First Report of Heterodera zeae Koshy, Swarup & Sethi, 1971 (corn cyst Nematode) Infecting Corn (Zea mays) in Spain.

Heterodera zeae Koshy, Swarup & Sethi, 1971 (corn cyst nematode) is an important disease of corn in several areas of the world, including India, Nepal, Pakistan, Egypt, USA, Greece and Portugal (Subbotin et al., 2010). It is a sedentary semi-endoparasite feeding on corn roots and other Poaceae plants and has been associated with significant yield losses in corn (Subbotin et al., 2010). During autumn 2022 a plant-parasitic nematode survey performed in corn at central-western area of Spain (Talavera de la Reina, Toledo), revealed a commercial field with stunted plants. Nematodes were extracted from soil by centrifugal-flotation method (Coolen, 1979). Corn roots inspection detected infections by immature and mature cysts, and soil revealed also mature live cysts and second-stage juveniles (J2s) with a population density of 1010 eggs and J2s/500 cm3 soil (including eggs from cysts). J2s and cysts were processed to pure glycerine using De Grisse's (1969) method. DNA was isolated from single live fresh J2s specimens for amplifying and sequencing of cytochrome c oxidase subunit II (COII) mitochondrial region using the primer pair species-specific H.Gly-COIIF_inFOR/P116F-1R (Riepsamen et al., 2011); D2 and D3 expansion domains of the 28S rRNA were amplified using the D2A/D3B primers (De Ley et al. 1999); internal transcribed spacer (ITS) region using primers TW81/AB28 (Subbotin et al., 2001); and cytochrome c oxidase subunit 1 (COI) gene was amplified using the primers JB3/JB5 (Bowles et al., 1992). Brown cysts were lemon-shaped with a protruding vulval cone with fenestra ambifenestrate, bullae prominent below underbridge and characteristically arranged in finger-like bullae (Fig. 1). J2 with slightly offset lip region (3-5 annuli), stylet strong with rounded stylet knobs, lateral field with four lines, and tail short and tapering conically. Measurements of cysts (n=10) included body length 559 (432-688) µm, body width 450 (340-522) µm, fenestral length 40 (36-43) µm, semifenestral width 19 (17-21) µm, and vulval slit 40 (35-44) µm. J2 measurements (n=10) included body length 477 (420-536) µm, stylet length 21 (20-22) µm, tail length 51 (47-56) µm, and tail hyaline region 23 (20-26) µm. Morphology and morphometrics of cysts and J2, fit with original description and others from several countries (Subbotin et al., 2010). Two J2s individuals were sequenced for COII region (OQ509010-OQ509011) showing 97.1-98.1% similarity with H. zeae from USA (HM462012). Six almost identical 28S rRNA sequences from J2s (OQ449649-OQ449654) were 99.2-99.4% similar to 28S rRNA sequences of H. zeae from Greece, Afghanistan and USA (GU145612, JN583885, DQ328695). Four identical ITS DNA fragments from J2s (OQ449655-OQ449658) were 97.0-97.8% similar to ITS sequences of H. zeae from Greece, and China (GU145616, MW785771, OP692770). Finally, six COI sequences of 400 bp obtained for J2s (OQ449699-OQ449704) were under 87% similarity to several COI sequences of Heterodera spp. in NCBI, being a new molecular barcoding for identifying this species. On the basis of these results, the cyst nematodes isolated from the corn plants from the central-western area of Spain (Talavera de la Reina, Toledo) were confirmed as H. zeae and up to our knowledge it is the first report in Spain. This is a well-known pest of corn, causing important losses in this crop (Subbotin et al., 2010) and it was previously regulated as a quarantine nematode in the Mediterranean region (EPPO).

First reports of Hemicycliophora poranga, Helicotylenchus dihystera and Tylenchorhynchus zeae (Tylenchomorpha) from Greece and further records of four other nematode species.

Nematode samplings in various areas and crops of Greece were carried out and the recovered nematode species were characterized using morphological and molecular data. Seven species of plant-parasitic nematodes were recovered, three of which are reported for the first time in Greece, including Hemicycliophora poranga, Helicotylenchus dihystera and Tylenchorhynchus zeae. Four other recovered species had already been reported in Greece, including Bitylenchus hispaniensis, Helicotylenchus microlobus, Nanidorus minor and Scutellonema brachyurus. D2-D3 segments of 28S rRNA gene for all of these nematode species are provided.

Distribution, Ecological Factors, Molecular Diversity, and Specific PCR for Major Species of Pin Nematodes (Paratylenchus spp.) in Prunus Plantations in Spain.

This study aimed to evaluate the prevalence and distribution of Paratylenchus species in the main areas of Prunus spp. production in Spain, their ecological constraints, and new molecular tools for the specific identification of major species. Pin nematodes are recognized as plant-parasitic nematodes with a wide host range and global distribution. Therefore, understanding the environmental and agronomic factors involved in their distribution is critical to design environmental and sustainable management strategies to reduce plant damage. A total of 219 sampling sites were surveyed and 12 Paratylenchus species were identified based on an integrative taxonomic approach (P. baldaccii, P. enigmaticus, P. goodeyi, P. hamatus, P. holdemani, P. indalus, P. israelensis, P. pedrami, P. tateae, P. tenuicaudatus, P. veruculatus, and P. zurgenerus). The most common pin nematode was P. hamatus, followed by P. tenuicaudatus. Nematode abundance was influenced by climatic characteristics, soil chemical properties, and agronomic management practices. Nine explanatory variables were selected as the most strongly associated with Paratylenchus distribution. Specifically, P. tenuicaudatus was significantly correlated with soil chemical characteristics, such as pH and carbon, sulfur, and sodium content, whereas P. goodeyi was closely related to fields with <10 years of almond cultivation. Species-specific PCRs were developed for P. hamatus and P. tenuicaudatus and their validity was evaluated studying the molecular variability of these species and against other Paratylenchus species.

First Report of Longidorus Leptocephalus Hooper, 1961 (Nematoda: Longidoridae) from Greece.

Longidorid nematodes comprise more than 500 species, and Longidorus and Xiphinema are the most diversified, prevalent, and cosmopolitan genera within plant-parasitic nematodes. The genus Longidorus comprise a group of species, some of which are vectors of plant viruses. New sampling for needle nematodes was carried out in a grapevine area in Thessaloniki, northern Greece, and one nematode species of Longidorus (L. leptocephalus) was recovered. Nematodes were extracted from soil samples by modified sieving and a decanting method. Extracted specimens were processed using glycerol, mounted on permanent slides, and subsequently identified morphologically. Nematode DNA was extracted from individual, live specimens, and PCR (Polymerase Chain Reaction) assays were performed for D2-D3 expansion segments of 28S rRNA, ITS1 rRNA, and partial mitochondrial COI regions. Morphology and morphometric data obtained from this population were consistent with the original description and reported populations of L. leptocephalus. To our knowledge, this is the first report of L. leptocephalus in Greece and the second in the Mediterranean Basin after the record of the species from Slovenia, extending the geographical distribution of this species in Europe.

First Report of Xiphinema Ifacolum Luc, 1961 (Dorylaimida: Longidoridae) from Nigeria.

A population of a species of dagger nematode (Xiphinema) belonging to the non-americanum group was recovered from the fields of kola nut (Cola sp.) in southern Nigeria. The morphological and morphometric data obtained from this population were consistent with the characteristics of the species Xiphinema ifacolum. In addition, molecular identification based on D2-D3 expansion segments of 28S rRNA and partial mitochondrial COI gene regions confirmed its identity. According to our knowledge, this is the first report of the species from Nigeria, and the second report from Africa, after the original description from Foulaya, Guinea.

New Distribution and Molecular Diversity of the Reniform Nematode Rotylenchulus macrosoma (Nematoda: Rotylenchulinae) in Europe.

Reniform nematodes of the genus Rotylenchulus are semi-endoparasites of numerous herbaceous and woody plant species roots and occur largely in regions with temperate, subtropical, and tropical climates. In this study, we provide new records of the nematode Rotylenchulus macrosoma in eight European countries (Czech Republic, France, Germany, Hungary, Italy, Romania, Serbia, and Portugal), in addition to the six Mediterranean countries (Greece, Israel, Jordan, Spain, Syria, and Turkey) where the nematode was previously reported. Four new host species (corn, pea, wheat, and an almond-peach hybrid rootstock) are added to the recorded host species (bean, chickpea, hazelnut, peanut, soybean, and wild and cultivated olive). Molecular analyses based on the cytochrome c oxidase subunit coxI and D2-D3 segments of 28S RNA markers showed high diversity and pronounced genetic structure among populations of Rotylenchulus macrosoma. However, the complexity of phylogeographic patterns in plant-parasitic nematodes may be related to the intrinsic heterogeneity in the distribution of soil organisms, a rare occurrence of a species, or the potential human impact associated with agricultural practices.

Morphological and molecular characterisation of Longidorus pauli (Nematoda: Longidoridae), first report from Greece.

Sampling for needle nematodes was carried out in a grapevine area in Thessaloniki, North Greece and two nematode species of Longidorus (L. pauli and L. pisi) were collected. Nematodes were extracted from 500 cm3 of soil by modified sieving and decanting method, processed to glycerol and mounted on permanent slides, and subsequently identified morphologically and molecularly. Nematode DNA was extracted from single individuals and PCR assays were conducted to amplify D2-D3 expansion segments of 28S rRNA, ITS1 rRNA, and partial mitochondrial coxI regions. Morphology and morphometry data obtained from these populations were consistent with L. pauli and L. pisi identifications. To our knowledge, this is the first report of L. pauli for Greece, and the second world report after the original description from Idleb, Syria, extending the geographical distribution of this species in the Mediterranean Basin.

First report of cultivated Cretan mountain tea (Sideritis syriaca) as a host of Meloidogyne hapla and M. javanica in Crete, with some additional records on the occurrence of Meloidogyne species in Greece.

Cultivated Cretan mountain tea or Malotira (Sideritis syriaca L.) was found to be infected by Meloidogyne hapla and Meloidogyne javanica in the island of Crete. The authors provide the first molecular characterization of M. hapla in Greece and the first report of Cretan mountain tea or Malotira as a host of Meloidogyne species worldwide. In addition, Meloidogyne hispanica was found infecting aloe (Andros island) and corn (Drama, North Greece) consisting the first reports of natural infection of these plants by M. hispanica in Europe. Furthermore, infection of corn by M. incognita and soybean by M. javanica (Drama, North Greece) are reported for the first time in Greece. Integrative taxonomical approach based on perineal pattern and EP/st ratio, as well as the region of the mitochondrial genome between the cytochrome oxidase subunit II (coxII) and 16S rRNA mitochondrial DNA (mtDNA) genes was used to differentiate Meloidogyne species.Cultivated Cretan mountain tea or Malotira (Sideritis syriaca L.) was found to be infected by Meloidogyne hapla and Meloidogyne javanica in the island of Crete. The authors provide the first molecular characterization of M. hapla in Greece and the first report of Cretan mountain tea or Malotira as a host of Meloidogyne species worldwide. In addition, Meloidogyne hispanica was found infecting aloe (Andros island) and corn (Drama, North Greece) consisting the first reports of natural infection of these plants by M. hispanica in Europe. Furthermore, infection of corn by M. incognita and soybean by M. javanica (Drama, North Greece) are reported for the first time in Greece. Integrative taxonomical approach based on perineal pattern and EP/st ratio, as well as the region of the mitochondrial genome between the cytochrome oxidase subunit II (coxII) and 16S rRNA mitochondrial DNA (mtDNA) genes was used to differentiate Meloidogyne species.

Integrative taxonomy of Xiphinema histriae and Xiphinema lapidosum from Spain.

Three populations of Xiphinema non-americanum group species were detected in agricultural and natural ecosystems, during routine surveys for plant-parasitic nematodes in Spain. Based on morphological and molecular analyses, the species were identified as Xiphinema histriae and Xiphinema lapidosum, being this the first record and molecular characterization of both species in Spain. The morphometrics and morphology of the Spanish populations agree with those of the original description and paratype specimens and the present study provided a first description of the second to fourth juvenile stages of both species. A detailed study on the morphology in the Spanish populations of X. histriae, as well as in paratypes, showed a pseudo-Z-organ with weakly muscularized wall and containing numerous small dense granular bodies, which was different to the original description by Lamberti et al. (1993). This new finding suggests that X. histriae must be considered a member of the morphospecies Group 5 of X. non-americanum. Phylogenetic analysis based on D2 to D3 expansion segments of 28S gene, ITS1 and partial CoxI gene indicated that X. histriae and X. lapidosum are phylogenetically related with other Xiphinema non-americanum group spp. reported from Spain. Considering the pathological and economic importance of this group of nematodes, the combination of morphological characters, measurements, and molecular analysis is crucial for accurate identification of these species.

First Report of Bitylenchus hispaniensis, Pratylenchoides alkani, and Helicotylenchus vulgaris in Association with Cultivated and Wild Olives in Crete, Greece and Molecular Identification of Helicotylenchus microlobus and Merlinius brevidens.

Nematode samplings in cultivated and wild olive in Crete, Greece, yielded the presence of Bitylenchus hispaniensis , Helicotylenchus microlobus , Helicotylenchus vulgaris , Merlinius brevidens , and Pratylenchoides alkani . With the exception of H. microlobus and M. brevidens , reports of these plant-parasitic nematode species constitute new records for Greece. Bitylenchus hispaniensis is also reported for first time in a country outside of Spain, where it was originally described. Pratylenchoides alkani is herein reported for the second time in the Mediterranean area and for the first time in association with olive. Two further populations of H. microlobus and H. vulgaris , from walnut and goji berry from Greece, were identified. Molecular data for all of these nematode species are provided, resulting in the first integrative identification of these Greek populations.

First Reports, Morphological, and Molecular Characterization of Longidorus caespiticola and Longidorus poessneckensis (Nematoda: Longidoridae) from Ukraine.

Seven needle nematode species of the genus Longidorus have been reported in Ukraine. Nematological surveys for needle nematodes were carried out in Ukraine between 2016 and 2017 and two nematode species of Longidorus (L. caespiticola and L. poessneckensis) were collected from natural and anthropogenically altered habitats on the territory of Opillia and Zakarpattia in Ukraine. Nematodes were extracted from 500 cm3 of soil by modified sieving and decanting method. Extracted specimens were processed to glycerol and mounted on permanent slides and subsequently identified morphologically and molecularly. Nematode DNA was extracted from single individuals and PCR assays were conducted as previously described for D2-D3 expansion segments of 28S rRNA. Sequence alignments for D2-D3 from L. caespiticola showed 97%-99% similarity to other sequences of L. caespiticola deposited in GenBank from Belgium, Bulgaria, Czech Republic, Russia, Slovenia, and Scotland. Similarly, D2-D3 sequence alignments from L. poessneckensis, showed 99% to other sequences of L. poessneckensis deposited in GenBank from Slovakia and Czech Republic. Morphology, morphometry, and molecular data obtained from these samples were consistent with L. caespiticola and L. poessneckensis identification. To our knowledge, these are the first reports of L. caespiticola and L. poessneckensis in Ukraine, extending the geographical distribution of these species.

Morphological and Molecular Identification of Longidorus euonymus and Helicotylenchus multicinctus from the Rhizosphere of Grapevine and Banana in Greece.

Plant-parasitic nematodes such as Longidorus euonymus and Helicotylenchus multicintctus are species widely distributed in central Europe as well as in Mediterranean area. In Greece, both species have been previously reported but no morphometrics or molecular data were available for these species. Nematode surveys in the rhizosphere of grapevines in Athens carried out in 2016 and 2017, yielded a Longidorus species identified as Longidorus euonymus. Similarly, a population of Helicotylenchus multicinctus was detected infecting banana roots from an outdoor crop in Tertsa, Crete. For both species, morphometrics and molecular data of Greek populations were provided, resulting in the first integrative identification of both nematode species based on morphometric and molecular markers, confirming the occurrence of these two nematodes in Greece as had been stated in earlier reports.

Molecular diversity of bacterial endosymbionts associated with dagger nematodes of the genus Xiphinema (Nematoda: Longidoridae) reveals a high degree of phylogenetic congruence with their host.

Bacterial endosymbionts have been detected in some groups of plant-parasitic nematodes, but few cases have been reported compared to other groups in the phylum Nematoda, such as animal-parasitic or free-living nematodes. This study was performed on a wide variety of plant-parasitic nematode families and species from different host plants and nematode populations. A total of 124 nematode populations (previously identified morphologically and molecularly) were screened for the presence of potential bacterial endosymbionts using the partial 16S rRNA gene and fluorescence in situ hybridization (FISH) and confocal microscopy. Potential bacterial endosymbionts were only detected in nematode species belonging to the genus Xiphinema and specifically in the X. americanum group. Fifty-seven partial 16S rRNA sequences were obtained from bacterial endosymbionts in this study. One group of sequences was closely related to the genus 'Candidatus Xiphinematobacter' (19 bacterial endosymbiont sequences were associated with seven nematode host species, including two that have already been described and three unknown bacterial endosymbionts). The second bacterial endosymbiont group (38 bacterial endosymbiont sequences associated with six nematode species) was related to the family Burkholderiaceae, which includes fungal and soil-plant bacterial endosymbionts. These endosymbionts were reported for the first time in the phylum Nematoda. Our findings suggest that there is a highly specific symbiotic relationship between nematode host and bacterial endosymbionts. Overall, these results were corroborated by a phylogeny of nematode host and bacterial endosymbionts that suggested that there was a high degree of phylogenetic congruence and long-term evolutionary persistence between hosts and endosymbionts.

First Report of Longidorus kuiperi and Rotylenchus eximius from Coastal Sand Dunes in Crete, Greece.

Plant-parasitic nematode species have been reported on several occasions from coastal sand dunes, including Longidorus and Rotylenchus species (Vovlas et al., 2008; De Luca et al., 2009; Mateille et al., 2014). In April 2016, 10 soil samples of 3 to 4 kg from the rhizosphere of Tamarix smyrnensis with different vegetation around (viz. Elymus farctus, Lycium schweinfurthii, Crithmum maritimum, and Arthrocnemum sp.) were collected for diagnosis of plant-parasitic nematodes. The area of sampling was a coastal sand dune near the archeological site of Komos, southwest of Crete, Greece. Low soil populations of a needle and a spiral nematode were detected (3 and 8 individuals/1,000 cm3 of soil, respectively), which prompted us to undertake a detailed morphological and molecular comparative study with previous reported data. Nematodes were extracted from soil with the wet sieving and decanting method (Cobb, 1918). Morphological and molecular analyses of females identified these species as Longidorus kuiperi Brinkman, Loof and Barbez, 1987, and Rotylenchus eximius Siddiqi, 1964. The morphology of L. kuiperi females (six specimens studied) was characterized by having a slender body; very broad lip region (27 ± 1.5 [25 to 30] µm in width); short, hemispherical tail; body length of (7.1 ± 0.8 [6.5 to 8.5] mm); vulva position at 47% to 55% of body length; odontostyle length of (105 ± 6.5 [90 to 115] µm); males very common (but less frequent than females [45% vs 55%]); tail region with 15 to 20 supplements and bulged terminal cuticle. The morphology of R. eximius females (four specimens studied) was characterized by having a hemispherical lip region clearly set off; with four annuli; body without longitudinal striations; lateral fields areolated in the pharyngeal region only; stylet 36 to 38 µm; and broadly rounded tail. The morphology of the isolated nematodes agreed with previous descriptions of L. kuiperi (Brinkman et al., 1987; De Luca et al., 2009), and R. eximius (Siddiqi, 1964; Castillo and Vovlas, 2005). A single individual was used for DNA extraction. Primers and polymerase chain reaction conditions used in this research were specified in Cantalapiedra et al. (2013) and Archidona-Yuste et al. (2016), and a single amplicon of 800 and 1,100 bp was obtained and sequenced, respectively. D2-D3 (KX398055-KX398056) and ITS sequence alignments (751 and 648 bp, respectively) from L. kuiperi (KX398057) showed 98% to 99% similarity, differing in 4, and from 6 to 12 nucleotides, respectively, to other sequences of L. kuiperi deposited in GenBank from Italy and Spain (AM911623, AM905267-AM905270, respectively), with a query coverage of 99%. Similarly, D2-D3 sequence alignment from R. eximius (KX398058) showed 100% to 99% to another sequence of R. eximius deposited in GenBank from Italy and Spain (EU280794, DQ328741), differing in zero to three nucleotides, respectively, and a query coverage of 81%. Phylogenetic analyses using Bayesian Inference placed L. kuiperi in a highly supported (100%) clade that included all L. kuiperi sequences deposited in GenBank (Archidona-Yuste et al., 2016), and R. eximius in a highly supported (100%) clade that included all R. eximius sequences deposited in GenBank (Cantalapiedra-Navarrete et al., 2013). All identification methods were consistent with L. kuiperi and R. eximius. To our knowledge, this is the first report of L. kuiperi and R. eximius in Greece. Consequently, all these data suggest that coastal sand dunes in Europe constitute environmental conditions optimal for colonization and development of L. kuiperi, as previously reported (De Luca et al., 2009). Similarly, R. eximius has been reported in several Mediterranean countries, including Italy, Morocco, Spain, and Tunisia (Castillo and Vovlas, 2005), and this report extend the geographical distribution of this species.

First Report of the Carolina Spiral Nematode, Scutellonema brachyurus, from Soil of a Garden in Crete, Greece.

Members of the genus Scutellonema can cause substantial crop losses to ornamental and cultivated plants directly by feeding ectoparasitically on plant roots (Bridge et al., 2005; Coyne et al., 2006). In May 2015, a soil sample from a house garden from Heraklion city in Crete, Greece, was sent for diagnosis of plant-parasitic nematodes. In this place, there had been cactus (Opuntia sp.) plants (probably imported), which were uprooted 3 to 4 years earlier. After that, the area was cropped with cucumber (Cucumis sativus L.) in spring-summer and leaf vegetables such as spinach (Spinacia oleracea L.) and chicory (Cichorium intybus L.) in autumn-winter. The soil was collected 1 mon after the end of chicory crop. A population density (ca. 30 individuals/100 cm(3) of soil) of spiral nematodes (Scutellonema sp.) was found by extracting soil with the wet sieving and decanting method (Cobb, 1918). Morphological and molecular analyses of females identified the species as Scutellonema brachyurus (Steiner, 1938) Andrássy, 1958. The morphology of females was characterized by a hemispherical lip region with four to six annuli, morphometric data for 12 females were L, 640 to 760 µm; a, 24.6 to 30.6; b, 5.8 to 7.4; c, 69.1 to 99.3; c´ 0.5 to 0.6; stylet, 24.5 to 27.5 µm with anterior part shorter than posterior; and spermatheca nonfunctional and male absent. The morphology agreed with the description of S. brachyurus (van den Berg et al., 2013). Alignment indicated that the D2-D3 and ITS sequences (KU059494 and KU059495, respectively) showed 99% and 100% to 99% similarity, respectively, to other sequences of S. brachyurus (type A) deposited in GenBank from the United States, Italy, and Korea (JX472037-JX472046, DQ328753, FJ485643; and JX472069, JX472070, JX472071, respectively), differing from one to six nucleotides. Phylogenetic analyses using Bayesian inference of these sequences placed the Scutellonema sp. in a highly supported (100%) clade that included all S. brachyurus (type A) sequences deposited in GenBank (van den Berg et al., 2013). All identification methods were consistent with S. brachyurus. To our knowledge, this is the first report of S. brachyurus for Greece. As the cucumbers and the leaf vegetables cultivated in the area were seed planted, we consider that the nematode originated most probably from the cactus plants which had been previously root ball planted. Scutellonema brachyurus may represent a threat for ornamental and cultivated plants production in Crete, Greece. The nematode has been already reported, mainly in greenhouses of six European countries (CABI and EPPO, 2006). In most of these cases, it is hypothesized that the nematode was introduced by imported plant material.

First Report of the Spiral Nematode Rotylenchus incultus (Nematoda: Hoplolaimidae) from Cultivated Olive in Tunisia, with Additional Molecular Data on Rotylenchus eximius.

Spiral nematode species of the genus Rotylenchus have been reported on olive (Olea europaea L.) in several Mediterranean countries (Castillo et al., 2010; Ali et al., 2014). Nematological surveys for plant-parasitic nematodes on olive trees were carried out in Tunisia between 2013 and 2014, and two nematode species of Rotylenchus were collected from the rhizosphere of olive cv. Chemlali in several localities of Tunisia (Tables 1,2 [Table: see text] [Table: see text] ). Twenty-two soil samples of 3 to 4 kg were collected with a shovel from the upper 50 cm of soil from arbitrarily chosen olive trees. Nematodes were extracted from 500 cm3 of soil by centrifugal flotation method (Coolen, 1979). Specimens were heat killed by adding hot 4% formaldehyde solution and processed to pure glycerin using the De Grisse's (1969) method. Measurements were done using a drawing tube attached to a Zeiss III compound microscope. Nematode DNA was extracted from single individuals and PCR assays were conducted as described by Castillo et al. (2003). Moderate-to-low soil populations of these spiral nematodes were detected (5.5-11.5, 1.5-5.0 individuals/500 cm3 of soil, respectively). This prompted us to undertake a detailed morphological and molecular comparative study with previous reported data. Morphological and molecular analyses of females identified these species as Rotylenchus eximius Siddiqi, 1964, and Rotylenchus incultus Sher, 1965. The morphology of R. eximius females (five specimens studied) was characterized by having a hemispherical lip region clearly off set, with four to five annuli, body without longitudinal striations, lateral fields areolated in the pharyngeal region only, stylet 32 to 36 µm long, and broadly rounded tail. The morphology of R. incultus females (51 females and 16 males; Table 2) was characterized by a hemispherical lip region with the basal annulus subdivided by irregular longitudinal striations, with three, rarely four annuli; stylet 21.5 to 27.5 µm long, female tail hemispherical with terminus regularly annulated; phasmids anterior to anus level (3-6 annuli above). The morphology of the isolated nematodes agreed with previous descriptions of R. eximius (Siddiqi, 1964; Castillo and Vovlas, 2005) and R. incultus (Sher, 1965; Castillo and Vovlas, 2005; Vovlas et al., 2008), respectively. A single individual was used for DNA extraction. Primers and PCR conditions used in this research were specified in Cantalapiedra-Navarrete et al. (2013), and a single amplicon of 800, 1,100, and 450 bp was obtained and sequenced for D2 to D3, ITS1, and cytochrome c oxidase subunit 1 (coxI), respectively. Sequence alignments for D2 to D3 (KX669231-KX669233), ITS1 (KX669238-KX669240), and coxI (KX669244-KX669245) from R. eximius, showed 99% to 97%, 98% to 94%, 93% similarity to other sequences of R. eximius deposited in GenBank (EU280794-DQ328741, EU373663-EU373664, JX015401-JX015402, respectively). Similarly, D2 to D3 (KX669234-KX669237), ITS1 (KX669241-KX669243), and coxI (KX669246-KX669249) sequence alignments from R. incultus, showed 99%, 99% to 95%, 99% to 90% similarity, respectively, to other sequences of R. incultus deposited in GenBank (EU280797, EU373672-EU373673, JX015403, respectively). The best fitted model of DNA evolution was obtained using jModelTest v. 2.1.7 (Darriba et al. 2012) with the Akaike information criterion. BI analyses were performed under the general time reversible (GTR) with invariable sites and a gamma-shaped distribution of substitution rates (GTR + I + G) model for ITS1 and coxI. Phylogenetic analyses of ITS1 and coxI using Bayesian inference (BI) placed R. eximius and R. incultus from Tunisia in subclades that included all R. eximius and R. incultus sequences deposited in GenBank (Fig. 1Fig. 1Phylogenetic relationships within Rotylenchus species found in Tunisia and other species from GenBank. Bayesian 50% majority rule consensus trees as inferred from ITS1 and coxI sequences alignments under the GTR + I + G model. Posterior probabilities more than 0.70 are given for appropriate clades. Newly obtained sequences in this study are in bold. Scale bar = expected changes per site.), which agrees with previous results (Cantalapiedra-Navarrete et al., 2013). Morphology, morphometry, and molecular and phylogenetic data obtained from these samples were consistent with R. eximius and R. incultus identification. To our knowledge, this is the first report of R. incultus in Tunisia. Consequently, all these data suggest that spiral nematode species of the genus Rotylenchus are predominant in olive as previously reported in other Mediterranean areas (Ali et al., 2014).

A Blind-Identification Test on Criconema annuliferum (de Man, 1921) Micoletzky, 1925 Species Complex Corroborate the Hyper-Cryptic Species Diversity Using Integrative Taxonomy.

Ring nematodes are obligate ectoparasites on crops and natural herbaceous and woody plants, and some species are of economic importance and cause damage to roots of several crops. Recent integrative taxonomical analyses recognized the existence of two cryptic species within the Criconema annuliferum morphotype in Spain. In this study, we corroborated that morphometric, morphological and a multi-locus analysis (including the ribosomal markers D2-D3 expansion segments of 28S rRNA, ITS rRNA, 18S RNA, and the mitochondrial DNA cytochrome oxidase I gene) identified a new lineage clearly separated from C. annuliferum, C. paraannuliferum and C. plesioannuliferum. The new lineage was described herein as Criconema pseudoannuliferum sp. nov., confirming that C. annuliferum species complex species complex comprises a hyper-cryptic species complex. This research analysed soil samples from the rhizosphere of maritime pine (Pinus pinaster Ait.) forests in Bermeja-Crestellina Mountain, located at the western part of Málaga province, southern Spain. The integrative taxonomical analyses revealed the occurrence of a new cryptic species identified using females, males and juveniles with detailed morphology, morphometry and molecular markers, described herein as Criconema pseudoannuliferum sp. nov. All molecular markers (D2-D3, ITS, 18S and COI) were obtained from the same individual that was also used for morphological and morphometric analyses. This research demonstrated the hidden diversity within the C. annuliferum species complex species complex can reach to four lineages under ribosomal and mitochondrial gene markers for one morphospecies group, which includes four species, viz. C. annuliferum, C. paraannuliferum, C. plesioannuliferum, and C. pseudoannuliferum sp. nov. Criconema pseudoannuliferum sp. nov. was detected in moderate soil density in two maritime pine forests (5 and 25 nematodes/500 cm3 of soil) suggesting that does not cause damage to maritime pine.

A Proposed New Species Complex within the Cosmopolitan Ring Nematode Criconema annuliferum (de Man, 1921) Micoletzky, 1925.

Ring nematodes are obligate ectoparasites on cultivated and wild herbaceous and woody plants, inhabiting many types of soil, but particularly sandy soils. This study explored the morphometrical and molecular diversity of ring nematodes resembling Criconema annuliferum in 222 soil samples from fruit crops in Spain, including almond, apricot, peach and plum, as well as populations from cultivated and wild olives, and common yew. Ring nematodes of the genus Criconema were detected in 12 samples from under Prunus spp. (5.5%), showing a low to moderate nematode soil densities in several localities from southeastern and northeastern Spain. The soil population densities of Criconema associated with Prunus spp. ranged from 1 nematode/500 cm3 of soil in apricot at Sástago (Zaragoza province) to 7950 and 42,491 nematodes/500 cm3 of soil in peach at Ricla and Calasparra (Murcia province), respectively. The integrative taxonomical analyses reveal the presence of two cryptic species identified using females, males (when available), and juveniles with detailed morphology, morphometry, and molecular markers (D2-D3, ITS, 18S, and COI), described herein as Criconema paraannuliferum sp. nov. and Criconema plesioannuliferum sp. nov. All molecular markers from each species were obtained from the same individuals, and these individuals were also used for morphological and morphometric analyses. Criconema paraannuliferum sp. nov. was found in a high soil density in two peach fields (7950 and 42,491 nematodes/500 cm3 of soil) showing the possibility of being pathogenic in some circumstances.

DNA Barcoding and Morphometry Reveal Further Cryptic Bio-Diversity within the Pin Nematode Genus Paratylenchus (Nematoda: Tylenchulidae).

Paratylenchus species are obligate ectoparasitic nematodes on cultivated and wild herbaceous and woody plants occupying numerous soil categories. Several species may cause damage to several crops (viz. P. dianthus, P. enigmaticus, P. microdorus, P. hamatus and P. epacris on carnation, lettuce, rose and walnut, respectively). This investigation proves and emphasizes the relevance of applying integrative taxonomy for the accurate detection of Paratylenchus species in mountainous wild environments in the Malaga province, Southern Spain. This research analyzed 45 soil samples of maritimus pine and one of green heather in southern Spain and identified fourteen Paratylenchus species, two of them are described herein as new species (P. paraaonli sp. nov., P. plesiostraeleni sp. nov.), six of them were first reports for Spain (P. canchicus, P. nainianus, P. neonanus, P. salubris, Paratylenchus sp. 2 SAS, and P. wuae), and six species (P. caravaquenus, P. microdorus, P. nanus, P. neoamblycephalus, P. sheri, and P. variabilis) have been already reported in Spain. Accordingly, these data increase the biodiversity of pin nematodes in Spain comprising a total of 47 species (33.1% out of 142 total species of this genus). Phylogenetic analyses based on ribosomal and mitochondrial markers (D2-D3, ITS, and partial COI) resulted in a consistent position for the newly described Paratylenchus species in this study (P. plesiostraeleni sp. nov., P. paraaonli sp. nov.). Paratylenchus plesiostraeleni sp. nov. grouped in a separated subclade as unequivocal species from the P. straeleni-complex species (including P. straeleni and P. parastraeleni), and P. paraaonli sp. nov. clustered with P. vitecus, but clearly separate from this species. This study indicates that Paratylenchus species diversity in natural environments may be higher than expected, and this study may help in accurate identifications.

Remarkable Cryptic Diversity of Paratylenchus spp. (Nematoda: Tylenchulidae) in Spain.

In previous studies, fifteen species of Paratylenchus, commonly known as pin nematodes, have been reported in Spain. These plant-parasitic nematodes are ectoparasites with a wide host range and global distribution. In this research, 27 populations from twelve Paratylenchus species from 18 municipalities in Spain were studied using morphological, morphometrical and molecular data. This integrative taxonomic approach allowed the identification of twelve species, four of them were considered new undescribed species and eight were already known described. The new species described here are P. caravaquenus sp. nov., P. indalus sp. nov., P. pedrami sp. nov. and P. zurgenerus sp. nov. As for the already known described species, five were considered as first reports for the country, specifically P.enigmaticus, P. hamatus, P. holdemani, P. israelensis, and P. veruculatus, while P. baldaccii, P. goodeyi and P. tenuicaudatus had already been recorded in Spain. This study provides detail morphological and molecular data, including the D2-D3 expansion segments of 28S rRNA, ITS rRNA, and partial mitochondrial COI regions for the identification of different Paratylenchus species found in Spain. These results confirm the extraordinary cryptic diversity in Spain and with examples of morphostatic speciation within the genus Paratylenchus.