Introduction

Root-knot nematodes (RKN) of the genus Meloidogyne are among nature’s most successful plant parasites, being distributed worldwide and encompassing more than 95 nominal species (Moens et al. 2010). These nematodes infect thousands of different herbaceous and woody monocotyledonous and dicotyledonous plants and cause severe losses to numerous agricultural crops and forest plants worldwide (Moens et al. 2010). Root-knot nematodes (Meloidogyne spp.) and root-lesion nematodes (Pratylenchus spp.) are the most damaging plant-parasitic nematodes of cultivated olive (Olea europaea L. subsp. europaea var. europaea), especially in nurseries (Castillo et al. 2010; Ali et al. 2014). Meloidogyne species known to damage olive trees are well adapted to temperate and subtropical areas and include Meloidogyne arenaria (Neal 1889) Chitwood 1949; Meloidogyne baetica Castillo et al. 2003a, Meloidogyne hapla Chitwood 1949, Meloidogyne incognita (Kofoid & White 1919) Chitwood 1949, M. javanica (Treub 1885) Chitwood 1949 and Meloidogyne lusitanica Abrantes and Santos 1991. In orchards, olive trees infected by Meloidogyne spp. show yellowing in the highest branches and defoliation (Castillo et al. 2010). These species occur on wild and cultivated olives in southern Spain, and also in nurseries (except for M. baetica) (Castillo et al. 2003b). However, plant-parasitic nematodes infecting wild olive have been scarcely studied in the Mediterranean Basin, except for the recent description of the root-lesion nematode Pratylenchus oleae Palomares-Rius et al. 2014 in Spain and Tunisia, and the reniform nematode Rotylenchulus macrosoma in Spain (Castillo et al. 2003a). Yet, wild olive populations have persisted in western and eastern parts of the Mediterranean Basin since the Late Tertiary (Besnard et al. 2013), and in particular in southern Spain and Morocco where Last Glacial Maximum refugia have been described for many organisms (Médail and Diadema 2009). Natural dispersal of native phytoparasitic nematodes could be limited (due to physical barriers and low migration ability) and communities of such regions are thus expected to be unique with high level of endemism.

Species determination of Meloidogyne is complex, difficult and time-consuming even for experts. The application of molecular methods to studies of RKN population structure and systematics have revealed that some long-assumed single species are in fact cryptic species that are morphologically indistinguishable but may be phylogenetically distant to one another (Gamel et al. 2014). Sequences of nuclear ribosomal (rDNA) and mitochondrial DNA (mtDNA) have been used for molecular characterisation and reconstruction of phylogenetic relationships within the genus Meloidogyne during the last decade (Powers and Harris 1993; Ziljstra et al. 2000; Adam et al. 2007; Adams et al. 2009; Castillo et al. 2009). The analysis of isozyme electrophoretic patterns, in particular esterase (Est) and malate dehydrogenase (Mdh), as well as several molecular approaches have proved to be a valuable tool for precise identification of Meloidogyne species (Blok and Powers 2009). Consequently, current availability of molecular techniques integrated with classical morphological approaches may help to provide tools for differentiating Meloidogyne species and can significantly improve and facilitate the routine identification of these nematodes.

Nematode surveys in wild olive soils in northern Morocco revealed very low population levels of a RKN. This RKN appeared morphologically close-related to M. kralli Jepson 1983, M. dunensis Palomares-Rius et al. 2007 and M. sewelli Mulvey and Anderson 1980, which prompted a comparative study among related species. Some reliable diagnostic approaches commonly used to identify and compare certain RKN species such as analyses of isozyme phenotypes and phylogenetic analyses of ribosomal and mitochondrial DNA sequences were included in the study.

This work describes a new Meloidogyne species associated with wild olive in northern Morocco as well as its phylogenetic relationship with other RKNs based on Bayesian inference (BI) and maximum likelihood (ML) analyses of sequences from the 18S, ITS1-5 · 8S-ITS2, D2-D3 of 28S rDNA, and the partial coxII-16S of rRNA and coxI gene of mtDNA. The undescribed root-knot nematode is herein described as Meloidogyne spartelensis n. sp., the species epithet referring to Cap Spartel where it was detected.

Material and methods

Soil sampling, nematode extraction, and Meloidogyne spp. detection and rearing

A nematode survey was conducted in March 2012 in wild olive (Olea europaea subsp. europaea var. sylvestris (Mill.) Lehr) soils in northern Morocco (Cape Spartel; 35.790583°N; 5.924983°W; altitude 16 m), near Tanger city. Soil sub-samples were collected with a shovel from the upper 20 cm of bare soil under each olive tree canopy and mixed to form one 500 dm3 reference sample. Nematodes were extracted from 250 cm3 of fresh soil using the elutriation procedure (Seinhorst 1962), and Meloidogyne juveniles and males were observed under a stereomicroscope (Leica M165C, ×60 magnification). Tomato (cv. Roma) and olive (cv. Picholine du Languedoc) plantlets were transplanted in the remaining native soil and reared under controlled greenhouse conditions in order to test the infection rate and plant host range of this RKN. No nematode survived on olive cv. Picholine du Languedoc. But several juveniles and males were extracted from the tomato roots placed in a mist chamber (Seinhorst 1950). The Meloidogyne population was again reared on tomato in a sandy-peat substrate (2/3-1/3).

Nematode morphological identification

For diagnosis and identification, females were collected from tomato galled roots, while males, eggs and second-stage juveniles (J2) of nematodes were extracted from the rhizosphere by centrifugal-flotation (Coolen 1979) and from feeder roots by blending in a 0.5 % NaOCl solution for 4 min (Hussey and Barker 1973). Specimens for light microscopy (LM) were killed with gentle heat, fixed in a 4 % solution of formaldehyde + propionic acid and processed to glycerin by Seinhorst’s rapid method (Seinhorst 1966). Specimens were examined using a Zeiss III compound microscope with Nomarski differential interference contrast at powers up to 1,000× magnification. Randomly selected specimens of each life-stage were measured. Measurements and drawings were made at the camera lucida on glycerine infiltrated specimens. All measurements were expressed in micrometers (μm). All other abbreviations used are as defined in Siddiqi (2000).

Fixed specimens were dehydrated in a gradient ethanol series, critical-point dried, sputter-coated with gold and observed by scanning electron microscopy (SEM) according to Abolafia et al. (2002).

Perineal patterns of mature females were prepared according to standard procedures (Hartman and Sasser 1985). Briefly, root tissues were teased apart with forceps and half spear to remove adult females. The lip and neck regions of the nematode were excised, and the posterior end was cleared in a solution of 45 % lactic acid to remove remaining body tissues. Then, the perineal pattern was trimmed and transferred to a drop of glycerin. At least 50 perineal patterns were examined for species identification.

Isozyme phenotype analysis

To obtain sufficient individuals of M. spartelensis n. sp. for electrophoretic analyses, the nematode population under study and a reference M. javanica population from olive trees sampled at Córdoba, Spain (Nico et al. 2002), were increased on tomato (cv. Roma) in a glasshouse at 25 ± 3 °C. For that, a single egg mass of M. spartelensis n. sp. was placed beneath the roots of individual tomato seedling in 12 cm pots filled with sterile loamy soil. Sixty days after inoculation, tomato plants were uprooted, their roots gently washed free of soil and the root tissues teased apart using forceps and transfer needles to remove adult females.

Five young egg laying females of Meloidogyne spartelensis n. sp. and of M. javanica (reference population) were macerated in microtubes containing 5 μl of 20 % (wt/vol) sucrose, 1 % (vol/vol) Triton X-100 and 0 · 01 % (wt/vol) bromophenol blue. Electrophoresis was carried out in 7 × 8 cm separating (pH 8 · 4) and stacking (pH 6 · 8) homogeneous gels, 7 and 4 % polyacrylamide, respectively, 0 · 75-mm thick, with Tris-glycine buffer in a Mini Protean II electrophoresis unit (BioRad). Gels were stained with the substrate α-naphthyl acetate for Est and with Fast Blue RR (Sigma-Aldrich) for Mdh. Band patterns and relative migration of the bands (Rm) were compared to M. javanica (Esbenshade and Triantaphyllou 1985).

Nematode molecular identification

For molecular analyses, one female nematode was temporary mounted in a drop of 1 M NaCl containing glass beads and after taking measurements and photomicrographs of diagnostic characters the slides were dismantled and DNA extracted. Nematode DNA was extracted from single females and PCR assays were conducted as described by Castillo et al. (2003b). The D2-D3 expansion segments of 28S rDNA was amplified using the D2A (5′-ACAAGTACCGTGAGGGAAAGTTG-3′) and D3B (5′-TCGGAAGGAACCAGCTACTA-3′) primers (De Ley et al. 1999). The ITS region was amplified using forward primers 18S (5′-TTGATTACGTCCCTGCCCTTT-3′) and 26S (5′-TTTCACTCGCCGTTACTAAGGGAATC-3′) (Vrain et al. 1992). The 18S region was amplified with MelF (5′-TACGGACTGAGATAATGGT-3′) and MelR (5′-GGTTCAAGCCACTGCGA-3′) as described in Tigano et al. (2005). The region of the mitochondrial genome between the cytochrome oxidase subunit II (coxII) and 16S rRNA mitochondrial DNA (mtDNA) genes was amplified using primers C2F3 (5′-GGTCAATGTTCAGAAATTTGTGG-3′) and 1108 (5′-TACCTTTGACCAATCACGCT-3′) (Powers and Harris 1993). A 450-pb portion of the cytochrome oxidase subunit I gene (coxI) of mtDNA was amplified using primers COX1F1 (5′-TTGRTTTTTTGGTCATCCTGARG-3′) and COX1R1 (5′-WSYMACWACATAATAAGTATCATG-3′). COX1F1 and COX1R1 were designed using PRIMER3 software (http://frodo.wi.mit.edu/primer3/input.htm) from nematode mtDNA sequences (Lazarova et al. 2006).

PCR products were purified after amplification using ExoSAP-IT (Affmetrix, USB products), quantified using a Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA) and used for direct sequencing in both directions using the primers referred above. The resulting products were purified and run on a DNA multicapillary sequencer (Model 3130XL genetic analyser; Applied Biosystems, Foster City, CA, USA), using the BigDye Terminator Sequencing Kit v.3.1 (Applied Biosystems, Foster City, CA, USA), at the Stab Vida sequencing facilities (Caparica, Portugal). CoxI PCR products were purified and sequenced in both directions using the primers referred above by Eurofins MWG Company (Germany). The resulting products were corrected and aligned using Geneious R7 v. 7.1.2 (www.geneious.com) and BioEdit (Hall 1999) softwares. The newly obtained sequences were submitted to the GenBank database under accession numbers indicated on the phylogenetic trees.

Phylogenetic analyses

D2-D3 expansion segments of 28S rDNA, ITS rDNA, partial 18S and coxII-16S rRNA sequences of different Meloidogyne species from GenBank were used for phylogenetic reconstruction. Outgroup taxa for each dataset were chosen according to previous published data (Castillo et al. 2009). The newly obtained and published sequences for each gene were aligned using MAFFT (Katoh et al. 2002) with default parameters. Sequence alignments were manually edited using BioEdit (Hall 1999). Phylogenetic analyses of the sequence data sets were performed based on maximum likelihood (ML) using PAUP * 4b10 (Swofford 2003) and Bayesian inference (BI) using MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003). The best fitted model of DNA evolution was obtained using jModelTest v. 2.1.1 (Darriba et al. 2012) with the Akaike Information Criterion (AIC). The Akaike-supported model, the base frequency, the proportion of invariable sites, and the gamma distribution shape parameters and substitution rates in the AIC were then used in phylogenetic analyses. BI analysis under a general time reversible of invariable sites and a gamma-shaped distribution (GTR + I + G) model for D2-D3 expansion segment of 28S rDNA, (GTR + G) model for ITS1, (GTR + I + G) model for partial 18S and (GTR + I + G) model for coxII-16S rRNA, were run with four chains for 2 × 106, 2 × 106, 3 × 106 and 1 × 106 generations, respectively. The Markov chains were sampled at intervals of 100 generations. Two runs were performed for each analysis. After discarding burn-in samples and evaluating convergence, the remaining samples were retained for further analyses. The topologies were used to generate a 50 % majority rule consensus tree. Posterior probabilities (PP) are given on appropriate clades. Trees were visualised using TreeView (Page 1996). In ML analysis the estimation of the support for each node was obtained by bootstrap analysis with 100 fast-step replicates.

Results

Meloidogyne spartelensis Footnote 1 n. sp. (Figs. 1, 2 and 3, Table 1)

Fig. 1
figure 1

Line drawings of Meloidogyne spartelensis n. sp. from Morocco. Second-stage juvenile (a-f). a Whole body. b Pharyngeal region. c Lip region. d-f Tail regions. Female (g-i). g Whole females. h Lip region. i Perineal patterns. Male (j-k). j Lip region. k Tail region

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Fig. 2
figure 2

Light micrographs of Meloidogyne spartelensis n. sp. from Morocco. Male (a-g). a Pharyngeal region. b Lip region. c Detail of stylet. d-f Tail region showing spicules and gubernaculum. g Lateral field at mid-body. Second-stage juvenile (h-n). h Whole Second-stage juvenile. i Pharyngeal region. j Lip region. k-n Tail regions. Mature female (o-s). o Whole female. p Female neck region. q-s perineal pattern. (Scale bars: a, d-f, k-n, p-s = 20 μm; b, c, g, j = 10 μm; h = 50 μm; o = 100 μm)

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Fig. 3
figure 3

Scanning electron microscope photographs of Meloidogyne spartelensis n. sp. from Morocco. Female (a-d). a anterior end in lateral view showing excretory pore. b Lip region in en face view. c-d Perineal pattern. Male (e-i). e Anterior end in ventro-lateral view. f Lip region in en face view. g Lateral field at mid-body. h-i Tail region. Second-stage juvenile (j-o). j-k Lip region in lateral view. l Lip region in en face view. m Lateral field at mid-body. n-o Tail region. Abbreviations: a = anus; am = amphid; ep = excretory pore; oa = oral aperture; v = vulva. (Scale bars: a, g, h, m = 5 μm; b, j, l = 1 μm; c-e, n, o = 10 μm; f, i, k = 2 μm;)

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Table 1 Morphometrics of adult males and second-stage juveniles (J2) of Meloidogyne spartelensis n. sp. All measurements in μm and in the format: mean ± s.d. (range)*
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Holotype (female in glycerin)

Body length L = 712 μm; maximum body width = 409 μm; a = 1.5; stylet length = 14.5 μm; dorsal pharyngeal gland opening (DGO) = 3.5 μm; excretory pore from anterior end = 21.5 μm; excretory pore distance from anterior end/length of stylet (EP/ST) = 1.5; vulva slit length = 22 μm; distance from vulva to anus = 26.5 μm.

Female paratypes

(n = 16) L = 621 ± 89 (489–772) μm; maximum body width = 386 ± 45 (267–455) μm; a = 1.6 ± 0.1 (1.4–1.8); stylet length 15.1 ± 0.3 (14.5–15.5) μm; excretory pore from anterior end 23.1 ± 2.2 (21.0–30.0) μm; EP/ST ratio (excretory pore to head end/stylet length) = 1.5 ± 0.1 (1.4–2.0); vulva slit = 22.0 ± 1.6 (18.0–25.0) μm; vulva-anus distance = 21.3 ± 2.2 (16.5–35.0) μm.

Female

Body usually completely embedded in galled tissue in tomato, pearly white body, varying in shape from ovoid to saccate and with a variable neck diam. and length (Figs. 1g to 2o). Lip region continuous with body contour. Head cap variable in shape, with labial disk and post labial annulus not elevated. In SEM view, the labial disc appears round-squared, slightly raised on the medial and lateral sectors, which are all fused together (Fig. 3a and b). Labial framework weakly sclerotized. Amphidial apertures elongated, located between labial disc and lateral lips. Stylet fairly long, with an almost straight, rarely curved, cone, cylindrical shaft and knobs oval and sloping posteriad, sometimes with concave anterior surfaces. Excretory pore usually at level of anterior end of procorpus. Pharyngeal gland with a large mononucleate dorsal lobe and two sub-ventral gland lobes, usually difficult to see. Perineal pattern mostly rounded to oval, with moderately high dorsal arch that is mostly rounded and sometimes squarish and generally low, with fine, sinuous cuticle striae, which become coarser in the vicinity of perivulval region; lateral field not clearly visible. However, in some specimens the lateral fields were slightly marked. In a few specimens, the striae form two wings or shoulders ending near the lateral field, which, in this case, are made more visible by fine and small zigzag striae. Phasmids distinct, located just above the level of anus. Vulva slit in the middle of the unstriated area, almost as long as the vulva-anus distance; anus fold clearly visible, but not always present (Figs. 1i and 2q to s). Punctations and striae absent in perineum. Commonly, large egg sac occurs outside the root gall, containing up to 350–450 eggs.

Male

Body vermiform, tapering anteriorly; tail rounded, with twisted posterior body portion. Lip region slightly set off from body and with a high head cap. Lip framework strong and sclerotised. Prominent slit-like amphidial openings between labial disc and lateral lips (Fig. 3e to f). In SEM view, the labial disc is slightly narrower and raised above the merged subventral and subdorsal medial lip sectors, with a centred oval prestoma into which opens a slit-like dorso-ventrally oriented stoma; lateral lips reduced to a very narrow strip, largely fused, in the middle part, with the post-labial annulus. Lip region high and lacking annulation. Stylet robust and straight, with cone and shaft broadening slightly in the distal part. Stylet knobs mostly rounded, laterally or obliquely directed, merging gradually with the base of the shaft. Lateral field consisting of four incisures with areolations along body but only few actually cross central field. Procorpus distinctly outlined, three times larger than metacorpus. Metacorpus ovoid, with a strong valve apparatus. Excretory duct curved. Excretory pore distinct and usually located four to six annuli posterior to hemizonid. Normally only one testis extending anteriorly. Spicules of variable length, arcuate and with two pores clearly visible at tip. Gubernaculum distinct. Phasmids at level of cloacal aperture and located in central lateral field, showing slit-like openings. All morphometric measurements of adult males are given in Table 1.

Second-stage juveniles

Body vermiform (Figs. 1a and 2h), tapering more towards posterior than anterior end. Lip region narrower than body and slightly set off (Figs. 1b and 2i). Head cap slightly elevated. Lip framework weakly developed (Figs. 1c and 2j). Labial disc and medial lips fused. In labial disc, a stoma-like slit located in an ovoid prestoma and surrounded by six inner labial sensilla. In SEM view, the labial disc appears oval to rectangular in shape, raised above medial lips, to which it merges in a dumbbell-shaped structure. Lip region smooth and lacking annulation (Fig. 3j to k). Amphidial apertures elongated and located between labial disc and lateral lips (Fig. 3l). Body annulated from anterior end to terminus. Lateral field consisting of four incisures, with areolations along body but only few extending across field. Stylet delicate, with cone straight, narrow, sharply pointed, shaft almost cylindrical, and knobs small, rounded, separate from each other, laterally directed. Pharynx with a long, cylindrical Procorpus (3.0-4.0 times length of metacorpus), round-oval metacorpus, short isthmus and rather long gland lobe, with three equally sized nuclei and overlapping intestine ventrally. Hemizonid located anterior to excretory pore, extending for ca. two body annuli. Excretory pore located posterior to nerve ring. Excretory duct curved and discernible when it reaches intestine. Rectum slightly dilated. Tail long, conoid, terminus usually pointed, with several constrictions in the hyaline region (Figs. 2 and 3). Tail annulation fine, regular in the proximal two third, becoming slightly coarser and irregular in the distal part. Hyaline tail terminus clearly defined and long, phasmids small, difficult to observe. All morphometric measurements carried out on second-stage juveniles are given in Table 1.

Associated host and locality

Meloidogyne spartelensis n. sp. was found in a loamy-clay soil around the roots of wild olive (Olea europaea subsp. europaea var. sylvestris) at Cape Spartel, near Tanger city, northern Morocco (35.790583°N; 5.924983°W, altitude 16 m).

Type material

Holotype female, female perineal patterns, J2 and paratype males, mounted on glass slides deposited in the authors’ nematode collection at the Institute for Sustainable Agriculture, CSIC, Córdoba, Spain. Additional males and J2 paratypes were distributed to the United States Department of Agriculture Nematode Collection, Beltsville, MD, WANECO collection, Wageningen, The Netherlands (http://www.waneco.eu/), and IRD collection, Montpellier, France. Specific D2-D3, ITS, 18S-rDNA and coxII-16S rRNA and coxI of mtDNA sequences are deposited in GenBank with accession numbers KP896292-KP896293, KP896294, KP896295, KP896297, and KP997290-KP997301 respectively.

Diagnosis and relationships

Meloidogyne spartelensis n. sp. is characterized by a stylet (14.5–15.5) μm long, a perineal pattern mostly rounded to oval, with moderately high dorsal arch that is mostly rounded and sometimes squarish and generally low, excretory pore usually at level of anterior end of procorpus (EP/ST ratio = 1.4–2.0), second-stage juveniles with smooth lip region, tail long, conoid, terminus pointed, with several constrictions in the hyaline region (19.5–46.0) μm long, males with stylet 17.5–19.5 μm long, and specific D2-D3, ITS, 18S-rDNA, partial coxII-16S rRNA and coxI sequences.

The female perineal pattern morphology of M. spartelensis n. sp. is roundish without marked lateral lines, which places it in Jepson’s Group 3 (Jepson 1987). While considering the species morphology, M. spartelensis n. sp. is related to the “graminis-group” studied by Jepson (1987), it is morphometrically closer to M. kralli (Jepson 1983). Females differ by EP/ST (1.4–2.0 vs. 2.0–3.0 in M. kralli). Juveniles were smaller b ratio (3.7 vs 6.5), longer stylet (13.0–14.5 vs 10.5–11.5 μm), and shorter DGO (2.1 vs 4.1). Males were longer (1497 vs 1076 μm), higher a ratio (42.5 vs 31.7), shorter DGO (3.3 vs 4.4) and longer distance from anterior end to excretory pore (145 vs 127.4). Partial 18S from M. kralli (KJ636370) is also different to M. spartelensis n. sp. in 3 % differences (24 nucleotides in difference).

Meloidogyne spartelensis n. sp. is also similar to M. dunensis, from which it differs mainly in morphology of female perineal patterns (lateral field clear visible on M. dunensis). Juvenile stylet is longer (13.0–14.5 vs 11.0–12.5 μm) and as its hyaline part of tail (19.5–46.0 vs 9.5–16.5 μm). In males, c ratio (107.8 vs 280.6) and spicules (25.0–30.5 vs 29.0–38.0 μm) were smaller. The new species is also morphologically close to M. sewelli, from which it differs mainly in higher female EP/ST ratio (1.4–2.0 vs 1.2), longer length of J2 stylet (13.0–14.5 vs. 11.0–12.0 μm), and in shorter length of male gubernaculum (6.5–7.5 vs 8.0–9.0 μm) and shorter DGO (2.5–4.0 vs 5.0–6.0). D2-D3, ITS and partial 18S sequences from M. dunensis (EF612713, EF612711 and EF612712, respectively) are also different to M. spartelensis n. sp. in 7 % differences (51 nucleotides), 6 % differences (25 nucleotides), and 2 % differences (16 nucleotides), respectively. Meloidogyne spartelensis n. sp. can also be compared with several other Meloidogyne species commonly infecting woody plants, such as M. arenaria, M. baetica, M. hispanica, M. incognita and M. javanica. Their main comparative diagnostic characters, useful to a rapid and easy identification, are summarized in Table 2. Meloidogyne spartelensis n. sp. also differs markedly from the other known European root-knot nematode species (Karssen and van Hoenselaar 1998; Karssen 2002; Castillo et al. 2003b).

Table 2 Morphological and morphometric differences among Meloidogyne species closely related to Meloidogyne spartelensis n. sp. and parasite species to olivea, b
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In addition, M. spartelensis sp. n. differs from related species in Est and Mdh phenotypes, as well as sequences of the ITS1-5 · 8S-ITS2 region, the small subunit 18S rDNA, the D2-D3 fragment of the 28S gene of rDNA, the coxII-16S rRNA, and coxI gene sequences (see below).

Isozyme analysis and molecular characterization

The isozyme electrophoretic analysis of five-specimen groups of young egg-laying females of M. spartelensis n. sp. revealed one strong Est band (Rm = 42.65) after prolonged staining (Fig. 4a) and one N1b Mdh band (Fig. 4b) that did not occur in the Est and Mdh phenotypes of M. javanica, which showed J3 and N1 phenotypes, respectively (Fig. 4a and b) or in other Est and Mdh phenotypes previously identified for other Meloidogyne spp. Malate dehydrogenase phenotype of M. spartelensis n. sp. clearly differs from that of M. kralli (with N1c bands) (Karssen 2002), showing a lower unique band for M. spartelensis n. sp in comparison to M. javanica and those of M. dunensis (with a weak VS1 and N1c bands, respectively, Table 2), with a lower esterase band and higher Mdh band, respectively, taking as reference M. javanica for M. spartelensis n. sp.

Fig. 4
figure 4

Esterase (a) and malate dehydrogenase (b) phenotype electrophoresis patterns of protein homogenates from five young, egg-laying females of Meloidogyne spartelensis n. sp. from Morocco (Ms), and five young, egg-laying females of M. javanica reference population (Mj)

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Amplification of the 18S, ITS1-5.8S-ITS2, D2-D3 region of 28S rDNA, and partial coxII-16S rRNA and coxI mtDNA from M. spartelensis n. sp. yielded single fragments of approximately 900, 700, 840, 500 and 400 bp respectively. The partial D2-D3, ITS1-5.8S-ITS2, 18S, and partial coxII-16S rRNA sequences of M. spartelensis n. sp. were clearly different from that present in the GenBank database. Substantial sequence divergence for D2-D3 region of 28S (7 % against M. dunensis, (EF612712)), ITS1-5.8S-ITS2 (11 % against M. hispanica Hirschmann 1986 (JX885742)), partial 18S (1 % against M. ardenensis Santos, 1968 (AY593894)), and partial coxII-16S rRNA (19 % against M. hapla, AY757889) sequences for the new species distinguish M. spartelensis n. sp. from other studied root-knot nematodes and support its separate specific status. CoxI gene sequences from M. spartelensis n. sp. (KP997290- KP997301) showed a similarity of 89 % with M. hapla (JX683718 and AY268113) and 86 % with M. javanica (KP202352), M. arenaria (KP202350) and M. incognita (KJ476151). Intraspecific variability for COI gene was of 99.6 % (1 nucleotide in 322).

Phylogenetic analysis (BI and ML) of M. spartelensis n. sp. based on D2-D3 expansion segments of 28S rDNA of a multiple edited alignment including 56 sequences and 765 bp showed one clade clearly separated and supported (Fig. 5). The phylogenetic analysis showed well supported groups at major and terminal clades in both analyses. The phylogenetic tree resolved a major clade excluding M. mali Itoh et al. 1969 (KF880399 and KF880400), M. camelliae Golden 1979 (KF542870), M. ichinohei Araki 1992 (EF029862), M. artiellia Franklin 1961 (AY150369) and M. baetica (AY150367). Meloidogyne spartelensis n. sp. is closely related to M. dunensis (EF612712) and M. silvestris Castillo et al. 2009 (EU570214) and in a higher clade level with M. hapla (KJ645433 and KJ598136).

Fig. 5
figure 5

Phylogenetic relationships within Meloidogyne species. Bayesian 50 % majority rule consensus trees as inferred from D2 and D3 expansion segments of 28S rDNA sequences alignments under the GTR + I + G model. Posterior probabilities more than 65 % are given for appropriate clades; bootstrap values greater than 50 % are given on appropriate clades in ML analysis. Newly obtained sequences in this study are in bold font

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Phylogenetic analysis (BI and ML) of M. spartelensis n. sp. based on ITS sequences of a multiple edited alignment including 71 sequences and 681 total characters showed one clade clearly separated and supported (Fig. 6). Similarly to D2-D3 expansion segments of 28S rDNA phylogeny M. artiellia (KC545880, JX393299 and AF248478), M. baetica (AY150366), M. camelliae (KF542872, JX912885 and KF542871), M. mali (JX978229) and M. panyuensis Liao et al. 2005 (AY394719) were not included in this major and well supported clade. Meloidogyne spartelensis n. sp. formed a well-supported clade with M. dunensis (EF612711).

Fig. 6
figure 6

Phylogenetic relationships within Meloidogyne species. Bayesian 50 % majority rule consensus trees as inferred from ITS rDNA gene sequence alignment under the GTR + G model. Posterior probabilities more than 65 % are given for appropriate clades (in bold letters); bootstrap values greater than 50 % are given on appropriate clades in ML analysis. Newly obtained sequence in this study are in bold font

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Phylogenetic analysis (BI and ML) of M. spartelensis n. sp. based on partial 18S sequences of a multiple edited alignment including 76 sequences and 1641 bp showed several nested clades clearly separated and supported (Fig. 7). A major clade including 62 sequences was well supported while M. mali (KJ636400, KC875395, KF895400 and JX978225) occupied a basal position to this clade. Meloidogyne artiellia (KC875391 and KC875392) and M. baetica (KP896296) formed a well-supported clade nested to the clades explained before. Finally, M. ichinohei (KJ636350, KC875386 and KJ636349), M. camelliae (JX912884) and M. coffeicola Lordello & Zamith 1960 (HE667739) formed a well-supported basal clade to the other species. Meloidogyne spartelensis n. sp. was related to M. microtyla Lordello & Zamith 1960 (AF442198), and M. ardenensis (AY593894) in a moderately supported clade that also includes M. graminis (Sledge & Golden 1964) Whitehead 1968 (JN241856) in a moderately supported clade.

Fig. 7
figure 7

Phylogenetic relationships within Meloidogyne species. Bayesian 50 % majority rule consensus trees as inferred from partial 18S gene sequence alignment under the GTR + I + G model. Posterior probabilities more than 65 % are given for appropriate clades (in bold letters); bootstrap values greater than 50 % are given on appropriate clades in ML analysis. Newly obtained sequences in this study are in bold font

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Phylogenetic analysis (BI and ML) of M. spartelensis n. sp. based on the partial coxII-16S rRNA of a multiple edited alignment including 62 sequences with 1633 bp in length showed two moderate supported clades (Fig. 8). One clade contained the majority of the species while the other was formed by M. camelliae (JX912887) and M. mali (KC112913). In the main clade, Meloidogyne spartelensis n. sp. is sister to two well-supported subclades: the first including M. arenaria, M. thailandica Handoo et al. 2005, M. morocciensis Handoo et al. 2005, M. javanica, M. incognita, M. floridensis Handoo et al. 2004, M. ethiopica Whitehead 1968, M. paranaensis Carneiro et al. 1996, M. arabicida López & Salazar 1989, M. enterolobii Yang & Eisenback 1983, M. cf. haplanaria Eisenback et al. 2003 plus other species not identified, and the second including M. hapla and M. partytila Kleynhans, 1986 in the other clade.

Fig. 8
figure 8

Phylogenetic relationships within Meloidogyne species. Bayesian 50 % majority rule consensus trees as inferred from partial coxII-16S rRNA genes sequences alignment under the GTR + I + G model. Posterior probabilities more than 65 % are given for appropriate clades (in bold letters); bootstrap values greater than 50 % are given on appropriate clades in ML analysis. Newly obtained sequence in this study are in bold font

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Discussion

The primary objective of this study was to identify and molecularly characterize a species of Meloidogyne detected in wild olive soils in northern Morocco. However, this species was not related morphologically to the other species associated with olive such as M. arenaria, M. artiellia, M. baetica, M. hapla, M. incognita, M. javanica, and M. lusitanica (Castillo et al. 2010; Ali et al. 2014) (see Table 2). The main character in its juveniles associated with the long tail and hyaline region indicates this species would be associated with the “graminis” group (Jepson 1987). However, its associated host plant (wild olive) is different to the main species in this group (mainly associated to monocotyledon plants). Other plants such as arundo canes, lentisc, Phillyrea, Mimosaceae schrubs, maritime pine and eucalyptus trees, etc. were also detected in the same site.

It also differs phylogenetically from this “graminis” group of species but is related to M. dunensis and M. silvestris associated in the Iberian peninsula with dicotyledonous plants and with woody plants respectively (Palomares-Rius et al. 2007; Castillo et al. 2009). The reproduction on tomato of this new species also expands its host-range, as for the polyphagous root-knot nematode species M. incognita, M. javanica and M. arenaria that are able to infect olive and other hosts. However, the other group of species that parasite olive (M. baetica and M. lusitanica) have a very restricted host-range and limited prevalence (El-Borai and Duncan 2005). Despite no M. spartelensis n. sp. surviving on olive cv. Picholine du Languedoc (may be due to cultivar incompatibility or low initial inoculum density), pathogenicity experiments should be carried out with M. spartelensis n. sp. in order to verify the olive host suitability.

Phylogenetic analysis of the used molecular markers (D2-D3 region, ITS, partial 18S, partial coxII-16S rRNA and coxI sequences) demonstrates that M. spartelensis n. sp. is different to the other species with molecular markers in GenBank. It is clearly phylogenetically related to M. dunensis, found in Spain (Cullera, Valencia Province) with sea rocket (Cakile maritima Scop.) in coastal dunes. Both species are able to infect tomato and possess long tails with similar morphological traits. Probably both species evolved from the same ancestor species. This species is also related to M. silvestris with only a woody host known (Ilex aquifolium L.) and also described in the Iberian Peninsula. These species group seems to be related to a diversification process in the Mediterranean basin with restricted areas of distribution in natural environments. This could be partially due to long persistence of Meloidogyne spp. in isolated regions (i.e., allopatric speciation), in particular during last glacial maximum. Indeed, the place where M. spartelensis n. sp. was detected belongs to the Rif-Mountain plant-refuge area (Médail and Diadema 2009) and to the Gibraltar strait biodiversity hot-spot (Rodriguez-Sanchez et al. 2008). In this specific area, wild olive consists in an ancestral wild gene pool with unique haplotypes (Besnard et al. 2013) supporting refuge conditions. The ability of some Meloidogyne species to infect olive tree seems to be acquired in several events in their evolution and not from a unique event, because species with restricted ability to infect other hosts, as M. baetica are related phylogenetically nor with them and nor the other group of highly polyphagous species (M. incognita, M. javanica and M. arenaria). Meloidogyne lusitanica did not have molecular markers in order to extend this supposition. However, this hypothesis is not completely tested with the lack of host test for M. baetica, M. lusitanica and M. spartelensis n. sp. Phylogenetic trees with the markers used showed the ancestry of certain species in the different markers studied, i.e., M. mali, M. camelliae, M. ichinohei, M. artiellia, M. panyuensis, M. coffeicola and M. baetica. This result is similar to other phylogenetic analysis with a more limited number of Meloidogyne spp. (Adams et al. 2009).

Meloidogyne spartelensis n. sp. also differs from Est and Mdh isozyme electrophoretic patterns to the other species closely related morphologically and specific to olive parasitism. Differences in the number of bands and their gel position give us the possibility to differentiate it. The analysis of isozyme electrophoretic patterns, in particular Est and Mdh, as well as several molecular approaches have been proved to be a valuable tool for precise identification of Meloidogyne species (Flores Romero and Navas 2005; Blok and Powers 2009). This accurate species description with molecular markers associated to the type population (D2-D3 region, ITS, partial 18S and partial coxII-16S rRNA; coxI of mtDNA) will provide accurate markers for the unequivocal species identification and monitoring the possibility of expansion or identification in other untested areas.

In summary, present study establishes the importance of using polyphasic identification highlighting the time consuming aspect and difficulty of a correct identification at species level within the Meloidogyne spp. and describes a new root-knot nematode associated with wild olive in the Mediterranean Basin area.