of 754 bp were generated for 21 of the Hsp90 amplicons. Consensus sequences from each assembly were used to construct multiple sequence alignments. No polymorphisms were detected in either the 18S rRNA or Hsp90 multiple sequence alignments suggesting that all nematodes in the sample were from a clonal population. The 18S rRNA sequence spanned nt  Karssen, 1996 which were 89, 85, and 84% identical to M. aegracyperi n. sp., respectively. Maximum likelihood trees comparing M. aegracyperi n. sp. sequence to comparison sequences from GenBank were created for each gene using the maximum likelihood method based on the Tamura 3-parameter model (Tamura, 1992). Initial trees for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach, and then selecting the topology with the highest log likelihood value. Evolutionary analyses were conducted in MEGA6 (Tamura et al., 2013). Both trees were robust with most branches, including M. aegracyperi n. sp. containing branches, having bootstrap support values well over 50%. Both trees also displayed similar topology with M. aegracyperi n. sp. sequences residing on an independent branch in between a branch containing M. naasi and/or M. graminicola, and a second branch containing sequences from M. fallax, M. chitwoodi Golden, O'Bannon, Santo, and Finley, 1980 and M. minor (18S rRNA tree in Fig. 8, Hsp90 tree in Fig. 9). While the 18S rRNA tree has more detail owing to more comparison sequences being available in Genbank, the Hsp90 tree showed higher bootstrap support for all branches owing to more variability in this gene, with the majority of the variability coming from intron regions (data not shown).
The new species was also molecularly characterized using the D2-D3 fragment of the 28S rRNA, the ITS region of the rRNA, and the COII-16S rRNA region. In this case, only a single amplicon was sequenced for each of these loci. The D2-D3 region of 28S rRNA gene yielded an amplicon of 766 bp, including the primer sequences. Blast searches showed as top hit

Type material
The original population was derived from the type locality and host. Holotype female and 6 females and 10 second-stage juvenile paratypes isolated from a single egg mass and maintained on purple nutsedge in a greenhouse were deposited in the USDA Nematode Collection (USDANC), Beltsville, Maryland. Paratypes (3 females and 10 J2) were deposited in the Canadian National Collection of Nematodes, Ottawa, Canada.

Differential diagnosis
The most important measurements of females, males, and second-stage juveniles of M. aegarcyperi n. sp. are compared with those of the most closely related species, M. naasi, M. fallax, M. minor, M. chitwoodi, and M. graminicola, in Table 3. Meloidogyne aegracyperi n. sp. is characterized by the small female (373 µm long × 306 µm in diameter) with a perineal pattern that occurs on a posterior protuberance that is at a 90 to 130° angle with the neck. In M. naasi, the female is larger (557 µm long × 330 µm in diameter) (Franklin, 1965) similar to that of M. graminicola (573 µm long × 419 µm in diameter) (Golden and Birchfield, 1965). The perineal pattern of M. naasi is often on a protuberance, and it is rounded to oval-shaped with striae that completely encircle the tail terminus, anus, and vulva; the tail terminus is usually smooth, but may contain rope-like striae that are parallel with the vulva (Franklin, 1965). The perineal pattern of M. graminicola is typically flat (Golden and Birchfield, 1965). The smooth region around the tail of M. aegracyperi n. sp. makes it different from that of M. naasi which usually contains rope-like striae that are perpendicular to the vulva. The edges of the stylet knobs are angular, the stylet is short (12 µm), and the DGO is long (3.6-6.1 µm) in M. aegracyperi n. sp., whereas in M. naasi the edges of the stylet are smooth, the stylet is longer (13 µm), and the DGO is shorter (2-4 µm) (Franklin, 1965). In M. graminicola, the stylet is shorter (11 µm) and the DGO is similar to that of M. naasi (3-4 µm) (Golden and Birchfield, 1965). Males are very rare in M. aegracyperi n. sp. which may be a useful diagnostic character since males are common in M. naasi and M. graminicola. The second-stage juvenile resembles that of M. naasi; however, the body is slightly shorter

Discussion
Meloidogyne aegracyperi n. sp. is morphologically similar to M. naasi, but it can be distinguished as a unique species based on features of the female, male, and second-stage juvenile. Superficially, the gross morphology of the second-stage juvenile and similar appearance of the perineal pattern of the female could cause a wrong identification of M. aegracyperi n. sp. as M. naasi. However, measurements of the stylets, DGO, body length, tail length, and hyaline tail terminus easily separate these two species. Meloidogyne aegracyperi n. sp. appear to be closely related phylogenetically to M. graminicola and M. naasi according to the trees that were drawn based on similarities of DNA sequences of the 18s Table 4. Hosts and non-host plants of Meloidogyne aegracyperi n. sp. compared to eight populations of its closest-related species: M. graminicola Birchfield, 1965 (Minton et al., 1987;Soomro and Hague, 1992a,b); and M. naasi Franklin, 1965, reported by Franklin (1965 from the type population in England; Radewald et al. (1970) and Allen et al. (1970) from California; from England, California, Illinois, Kansas, and Kentucky.