Xiphinema barooghii, Vazifeh & Niknam & Jabbari & Naghavi, 2019

Vazifeh, Nasir, Niknam, Gholamreza, Jabbari, Habibeh & Naghavi, Arezoo, 2019, Description of a new dagger nematode, Xiphinema barooghii n. sp. (Nematoda: Longidoridae) and additional data on the three known species of the genus from northwest of Iran, Journal of Nematology 51 (1), pp. 1-17 : 3-12

Xiphinema barooghii n. sp.

( Figs. 1–3 ; Tables 1–3 ).

Description

Female

It is characterized by having a cylindrical body, gradually tapered towards both ends, ventrally curved, open C to G-shape upon fixation. It has a two-layered cuticle and very fine transverse striations are visible more in tail region, 3.0–4.0 μm wide at mid-body and 11–16 μm at the tail tip. Lateral pores are present along the body, with four dorsal and five ventral located between anterior end and guiding ring. Lateral chords of 14–17 μm or those occupying one-fifth of the mid-body diameter are present. The lip region is flat and the cephalic region is rounded, separated from body contour by a shallow depression, 1.7–2.5 times as broad as high and one-fourth to one-fifth (21–28%) of body diameter at neck base. Amphidial fovea is cup shaped, with aperture occupying 52–59% of the corresponding lip region diameter, located slightly anterior to depression of head, remainder of body and pouches typical of the genus. Odontostyle is long and slender, furcates at junction with odontophore, 8.8–9.4 times lip region diameter or 1.5–1.8 times odontophore length. Odontophore with well-developed basal flanges, 14–18 μm wide, exists. A double guiding ring and a guiding sheath of 3–30μm length, depending on the degree of protraction/retraction of stylet, is present. Esophagus is slender with a weak muscular narrow part extending to a cylindrical terminal esophageal bulb with three nuclei. The esophageal basal bulb is 128–153 μm long, occupying about 24–29% of total esophagus length and 24–31 μm width. The nucleus of dorsal esophageal gland ( DN) is located at the beginning of basal bulb (8–11%), 3.7–5.1 μm in diameter, dorsal gland esophageal orifice ( DGEO), 4.7–6.5 μm from anterior end, and two smaller ventrosublateral nuclei ( SVN) located at 52–57% of the terminal bulb length. The esophageal intestinal valve is rounded conoid with 12–14 μm length. The tip of reserve odontostyle (vestigium) is observed in isthmus in some specimens. The nerve ring is positioned at 55 68% length from anterior end and intestine is simple. The female reproductive system is didelphic–amphidelphic with equally developed genital branches (428–575) μm and (387–512) μm long, respectively. Each branch is composed of a 64–117 μm long reflexed ovary, not reaching the oviduct–uterus junction; oocytes are arranged first in several rows and then in a single row; oviduct is 70–160 μm long with developed pars dilatata oviductus near the sphincter, joining the ovary terminally; oviduct-uterus junction is marked by a poorly developed sphincter and a 256–297 μm long bipartite uterus composed of pars dilatata uteri close to sphincter and a tubular part containing spines 2–5 μm long, spindle-shaped and scattered between the enlarged distal portion and the ovejector, with a lack of sperm in the genital tract. Ovejector is well developed (71- 84×15- 28 μm); vagina is perpendicular to body axis, 31–37 μm long or 42–52% of corresponding body diameter in lateral view and surrounded by robust muscles. Vulva, a transverse slit, pre-equatorial in position, is present. Pre-rectum variable is 503–700 μm in length and the rectum length is 0.7–1.1 times anal body diameter. A short tail, conoid and dorsally convex, ventrally directed with a small terminal peg, 4.5–7.5 μm long, and a distinct terminal blind canal, exists. Three to four caudal pores are present on each side.

Male

Not found.

Juveniles

All four juvenile stages were identified using morphological characters such as body length, length of replacement and functional odontostyle ( Robbins et al., 1996). The scatter diagram representing the relationships between body length, functional and replacement odontostyle of females and juveniles

JOURNAL OF NEMATOLOGY

is given in Figure 4 . Juveniles are similar to adults in gross morphology, except for their smaller size, longer tail, and undeveloped reproductive organs. Jl is characterized by the lip region being separated from body contour by a deep depression, replacement odontostyle tip being close to base of functional odontostyle and located at the level of odontophore, and tail conoid and dorsally convex, directed ventrally, has a depression on dorsal and ventral sides at hyaline level, with a curved finger like cuticular extension and blind canal at the end. The lip region in J2 is separated from body contour by a depression but in J3 and J4, it is similar to that of female, i.e., flat with the cephalic region rounded and separated from body contour by a shallow depression. In J2–J4, replacement odontostyle is located at some distance from odontophore.

Continued

Rectum length 14.0 ± 0.6 19 26.0 ± 1.4 33.0 ± 1.5 49 43.0 ± 5.4 (13.0 - 15.0) (22.0 - 28.0) (31.0 - 35.0) (35.0 - 51.0)

Tail length 61.0 ± 1.3 60 49.0 ± 1.1 46.0 ± 1.5 30 33.0 ± 2.6 (59.0 - 63.0) (48.0 - 51.0) (44.0 - 48.0) (30.0 - 38.0)

Hyaline part of tail 8.4 ± 0.3 13 13.0 ± 1.3 14.2 ± 0.5 13 13.0 ± 0.8 (8.0 - 10.0) (12.0 - 15.0) (13.0 - 15.0) (11.0 - 16.0)

In J2, the tail is conoid and dorsally convex, slightly bent ventrally and with a dorsal depression at hyaline region level; in J3, tail is conoid, dorsally convex, ventrally more or less flat with a slightly developed mucro and tail of J4 is similar to that of female.

Diagnosis and relationships

Xiphinema barooghii n. sp. belongs to morphospecies group 6 sensu Loof and Luc (1990). It is an apparently parthenogenetic species characterised by a medium-to-moderate long body of 3.67–4.25 mm; a C to G-shape upon fixation; a flat lip region, a rounded cephalic region, separated from body contour by a shallow depression of 14–15 μm width; along and slender odontostyle having 132–139 μm length; a guide ring being located at 117–132 μm from anterior end; a female reproductive system being didelphic with two opposite almost equally developed reproductive branches with spines in the tubular part of the uterus; a short tail, conoid and dorsally convex, ventrally

Continued

X. gersoni Almonte, Huelva province, Spain X. georgianum Florida, USA

Eucalyptus KC567180 – DQ299497

X. globosum Valdeinfierno in the Los Alcornocales Regional Black alder, Alnus glutinosa GU549474 Park, Alcalá de los Gazules, Cádiz province, L. Gaertn., and river bank southern Spain grapevine, Vitis riparia

X. granatum Saveh , Markazi province, Iran Pomegranate trees (Punica JQ240273 granatum L.)

X. hangzhouense Hangzhou , Zhejiang Province, China Magnolia grandiflora L. MF538772

X. herakliense Vathy Rema, Heraklion province, Crete, Olive tree (Olea europaea KM586345

Greece subsp. sylvestris )

X. herakliense Vathy Rema, Heraklion province, Crete, Olive tree (Olea europaea KM586346 Greece subsp. sylvestris )

X. herakliense Agiofarago , south west Heraklion province, Olive KM586347 Crete, Greece

X. herakliense Agiofarago , south west Heraklion province, Olive KM586348 Crete, Greece

X. herakliense Agiofarago , south west Heraklion province, Olive KM586349 Crete, Greece

X. herakliense Agiofarago , south west Heraklion province, Olive KM586350 Crete, Greece

X. herakliense Hersonisos , northeast Heraklion province, Olive (Olea europaea subsp. KM586351

Crete, Greece europaea L.)

X. herakliense Hersonisos , northeast Heraklion province, Olive (Olea europaea subsp. KM586352

Crete, Greece europaea L.)

X. hispanum Andujar, Jaen province, Spain Cistus albidus L. GU725074

X. hispidum Bollullos par del Condado, Huelva province, Grapevine (Vitis vinifera L.) HM921346

Spain

X. hunaniense Shenzhen, China – KP793046

X. hunaniense Shenzhen, China X. index CÓrdoba province, Spain

X. index Kentri , Greece

X. index CÓrdoba province, Spain X. index CÓrdoba province, Spain

X. index CÓrdoba province, Spain X. index Cádiz province, Spain

– KP793048 Grapevine HM921398

Olive KJ802882 Grapevine HM921399 Grapevine HM921400

Grapevine HM921401 Grapevine HM921402

X. index Roodghat area, Sufiyan, East-Azarbaijan Apple (Malus domestica L.) MH 879782 province, northwest of Iran variety Red delicious

X. ingens Chogha Kaboud village, Harsin, Kermanshah Astragalus sp. KJ956388 province, Iran

X. insigne – – AY601619

X. israeliae Roufas, Greece

X. israeliae Agiofarago, Greece

X. italiae Cabra, CÓrdoba province, Spain

X. italiae

X. iznajarense Iznaajar, Cordoba province, Spain

X. japonicum Japan

Olive KJ802883 Wild olive KJ802884 Grapevine KC567182

AY601613

Cultivated olive KX244892

Podocarpus macrophyllus L. KY131240

Continued

X. laevistriatum Florida, USA X. lambertii India

– DQ299505 – HM163211

X. lupini Bollullos par del Condado, Huelva province, Grapevine (Vitis vinifera L.) HM921352 Spain

X. lupini Hinojos, Huelva province, Spain X. macroacanthum Southern Italy

X. macrodora La Granjuela, CÓrdoba province, Spain

X. mengibarense Mengibar, Jaen province, Spain X. meridianum Sbitla, Kasserine, Tunisia

X. naturale Florida, USA

X. nuragicum Marchena, Seville province, Spain Grapevine KC567183 Olive orchards HF546080 Cultivated olive KU171040

Cultivated olive KX244894 Cultivated olive KX062679

– DQ299515

Olea europaea sp. europaea GU725071 L.

X. nuragicum Puente Genil, Cordoba province, Spain Vitis vinifera L. GU725067 X. oleae Tarifa, Cádiz province, Spain Wild olive KU171037

X. poasense Toro Amarillo, Valverde Vega , San Carlos Eucalyptus, cypress and MF461347

Alajuela, Costa Rica fountain grass

X. pseudocoxi Alcaracejos, Cordoba province, Spain Wild olive KX244915

X. pyrenaicum Cahors, Midi-Pyrenees province, France Vitis vinifera L. GU725073

X. rivesi Bollullos par del Condado, Huelva province, Grapevine (Vitis vinifera L.) HM921358

Spain

X. robbinsi Sbitla , Kasserine, Tunisia Cultivated olive KX062683

X. robbinsi Abida , Kairouan, Tunisia Cultivated olive KX062685

X. santos – X. savanicola –

– AY601587 – AY601620

X. setariae – – AY601621

X. Coto Rios, Jaen province, Spain Quercus faginea L. GU725076 sphaerocephalum

X. tarjanense DQ299511

X. tica Chirraca, San Ignacio de Acosta, San José, Grapevine KY623485

Costa Rica

X. turcicum Sanlúcar de Barrameda, Cádiz province, Grapevine KC567185

Spain

X. turdetanensis Sanlúcar de Barrameda, Cádiz province, Stone pine KC567186 Spain

X. vuittenezi Czech Republic

X. vuittenezi –

X. vulgare Florida, USA

X. zagrosense Madavan village, Kohgiluyeh and Boyer-Ahmad province, Iran

directed with a small terminal peg, 4.5–7.5 μm long, and a distinct terminal blind canal; lack of males in population, having four juvenile stages and J1 tail having a depression on dorsal and ventral sides at hyaline level, with a curved finger like cuticular extension at the end.

– EF614266

– AY601614

– DQ299514

Grasses JN153101

The identification codes for the new species, according to the polytomous key of Loof and Luc (1990), are: A4, B3, C5a, D6, E5, F4, G3, H2, I3, J4, K2, L1.

Based on the molecular and morphological similarities, the new species is closely related to X. aceri ( Chizhov et al., 1986); X. granatum ( Pedram, Pourjam, Palomares-Rius, Ghaemi, Cantalapiedra-Navarrete and Castillo, 2012) ; X. herakliense ( Tzortzakakis et al., 2015) ; X. zagrosense ( Ghaemi et al., 2012) , and X. vuittenezi ( Luc et al., 1964) , but it can be separated using the morphometric data, characters of the juveniles, especially the shape of their tail in the first stage and partial sequences of 28S rDNA (except X. aceri as it currently lacks molecular data of D2–D3 expansion part of 28S rDNA).

Compared to X. aceri, the new species has a shorter body (3.67–4.25 vs 4.90–5.50mm), smaller a (53–63 vs 75–83) and V values (46–48 vs 48–51), shorter odontophore (75–85 vs 83–101 μm) and juvenile characters as well, e.g., shorter tail length (59–63 vs 67.2–68.8 μm) and tail characters in J1. Xiphinema barooghii n. sp. differs mainly from X. granatum by having lower a (53–63 vs 74–99), b (7.0–8.5 vs 8.5– 11.0) and c¢ (0.7–0.9 vs 1.2–1.5) values, longer odontostyle (132–139 vs 118–132 μm), odontophore (75–85 vs 65–74 μm) and spear (215–225 vs 189–204μ m), esophagus (493–553 vs 360–460μm) and esophageal basal bulb length (128–153 vs 77–104 μm), posteriorly located guiding ring (117–132 vs 100–116 μm), absence of males, uterus with spines vs devoid of any Z- differentiation or spines and juvenile characters. In addition, X. barooghii n. sp. is similar to X. herakliense but differs by a longer esophageal basal bulb (128–153 vs 94–121 μm), absence vs presence of a pseudo-Z-organ and crystalloid bodies in the uterus. Furthermore, X. herakliense is an amphimictic species and has functional males and sperm in the female reproductive system compared to the absence of males in the new species. The new species differs from X. zagrosense by a shorter odontostyle (132–139 vs 151–169μm), odontophore (75–85 vs 94–105 μm) and spear length (215–225 vs 246–274μm), slightly smaller lip region width (14–15 vs 15–18μm), the shape of tail (conoid, dorsally convex, ventrally directed with a small terminal peg and distinct terminal blind canal vs conoid and dorsally convex, with rounded end lacking a mucro or cuticular projection) and differences in juvenile characters. Finally, X. barooghii n. sp. can be differentiated from X. vuittenezi (according to original description) by a slightly longer body in the females (3.67–4.25 vs 2.63–3.83 mm), longer spear (215–225 vs 183–212μm), wider body diameter at neck base (52–69 vs 44 μm) and mid - body or vulva level (60–79 vs 39–58μm), cuticle two vs three layered, cephalic region separated from body contour by a shallow depression and 21–28% of body diameter at neck base vs clearly depression and 31% of body diameter at neck base, greater length and width of esophageal basal bulb, 128–153 × 24–31 vs 109–126 × 18–23 μm, cardia 12–14 μm long and rounded conoid vs 6.5 μm (calculated from the image) conoid. In addition, there are a number of differences in juvenile characters, in J1: greater body (1.14–1.16 vs 0.78–1.03mm), odontostyle (58–59 vs 47–53 μm), odontophore (44–47 vs 34–40 μm), spear (103–105 vs 81–93 μm), replacement odontostyle (71–74 vs 62–71μm) length, oral aperture to guide ring (50–52 vs 43–50 μm), tail length (59–63 vs 40–52 μm) and shape (presence a depression on dorsal and ventral sides at hyaline level, with a curved finger like cuticular extension at the end vs absence); in J2: longer body (1.73 vs 1.07–1.49mm), odontophore (54 vs 42–50 μm), spear (124 vs 107–121 μm), replacement odontostyle (93 vs 80–87μm), oral aperture to guide ring (65 vs 56–59 μm) and tail length (60 vs 43–50 μm), larger body diam. at mid - body (42 vs 22–34 μm) at anus level (26 vs 15–23 μm); in J3: higher body length (2.01–2.31 vs 1.47–1.95mm), odontostyle (93–100 vs 79–89 μm) and spear (150–167 vs 130–146 μm) length and a value (51–62 vs 37–48); in J4: longer body (2.87–3.34 vs 2.01–2.74mm), replacement odontostyle (135–139 vs 121–135 μm) and a value (51–64 vs 44–50).

Type habitat and locality

Soil samples were collected from the rhizosphere of common wheat (Triticum aestivum L.) in Roodghat area, Sufiyan, East - Azarbaijan province, northwest of Iran, during 2016 and 2017 (GPS coordinates: N 38°22′ 10′′, E 46° 07′ 26′′, altitude 1808 m a.s.l.).

Type material

Holotype and paratype females and juveniles were deposited at Nematology Collection of the Department of Plant Protection, Faculty of Agriculture, University of Tabriz, Tabriz, Iran. Two paratype females were also deposited at Nematode Collection of the University of Jaen, Spain.

Etymology

The new species is named in honor of Dr. Hassan Barooghi, the late Entomologist and Associate Professor in Department of Plant Protection, University of Tabriz, Tabriz, Iran.

Molecular characterization and phylogeny

For molecular analysis, one D2–D3 28S rDNA sequence, 800 bp long, was obtained (GenBank accession no. MH 884067). The evolutionary relationships of the new species, Xiphinema barooghii n. sp., are shown in Figure 5 . The tree is reconstructed from 93 sequences, out of which 84 sequences belong to species of Xiphinema non-americanum group, 8 from X. americanum group and Longidorus helveticus sequence as out group taxon. The X. non-americanum species included in the analysis had representatives from all morphogroups as defined by Loof and Luc (1990). The species from GenBank with the highest match in Nblast search with X. barooghii n. sp. were selected for phylogenetic analysis and the new species showed 98, 97, 97, 96, 95, 94 and 91 percent of similarity to X. herakliense (KM586348), X. zagrosense (JN153101), X. israeliae (KJ802883), X. barense (KM199691), X. vuittenezi (EF614266), X. granatum (JQ240273) and X. robbinsi (KX062685), respectively, and 15, 20, 22, 29, 30, 41 and 53 nucleotides differences, respectively, as compared to the new species. The average nucleotide composition is as follows: 24.02% A, 23.41% C, 28.85% G and 23.73% T.

Xiphinema barooghii n. sp. is phylogenetically related to X. herakliense , X. granatum and X. vuittenezi from morphospecies Groups 5, 6 and 8, well positioned within, but clearly separated from them. In this subclade, all four species share a conoid and dorsally convex female tail with a central subdigitate peg. In this regard, our data did not demonstrate a correlation between morphospecies and their grouping in phylogenetic analysis using molecular markers, confirming the findings by Gutiérrez-Gutiérrez et al. (2013), Roshan-Bakhsh et al. (2014), De Luca et al. (2014), and Tzortzakakis et al. (2015).

In summary, molecular characterisation and phylogenetic analysis of D2–D3 region sequence and morphological and morphometric analyses clearly supported the status of Xiphinema barooghii n. sp. as a new taxon within the X. non-americanum group.

Figures

Figure 1: Xiphinema barooghii n. sp. (A) Amphidial pouch; (B) Entire body; (C) Neck region; (D) Female tail; (E) Anterior end; (F) Posterior genital branch; (G–J) Tail of juveniles from J1–J4, respectively.

Figure 2: Xiphinema barooghii n. sp. Female (A, B) anterior end; (C, G) uterine differentiation spines; (D) entire body; (E) reproductive system; (F) vagina; (H) tail; (I) caudal pores in lateral optical view; (J) amphidial pouch; (K) pharyngeal expansion. (Scale bars: A–K=10μm, D=70μm).

Figure 3: Juvenile stages of Xiphinema barooghii n. sp. Anterior region and tail shape of (A, B) first; (C, D) second; (E, F) third and (G, H) fourth juvenile stages, respectively (Scale bars =10μm).

Figure 4: Xiphinema barooghii n. sp. Graph of correlation of functional and replacement odontostyle to body length in all developmental stages from Jl to mature females.

Figure 5: Bayesian tree inferred under the GTR + I + G model from 28S rDNA D2–D3 expansion domains of X. barooghii n. sp. and X. index (−lnL= 9,085.8555; AIC =18,191.7109; freqA =0.2402; freqC =0.2341; freqG= 0.2885; freqT =0.2373; R (a)=1.0503;R (b)=2.8325; R (c)=2.6784;R(d)=0.5047;R(e)= 4.0878;R (f) =1.0000). Posterior probabilities are given for appropriate clades. Newly obtained sequences are indicated by bold letters.