Taxonomy and Identification of Principal Foliar Nematode Species (Aphelenchoides and Litylenchus)

Nematodes are Earth’s most numerous multicellular animals and include species that feed on bacteria, fungi, plants, insects, and animals. Foliar nematodes are mostly pathogens of ornamental crops in greenhouses, nurseries, forest trees, and field crops. Nematode identification has traditionally relied on morphological and anatomical characters using light microscopy and, in some cases, scanning electron microscopy (SEM). This review focuses on morphometrical and brief molecular details and key characteristics of some of the most widely distributed and economically important foliar nematodes that can aid in their identification. Aphelenchoides genus includes some of the most widely distributed nematodes that can cause crop damages and losses to agricultural, horticultural, and forestry crops. Morphological details of the most common species of Aphelenchoides (A. besseyi, A. bicaudatus, A. fragariae, A. ritzemabosi) are given with brief molecular details, including distribution, identification, conclusion, and future directions, as well as an updated list of the nominal species with its synonyms. Litylenchus is a relatively new genus described in 2011 and includes two species and one subspecies. Species included in the Litylenchus are important emerging foliar pathogens parasitizing trees and bushes, especially beech trees in the United States of America. Brief morphological details of all Litylenchus species are provided.


Introduction
Foliar nematodes are mostly pathogens of ornamental crops in greenhouses, nurseries, and forest trees, as well as field crops [1]. Foliar nematodes include several nematode genera among which Aphelenchoides, Anguina, Ditylenchus, and Litylenchus. Foliar nematodes have been documented as associated with more than 1100 different species of plants, belonging to 126 botanical families, to include dicots, monocots, gymnosperms and angiosperms, ferns and mosses [2]. Aphelenchoides, as well as nematodes of genus Litylenchus, are phytoparasites known to infect leaves, stems, and buds [3]. The damage caused by the foliar nematodes can cause marketability problems in ornamentals because they interfere with the appearance of the plant or they can reduce yield in food crops [2].

General Techniques
For morphological observation, adult specimens of foliar nematodes can be extracted from fresh leaves. The best method for extracting nematodes from fresh leaves is by using the Baermann Funnel method. Another simple extraction method of nematodes from rice seeds was described by Hoshino and Togashi [4]. They cut the rice seeds longitudinally in two, then transferred the pieces into single plastic pipette tips, which were placed upright in glass vials with water. The pipet tips are transferred to new vials 2, 4, 8, and 24 h later the rate of nematode extraction can be observed. Nematodes are

•
Six fused, non-annulated, similar lips, slightly offset from body; • Male tail without bursa, with one pair of approximately adanal and two pairs of postanal, ventro-submedian, caudal papillae; • Spicules paired and shaped like rose thorns; • Tails of both sexes never elongate filiform but short, tapering, conical, and frequently ending in one or more mucrones.
A polytomous key was developed and tested on 14 populations by Hockland [18] and the primary key characters were identified as: • The length of the post-vulval sac; • The shape of the tail terminus and tail; • Body length; • Ratios 'a' and 'c'.
A more detailed characterization of the genus was first given by Allen [19]: Female and male tail never elongate filiform. A recent characterization of Aphelenchoides was given by Wheeler and Crow [20]: • Stylet with small basal knobs; • Males are common; • Vulva located near 2/3 the body length from the anterior; • Prodelphic (anteriorly outstretched) ovary and a post-uterine sac; • Males have prominent, thorn-shaped spicules (paired, cuticularized copulatory structures).
According to Hunt [21] members of the Aphelenchoides genus can be diagnosed by the following morphological characteristics: • Body length between 0.4 to 1.2 mm (commonly from 0.4 to 0.8 mm); • Females become straight to ventrally arcuate when heat relaxed while males assume a "walking-stick shape"; Plants 2020, 9,1490 4 of 31 • Cuticle finely annulated, two to four (rarely six) incisures in the lateral field; • Stylet slender with basal knobs (sometimes indistinct), length between 10-12 µm; • Long and slender procorpus; well-developed spherical to rounded-rectangular shaped metacorpus, with central valve plates; esophageal gland lobe long, with dorsal overlap of the intestine; • Vulva usually between 60 and 75% of the body length; • Ovary monoprodelphic, typically outstretched, but may reflex; • Post-vulval sac present most of the times; • Oocytes in one or more rows; • Post-uterine sac present (sometimes absent) and most of the times contains spermatozoa; • Tail shape is conoid to variable; males have a tail more strongly curved ventrally and papillae variable; • Tail terminus with one or more mucros or without mucros; • Spicules well-developed, thorn-shaped, paired and separate without bursa.

Genus Aphelenchoides Fischer, 1894
Emended Diagnosis [22] These nematodes are small and slender, averaging around one millimeter in length and a width less than 20 microns. One characteristic of thee Aphelenchidae nematodes family is that they have a larger median bulb as compared to other plant parasitic nematodes in the order Tylenchida. The dorsal esophageal gland orifice connects to the esophageal lumen at the base of the stylet in most plant-parasitic nematodes, but in Aphelenchida, this duct empties into the esophageal lumen within the median bulb. In Aphelenchoides, males are more common, and they reproduce primarily by amphimixis. In most species, the vulva of the female is located near 2/3 the body length from the anterior. Females have a single, prodelphic (anteriorly outstretched) ovary and a post-uterine sac, while males have prominent, thorn-shaped spicules (paired, cuticularized copulatory structures). There is a considerable variation in the shape of the tail terminus within populations of species of the genus Aphelenchoides. The tail terminus can be used to divide Aphelenchoides species into four groups [22]. The four groups are: a.
Tail without any outgrowth or mucro; b.
Tail with one or sometimes two mucronate structures on tail end; c.
Star shaped tail with four mucronate structures; d.
Tail end with outgrowth other than spine or star.

Systematic Position
The number of valid nominal species in the Aphelenchoidea is still debatable. However, modern molecular technology may help solve this problem soon. According to Hunt [23] there are 453 'valid' species in Aphelenchoidea, of which 33 belong to the Aphelenchidae and 420 to the Aphelenchoididae. From Aphelenchoididae family, Aphelenchoides genera has the most species, namely 153 [23]. A more recent 2015 study conducted by Sánchez-Monge et al. [2] assigned approximately 200 species to the genus. However, after conducting a through literature review, the authors have identified 182 valid nominal species assigned to the Aphelenchoides genus.

Diagnostic Characters
Some diagnostic characteristics of Aphelenchoides are presented below [3]: Tails of both sexes never elongate-filiform, but usually more or less tapering, conical, and frequently ending in one or more mucrons.

Genus Synonyms
Emended list of Aphelenchoides species and synonyms: Aphelenchoides besseyi (Figure 1) is an economically important pathogen of rice and has been reported from many countries. However, it is not commonly found in ornamentals [38,39], with the exception of some reports on tuberose [36], begonia [40], gerbera [41], hydrangea [27], tuberose [42], and even on bird nest fern [43]. A. besseyi distribution is mostly in warmer climates, whereas A. ritzemabosi and A. fragariae are more commonly associated with temperate climates, while found in both tropical and temperate localities [1]. Plants 2020, 9, x FOR PEER REVIEW 11 of 34

Description
Female: female specimens share a slender body, slightly arcuate ventrally when relaxed, anteriorly tapering from the level of esophageal glands to the head, which is one half of the body width. Four lateral lines (occasionally six noted) are present in the lateral field ( Figure 2). In en face view, the pore-like amphids are on outer margins of lateral lips; four papillae, one on each submedian lip ( Figure 2). Lip region is non-striated and set off from body by a constriction as wide as or slightly wider than adjacent body; labial framework weakly developed; cheilorhabdions well sclerotized. Basal knobs of spear distinct, 2 µm across. Procorpus cylindrical; median esophageal bulb one and a half times to twice as long as wide, with refractive valvular apparatus slightly posterior to center. Esophageal glands extending over intestine 5 to 8 body widths. Excretory pore at 58 to 83 µm from anterior end, level with or slightly anterior to nerve ring. Hemizonid distinct in specimens from rice seeds (but not from cultured specimens), 11 to 15 µm behind excretory pore; hemizonion 20 to 30 µm behind hemizonid, usually difficult to see. Tail straight, slender, regularly tapering to a narrowly rounded end, 3-5 to 5 anal body diameters long; mucro with 3 to 4 processes. Ovary not extending to esophageal glands; oocytes in 2 to 4 rows; spermatheca very conspicuous, elongate oval, full of rounded sperms showing a central nucleolus usually surrounded by a circle of black dots of unknown nature. Post-vulval uterine sac short, slender and extending up to one fourth of the distance from vulva to anus (2.5 to 3 body diameters) often found empty and collapsed but more conspicuous and rounded in nematodes from cultured specimens. Vulval lips slightly protruding after Fortuner [45].
Male: tail end usually curved by 90 • (a greater curvature has also been found) in specimens killed in 3% formaldehyde; mucro of diverse shape, with 2 to 4 processes. Spicule length between 17 to 21 µm along dorsal limb. Different morphometric characters, such as the shape of the head, the position of the excretory pore in relation to the nerve ring and the shape and length of the post-vulval uterine sac were found to be variable between populations [45].

Distribution
According to Devran et al. [46], A. besseyi was on the quarantine lists of nine countries in 1982 and up to 70 countries in 2002. Centre for Agriculture and Biosciences International (CABI), Invasive Species Compendium [47] lists A. besseyi being present in 75 countries around the world. The quarantine pests lists A. besseyi as the second most prevalent nematode after Globodera rostochiensis [39].

Distribution
According to Devran et al. [46], A. besseyi was on the quarantine lists of nine countries in 1982 and up to 70 countries in 2002. Centre for Agriculture and Biosciences International (CABI), Invasive Species Compendium [47] lists A. besseyi being present in 75 countries around the world. The quarantine pests lists A. besseyi as the second most prevalent nematode after Globodera rostochiensis [39].

Description
Female: have a slender body, attenuated slightly anteriorly, and more prominently toward posterior end ( Figure 3). When relaxed by gentle heat the position of the body is straight and only the tail region is slightly curved. Cuticle is finely striated, with annuli measuring between 0.47-0.58 µm wide and 0.39-0.51 µm thick. Lateral field has two lateral lines. Head distinctly set off from body. Lip region rounded, offset with no annules. Stylet weak, with small basal swellings. Metacorpus rounded, occupying approximately 73% of body width. Nerve ring is located about 1/2 body width behind metacorpus. Excretory pore opposite anterior margin of nerve ring. Vulva a transverse slit and slightly protruding, about 66% of body length from anterior end. Post-vulvar uterine sac extending for one-fifth of distance from vulva to end of tail. Rectum prominent, straight, near ventral body wall, and in length approximately three-fourths of anal body width. Tail gradually tapering to terminus, which is unevenly bifurcated with one prong longer than the other.  Females of A. bicaudatus ( Figure 4) can be differentiated from other members of the genus by having an unevenly bifurcated tail tip with prongs of different lengths [51].

Distribution
A. bicaudatus was recorded in most of the tropical and subtropical regions of the world as well as some warmer temperate areas [21]. More specifically, it was reported in the following countries: Australia, Brunei, France, Japan, USA, Russia, Venezuela [28], South Korea [52], Taiwan [32].

Distribution
A. bicaudatus was recorded in most of the tropical and subtropical regions of the world as well as some warmer temperate areas [21]. More specifically, it was reported in the following countries: Australia, Brunei, France, Japan, USA, Russia, Venezuela [28], South Korea [52], Taiwan [32].

Strawberry Crimp Nematode (Aphelenchoides fragariae (Ritzema Bos, 1891) Christie, 1932)
Aphelenchoides fragariae was originally described by Ritzema Bos (1891) in specimens recovered from strawberry plants sent to him from England ( Figure 5). When compared to all the Aphelenchoides species mentioned previously, it has the widest distribution as well as hosts range (more than 600 species), to include ferns, herbaceous perennials and bedding plants [2,33,53]. A. fragariae is an ectoand endo-parasite of the above ground parts of a plant, but it can also be mycetophagous [2,21,33]. The nematodes enter the plant leaves through stomata or wounds [1,47]. In the leaves, nematodes feed on mesophyll cells which causes characteristic vein delimited lesions [1,47]. A. fragariae survives overwinter in soil, dormant buds, dry leaves, but not in roots [18,47]. Research showed that A. fragariae nematodes can tolerate temperature as high as 40 • C and as low as −80 • C once in leaf tissues [18].

Description
Body very slender (a = 45-63 µm), straight or arcuate when relaxed. Cuticle marked by fine transverse striae about 0.9 µm apart; lateral field with two incisures, 1/7th of body-width. Cephalic region, smooth, anteriorly flattened with straight to curved side margins, almost continuous with neck contour. Lips without annulation. Stylet slender, approximately 10 µm long, with small but distinct basal knob. Median esophageal bulb well developed, oval. Nerve ring about one body width behind median bulb. Excretory pore level at or close behind nerve ring. Esophageal glands stretched five body widths behind the medium bulb, joining esophagus immediately behind the medium bulb. Tail elongate-conoid, terminus bearing a terminal peg which is simple, spike-like.
Female: vulva a transverse slit, at approximately 64-71% of body. Spermatheca elongate-oval. Posterior uterine sac more than half the vulva-anus distance, often containing sperm. Ovary single, with oocytes in a single row. Tail terminus with a single mucronate points point enlarged at the base.

Distribution
A. fragariae has a widespread distribution in Europe, Russia, Japan and North America [21]. According to the CABI Invasive Species Compendium [47], A. fragariae is currently reported to be present in 37 countries.

Chrysanthemum Nematode (Aphelenchoides ritzemabosi (Schwartz, 1911) Steiner and Buhrer)
Aphelenchoides ritzemabosi (Schwartz, 1911) Steiner and Buhrer, also known as the Chrysanthemum foliar nematode, is a common plant-parasite infecting more than 300 plant species, second only to A. fragariae [2] in the Aphelenchoides genus based on the number plants they parasitize.

Measurements
After Allen [13]. Female: nematodes with slender body, with fine transverse striae on the cuticle. Four lines present in the lateral field. Lip region set off, wider than neck at base of lips with no annulations. Hexaradiate framework weakly sclerotized. Stylet approximately 12 µm long, with small but well-developed basal knobs. Median esophageal bulb well developed, oval in shape. Nerve ring 1.5 body widths behind median bulb. Excretory pore located behind nerve ring, approximately 0.5-2 body widths posterior to nerve ring. Esophageal glands extending 4 body widths over the intestine, joining esophagus immediately behind median bulb. Oocytes in multiple rows, several in a cross-section at middle of ovary. Posterior uterine branch extending for more than half the vulva-anus distance, usually containing sperms. Tail elongated-conoid. Terminus peg-like armed with two-four small mucronate points pointing posteriorly.
Male: males are common, having a tail curvature at about 180 degrees when relaxed. Testis single. Three pairs of ventro-submedian papillae. First pair adanal, second midway on tail, third near end. Spicules smoothly ventrally curved, the ventral piece without a ventral process at the distal end; dorsal limb 20-22 µm long. Terminus peg-like armed with two-four small mucronate points.

Distribution
Aphelenchoides ritzemabosi is a major pest of chrysanthemum in Europe, Russia, North America, South Africa, New Zealand, Australia, and Brazil [25]. According to the CABI Invasive Species Compendium [55], A. ritzemabosi is currently reported to be present in 35 countries around the world.

Identification
Accurate identification of foliar nematodes (Aphelenchoides spp.) is crucial for effective disease control. Major efforts should be geared towards rapid and accurate classification of the pathogens so that appropriate control measures could be taken. In addition, timely and accurate diagnosis is also needed to make sound decisions regarding quarantine of imported and exported plant material and commodities. Nevertheless, the identification of foliar nematodes to species level remains a challenging endeavor. The diagnosis and/or relationship between conserved morphology, variable morphometrics, host effects, intraspecific variation, existence of cryptic species, and the ever-increasing number of described species, still vary significantly. To add to the confusion, there is verification of mixed populations and/or detection of rare species which require(s) identification techniques, including morphology of adult females; male, and labial region shape, and stylet morphology; V% age, body length, and shape of tail and tail terminus, and, in some cases, biochemical or molecular methodologies. Because of an increasing number of described species, the value of many of these characters often show large intraspecific variation. Isozyme electrophoresis has discriminated a number of these otherwise cryptic species. Currently used PCR-based molecular methodologies offer hope for a future relying on bigger genebanks that could be used by scientists for a more accurate specie identification. Integrated morphology and molecular approaches are essential to future improved identification of Anguinata nematodes. Detailed diagnostic characters differentiating various species of foliar nematodes have been given by authors such as Allen [19], Hunt [21], Shahina [22].

Genus Litylenchus Zhao, Davies, Alexander and Riley, 2011
Genus Litylenchus Zhao, Davies, Alexander and Riley, 2011 is a new genus with much smaller number of species when compared to Aphelenchoides genus. Litylenchus crenatae Kanzaki, 2019, Litylenchus crenatae mccannii Carta 2020, are emerging foliar pathogens of major economic importance. Nematodes from this genus parasitize trees (Fagus grandifolia) and bushes (Coprosma repens). Litylenchus crenatae mccannii described by Carta et al. [11] seems to be a very aggressive subspecies with devastating effects on beech trees (Fagus grandifolia). Even though Litylenchus crenatae mccannii was initially found infesting beech trees in Ohio [11], it was also reported in several other states and provinces, to include Pennsylvania, New York, Ontario, Canada [56], Connecticut [57], New Jersey, Rhode Island, and West Virginia (unpublished data).

Systematic Position
Based on phylogenetic analyses, Litylenchus genus [35] is close to Subanguina. However, the two genera have many morphological differences as highlighted below: List of Litylenchus species and synonyms: Type species: 1.

Litylenchus coprosma
Other species
Semi-obese female: when killed by heat body is almost straight, semi-obese. Maximum body width is at mid-body. Body cuticle finely striated, almost smooth. Four lines can be observed in lateral field extending almost to tail terminus. Head offset, cephalic framework, and stylet as described for male. Excretory pore located ca 3-3.5 body diameter from anterior, opening near anterior end of terminal bulb, duct with obvious cuticular lining. Hemizonid, pharynx, pharyngeal glands, and pharyngo-intestinal junction as described for male. Nerve ring is located approximately 100 µm from anterior extremity. Deirids and phasmids not seen. Gonads are monodelphic, prodelphic, outstretched, crustaformeria forming a quadricolumella. Oocytes arranged in single row. Oviduct with several cells forming a valve just anterior to elongate, sac-like spermatheca. Vulva located 7-11 anal body diameter anterior to anus (80-85% of body length). Vulval slit occupying almost half body diameter when viewed laterally, vagina almost perpendicular to body wall. Post-uterine sac extending 20-70% of distance from vulva to anus, approximately 2.7 anal body long, sometimes with sperms, lacking cellular relicts of posterior ovary. Rectum difficult to see, anus pore-like, opening in a cuticular depression. Tail approximately 4-5 anal body diameter long, conoid, straight, with a variable tail terminus, may be bluntly rounded, more or less bifurcate, or appear bilobed. Mucro not observed.
Slender female: very similar to the semi-obese females, but slender. Head capsule is a little bit bigger, 59-77% of body diameter at level of stylet knobs compared to the semi-obese females, where the head capsule is between 48-62%. Quadricolumella cells are smaller than in semi-obese female.
Male: when killed, the nematodes assume a smoothly ventrally arcuate shape, body cylindrical, narrowing to a bluntly rounded conoid tail. Body cuticle smooth with three incisures in the lateral field visible in the region of procorpus increasing to four incisures at mid-body and extending almost to tail tip. Head is set off from the body, smooth, and not annulated. Lightly sclerotized cephalic framework with six sectors.

Distribution
Litylenchus coprosma was reported in New Zealand from Coprosma repens [35] and from Coprosma robusta [26]. En-face view shows amphidial apertures appearing as small lateral slits. Stylet robust, with welldeveloped rounded knobs, conus comprising ca 40% of stylet length, diameter narrowing sharply to be distinctly less than that of shaft. The opening of dorsal esophageal gland is located just posterior to stylet knobs. Nerve ring is located 70-110 µm from anterior extremity, surrounding isthmus, ca one body diameter long. Excretory pore is located ca 5-6 body diameter from anterior end, opening posterior to nerve ring. Hemizonid located immediately anterior to excretory pore. Procorpus cylindrical, fusiform, non-muscular median bulb which is approximately one body diameter long and narrowing sharply to isthmus which is slender, cylindroid, marked off from terminal bulb, pharyngeal glands enclosed in a pyriform terminal bulb containing three large nuclei. Esophago-intestinal junction is immediately posterior to terminal bulb and covered by it in some specimens, valve present, without hyaline cells. Deirids and phasmids were not observed. Testis outstretched, reflexed in some specimens, reaching to nerve ring in some specimens, with spermatocytes arranged in a single row. Spicule paired, similar, arcuate, 2-3 µm wide at anterior end, gradually narrowing towards tip. Capitulum absent. Gubernaculum simple and arcuate. Tail conoid with a variable in shape tail terminus, usually bluntly rounded, but may have terminal process; no mucron observed. Bursa membranous, crenate in some, arising ca 1-2 cloacal body diameter anterior to cloacal aperture, extending nearly (90-95% of tail length) to tail tip.

Description
Female (Figure 9a): when killed, the nematodes assume a smoothly ventrally arcuate shape, body cylindrical, vermiform to semi-obese. Anterior part and cuticular morphology similar to mature male. Female gonad single, anteriorly outstretched reaching to level of pharyngeal glands. Oocytes are arranged in single row in entire ovary. Oviduct is short and spermatheca is elongated oval filled with large sperm, posteriorly connected to crustaformeria, which consists of four rows of four large and rounded cells, i.e., forming a quadricolumella, posteriorly connected to uterus by a cluster of small cells. Uterus, a thick-walled tube, sometimes containing an egg. Vagina at right angles to body axis or slightly inclined anteriorly. Vulva, a horizontal slit. Post uterine sac present, well-developed, with a thin wall and a short appendage comprising several rounded cells at distal end. Rectum is about less than one anal body diameter in length, with muscular constriction at intestine-rectal junction. Tail is short and broad, abruptly narrowing at the end with a conoid and bluntly pointed terminus, sometimes appearing like a conical blunt mucron.   Male ( Figure 9b): when killed, the nematodes assume a smoothly ventrally arcuate shape, body cylindrical, not clearly obese or semi-obese. Body cuticle annulated with six incisures in the lateral field at the anterior part of body, 6-8 incisures around mid-body, and posteriorly connected to bursa. Deirids present in middle of lateral field slightly posterior to hemizonid and excretory pore. Lip region slightly offset from body, with a truncated shape, separated by a very shallow constriction.
Stylet with narrow lumen and a shaft with prominent rounded basal knobs (3.6 µm in diameter). Dorsal esophageal gland is located posterior to stylet knobs. Procorpus is cylindrical. Median esophageal bulb is weakly developed, with small metacarpal valve at mid-bulb length. Isthmus is cylindrical, but narrower than the procorpus, enveloped by the nerve ring in its mid-length. Broad and glandular gland lobe with three large nuclei were observed ( Figure 10). Hemizonid found at level the beginning of expansion of pharynx. Excretory pore located slightly posterior to hemizonid, with clear secretory-excretory duct. Nuclei of the esophageal overlap observed between hemizonid and pharyngo-intestinal junction, two being just anterior to the third, and latter located slightly anterior to junction. Gonad single, anteriorly outstretched reaching to level of pharyngeal glands. Testis outstretched with spermatocytes arranged in single row from anterior to middle part of testis and in multiple rows in posterior section. Vas deferens is visible, consisting of rounded cells, sometimes containing well-developed sperm. Spicules paired, smoothly arcuate ventrally, forming a smoothly curved horn-like blade with bluntly pointed distal end in lateral view (V-shaped). Gubernaculum simple, crescent or bow-shaped in lateral view. Bursa peloderan, well developed arising three cloacal body diameter anterior to cloacal opening and terminating near tail tip. Tail is conoid, bluntly pointed in lateral view.

Description
Females have long and slender bodies, a lip region slightly offset with 5 annules. Stylet measures 9.7 ± 0.9 µm in young females with 5% of the pharynx length, and 7-10% of the pharynx length in males. Median bulb is weak without an obvious valve. The vulval region is kinked and irregular and the anterior gonad is relatively long, nearly five times the length of the post uterine sac. The post uterine sac is about three times the vulval body width and one quarter of the vulval anal distance. The rectum is approximately one quarter of the tail length and the anus is pore-like and obscure in most specimens. Tail is conical, slender and asymmetrically pointed, with a gradually tapering and the tail tip often with mucronate extension (Figure 11). There is a shape variation in tails of immature and mature females. Plants 2020, 9, x FOR PEER REVIEW 28 of 34 Molecularly, Litylenchus crenatae mccannii from Ohio, Pennsylvania, and the neighboring province of Ontario, Canada, showed some differences in morphometric averages among females when compared to the Japanese population described by Kanzaki et al. [32]. Ribosomal DNA marker sequences were nearly identical to the population from Japan [11]. The 18S rDNA and internal transcribed spacer (ITS) rDNA sequences for Litylenchus crenatae from Japan are 99.9% and 99.7% similar, respectively, to Litylenchus crenatae mccannii from North America. A sequence for the COI marker was also generated, although it was not available in the Japanese population [11]. The marker sequences derived from Litylenchus crenatae mccannii specimens, 104H78 and 104H82 were deposited to GenBank with accession numbers for rDNA (MK292137, MK292138) and COI (MN524968, and MN524969).
Phylogenetic trees for 18S rDNA of Aphelenchoides and Litylenchus are shown in Figures 12 and  13. Female: Litylenchus crenatae mccannii ssp. n. young female population from North America can be differentiated from the Litylenchus crenatae described from Japan by:

•
Having longer stylet 9.7 ± 0.9 µm (8.6-11.2) vs. 8 Male: males of Litylenchus crenatae mccannii ssp. n. are very similar to Litylenchus crenatae males described from Japan. Carta et al. [11] noted some differences between the North America and the Japan population such as:
Molecularly, Litylenchus crenatae mccannii from Ohio, Pennsylvania, and the neighboring province of Ontario, Canada, showed some differences in morphometric averages among females when compared to the Japanese population described by Kanzaki et al. [32]. Ribosomal DNA marker sequences were nearly identical to the population from Japan [11]. The 18S rDNA and internal transcribed spacer (ITS) rDNA sequences for Litylenchus crenatae from Japan are 99.9% and 99.7% similar, respectively, to Litylenchus crenatae mccannii from North America. A sequence for the COI marker was also generated, although it was not available in the Japanese population [11]. The marker sequences derived from Litylenchus crenatae mccannii specimens, 104H78 and 104H82 were deposited to GenBank with accession numbers for rDNA (MK292137, MK292138) and COI (MN524968, and MN524969).
Phylogenetic trees for 18S rDNA of Aphelenchoides and Litylenchus are shown in Figures 12 and 13.

Conclusions and Future Prospects
Until recently, morphology used to be the only way to differentiate nematodes. With recent developments of molecular approaches in taxonomy gaining more widespread use, molecular identification has the potential to become an indispensable tool in the near future. As the GenBank continues to expand, molecular identification can become a reliable resource for nematode identification. Classical morphology continues to play a very important role in nematode identification, being reliable, cheap and quick. Molecular approaches can complement classical morphology and are crucial for species with similar morphological characters. A blend of both morphological (including SEM), morphometric, and molecular data is essential for future new foliar nematode species. The prospects in foliar nematode taxonomy and diagnostics are dependent on molecular-based methodologies that will discriminate not only species but also at the level of host races and pathotypes. This finer discrimination provides opportunities for more focused management strategies. These techniques can provide rapid diagnostics and help resolve the present problems associated with morphologically conservative organisms. When widely employed, these characterization techniques will allow differentiation between nominal species, also enhancing our  [59] from multiple sequence alignment made with Clustal Omega (EMBL-EBI, https://www.ebi.ac.uk/Tools/ msa/clustalo/) within Geneious Prime Version 2020.2.4 (Biomatters, Ltd., Auckland, NZ).

Conclusions and Future Prospects
Until recently, morphology used to be the only way to differentiate nematodes. With recent developments of molecular approaches in taxonomy gaining more widespread use, molecular identification has the potential to become an indispensable tool in the near future. As the GenBank continues to expand, molecular identification can become a reliable resource for nematode identification. Classical morphology continues to play a very important role in nematode identification, being reliable, cheap and quick. Molecular approaches can complement classical morphology and are crucial for species with similar morphological characters. A blend of both morphological (including SEM), morphometric, and molecular data is essential for future new foliar nematode species. The prospects in foliar nematode taxonomy and diagnostics are dependent on molecular-based methodologies that will discriminate not only species but also at the level of host races and pathotypes. This finer discrimination provides opportunities for more focused management strategies. These techniques can provide rapid diagnostics and help resolve the present problems associated with morphologically conservative organisms. When widely employed, these characterization techniques will allow differentiation between nominal species, also enhancing our understanding of the phylogeny of the genus and its relationship with other plant-parasitic nematodes.