The more we search, the more we find: discovering and expanding the biodiversity in the ring nematode genus Xenocriconemella De Grisse and Loof, 1965 (Nematoda: Criconematidae).

The ring nematode genus Xenocriconemella De Grisse and Loof, 1965 comprises only one nominal species, Xenocriconemella macrodora (Taylor, 1936) De Grisse and Loof, 1965. The initial objective of the present study was to investigate the morphological-morphometric and molecular diversity of 28 X. macrodora populations in the Iberian Peninsula associated with tree forests (mainly Quercus spp.). However, a detailed integrative taxonomic analysis (morphological-morphometric and molecular data) from each population and analysis of this data using principal component analysis (PCA) for morphometric data (including these 28 populations and other 25 X. macrodora populations around the world) and molecular and phylogenetic species delimitation methods revealed that X. macrodora forms a species complex. This species complex is composed by species that are morphometricly and morphologically similar, but clearly different at the molecular level. Three new species are described applying integrative taxonomy, namely as Xenocriconemella iberica sp. nov., Xenocriconemella paraiberica sp. nov. and Xenocriconemella pradense sp. nov. However, the molecular diversity of this species in USA and Italy confirmed that additional species are likely present in this species complex, and the diversity of this group may be higher than expected. The study of X. macrodora topotypes can clarify the position of this species using molecular markers under an integrative approach.

Integrative taxonomy and phylogenetic relationships between representatives of genera within Merliniinae (Nematoda: Merliniidae), with new data on fourteen known and one unknown species.

The application of integrative taxonomic approaches is useful to species delineation based on a combination of distinct types of characters, here morphological features and ribosomal DNA sequences. In this study, we surveyed ectoparasitic nematodes of the subfamily Merliniinae in cultivated and natural environments in Iran. Results of morphological and morphometrical studies, light and scanning electron microscopic observations, and molecular analyses allowed us the identification of fourteen known and one unknown species including representatives of the genera Amplimerlinius (five species), Geocenamus (one species), Merlinius (three species), Nagelus (two species), Paramerlinius (one species), Scutylenchus (two species), and Telomerlinius (one species). The unknown species, Scutylenchus sp., characterized by having 35-50 incisures at mid-body; lateral field with 6 longitudinal incisures; lip region slightly offset by a constriction, flattened at front end; bearing 5-7 annuli; cephalic framework not refractive; stylet robust, 18.3-27 µm long; post anal intestinal sac absent; tail elongate conical, dorsally convex, with 24 (19-28) annuli in ventral side, ending to a smooth terminus and males common; spicules 24.5-31 µm long. The phylogenetic analyses were carried out using molecular data from nuclear ribosomal DNA (rDNA) genes viz. D2-D3 expansion segments of the large ribosomal subunit (28S rRNA), partial small ribosomal subunit (18S rRNA), and internal transcribed spacer (ITS). The molecular variability of D2-D3 expansion segments of the 28S rRNA and partial 18S rRNA was low in this family in comparison to the ITS region, which could be a more helpful molecular marker in species and genus identification.

A new needle nematode, Longidorus zanjanensis sp. nov. (Nematoda: Longidoridae) from north-western Iran.

During a survey of soil nematodes in 2022, a new species of the genus Longidorus, described here as Longidorus zanjanensis sp. nov., was discovered in the rhizosphere of Astragalus sp. in Zanjan Province, Iran. The new needle nematode is described and illustrated based on morphological, morphometric, and molecular traits. Further, its females are characterized by having a long body ranging 5.6-7.7 mm long, lip region anteriorly flattened and almost continuous or slightly offset by a depression with body contour, ca 16.5-18.5 µm wide, amphidial fovea pouch-like without basal lobes, guiding ring at 35-41 µm distance from the anterior end, and an odontostyle and odontophore ranging 102-115 and 47-75 µm long, respectively. The pharyngeal bulb is 123-153 µm long, female reproductive system didelphic-amphidelphic containing sperm, vulva almost equatorial, located at 46.7-51.4% of body length, tail short, rounded to bluntly conoid, bearing two pairs of caudal pores and terminus widely rounded with distinct radial lines in hyaline region (39-50 µm long, c = 122.4-189.4, c' = 0.6-0.8). Males are common, making up to 60% of the adults, and are functional, with spicules 68.0-80.0 µm long, as well as having 8-14 ventromedian copulatory supplements. All four juvenile life developmental stages were present, with the tail of first-stage juvenile conoid shape, dorso-ventrally curved with rounded terminus. The polytomous codes delimiting the new species are: A4-B3-C3-D3-E1-F34-G12-H1-I2-J1-K6. Morphologically, the new species comes close to eight known species of the genus, namely L. apulus, L. armeniacae, L. crassus, L. kheirii, L. soosanae, L. proximus, L. pauli, and L. ferrisi. The morphological differences between the new species and the aforementioned species are discussed. Molecular phylogenetic analyses based on D2-D3 of large subunit (LSU) and internal transcribed spacer 1 (ITS1) rRNA sequences indicate that Longidorus zanjanensis sp. nov. is closely related to L. hyrcanus, L. soosanae, and L. elongatus.

Anatomical and molecular characterization of some rhigonematid parasites of millipedes in Nigeria, with new insights into their phylogeny.

Parasitic nematodes of millipedes from Nigeria are molecularly characterized for the first time. During nematode surveys on live giant African millipedes from several localities in Nigeria, 4 species of rhigonematids were identified by application of integrative taxonomical approaches (morpho-anatomy and molecular markers), including Brumptaemilius sp., Gilsonema gabonensis, Obainia pachnephorus, and Rhigonema disparovis. The results of morphometric and molecular analyses of D2-D3 28S, ITS, partial 18S rRNA, and cytochrome oxidase c subunit 1 (COI) gene sequences further characterized the rhigonematid species, and clearly separated them from other related species. Phylogenetic relationships based on 28S and 18S rRNA genes suggest that genera within Ransomnematoidea (Ransomnema, Heth, Carnoya, Brumptaemilius, Cattiena, Insulanema, Gilsonema) and Rhigonematoidea (Rhigonema, Obainia, Xystrognathus, Trachyglossoides, Ichthyocephaloides) clustered rather closer than could be expected in view of their morphological differences. Phylogenetic relationships based on ITS and COI are congruent with those of other ribosomal genes; however, they are not conclusive due to the scarcity of available sequences of these genes for these genera in NCBI.

A new needle nematode, Longidorus maginicus n. sp. (Nematoda: Longidoridae) from southern Spain.

During nematode surveys in natural vegetation in Sierra Mágina, Jaén province, southern Spain, a Longidorus species closely resembling Longidorus carpetanensis was found, but application of integrative taxonomic approaches clearly demonstrated that it is a new species described herein as Longidorus maginicus n. sp. The new species is amphimictic, characterized by a moderately long body (4.2-5.2 mm); lip region anteriorly flattened, slightly separated from the rest of body by a depression, 9.0-11.0 µm wide and 3.5-6.0 µm high; amphidial fovea not lobed; relatively short odontostyle (61.0-70.5 µm); guiding ring located 23.5-27.0 µm from anterior end; vulva located at 42.0%-51.3% of body length; female tail 39.0-61.0 µm long, conoid, dorsally convex with rounded terminus (c' = 1.3-2.1), with two or three pairs of caudal pores; and males common (1:2 ratio males:females), with moderately long spicules (39.0-48.5 µm) and 1 + 6-9 ventromedian supplements and three juvenile developmental stages. According to the polytomous key, codes for the new species are (codes in parentheses are exceptions): A2-B1-C2-D2-E1-F2(3)-G2-H5(4)-I2-J1-K6. The results of molecular analysis of D2-D3 28S, internal transcribed spacer region, partial 18S rDNA, and cytochrome oxidase c subunit 1 (coxI) gene sequences further characterized the new species status, and separated it from L. carpetanensis and other related species.

Taxonomical considerations and molecular phylogeny of the closely related genera Bitylenchus, Sauertylenchus and Tylenchorhynchus (Nematoda: Telotylenchinae), with one new and four known species from Iran.

During several nematological surveys in cultivated and natural habitats in Khuzestan and Zanjan provinces of Iran, a new species, Bitylenchus parvulus n. sp., two new records for Iran - namely, Tylenchorhynchus agri and Tylenchorhynchus graciliformis - and a population of Bitylenchus parvus and one of Sauertylenchus maximus were recovered and characterized based upon morphological and molecular approaches. The new species is characterized by lip region with five to seven annuli, stylet 17.7 (17.0-18.5) µm long, sub-cylindrical tail narrowing abruptly near terminus giving a bluntly digitate shape to the tail tip, cuticle near anterior part of vulva wrinkled and post-rectal sac occupies whole of tail cavity. The phylogenetic analyses were carried out using molecular data from D2-D3 expansion segments of large ribosomal subunit (28S rRNA) for all studied species and the partial small ribosomal subunit (18S rRNA) for the new species. The representatives of Bitylenchus and Sauertylenchus formed distinct clades from Tylenchorhynchus members, supporting the hypothesis in which Bitylenchus and Sauertylenchus could be considered as valid genera, but rejecting the 'large-genus' concept for Tylenchorhynchus. Also, Sauertylenchus ibericus was proposed as a junior synonym of S. maximus based on the results from morphological and phylogenetic analysis. Furthermore, an identification key for all known species included in the three genera Bitylenchus, Tylenchorhynchus and Sauertylenchus is presented herein. The number of transverse annuli on the lip region and presence/absence of post-rectal sac were considered as the main diagnostic characters for classifying the species into seven groups, and other morphological and morphometric characters were subsequently used for distinguishing species in each group.

The utility of mtDNA and rDNA for barcoding and phylogeny of plant-parasitic nematodes from Longidoridae (Nematoda, Enoplea).

The traditional identification of plant-parasitic nematode species by morphology and morphometric studies is very difficult because of high morphological variability that can lead to considerable overlap of many characteristics and their ambiguous interpretation. For this reason, it is essential to implement approaches to ensure accurate species identification. DNA barcoding aids in identification and advances species discovery. This study sought to unravel the use of the mitochondrial marker cytochrome c oxidase subunit 1 (coxI) as barcode for Longidoridae species identification, and as a phylogenetic marker. The results showed that mitochondrial and ribosomal markers could be used as barcoding markers, except for some species from the Xiphinema americanum group. The ITS1 region showed a promising role in barcoding for species identification because of the clear molecular variability among species. Some species presented important molecular variability in coxI. The analysis of the newly provided sequences and the sequences deposited in GenBank showed plausible misidentifications, and the use of voucher species and topotype specimens is a priority for this group of nematodes. The use of coxI and D2 and D3 expansion segments of the 28S rRNA gene did not clarify the phylogeny at the genus level.

Mitochondrial genome diversity in dagger and needle nematodes (Nematoda: Longidoridae).

Dagger and needle nematodes included in the family Longidoridae (viz. Longidorus, Paralongidorus, and Xiphinema) are highly polyphagous plant-parasitic nematodes in wild and cultivated plants and some of them are plant-virus vectors (nepovirus). The mitochondrial (mt) genomes of the dagger and needle nematodes, Xiphinema rivesi, Xiphinema pachtaicum, Longidorus vineacola and Paralongidorus litoralis were sequenced in this study. The four circular mt genomes have an estimated size of 12.6, 12.5, 13.5 and 12.7 kb, respectively. Up to date, the mt genome of X. pachtaicum is the smallest genome found in Nematoda. The four mt genomes contain 12 protein-coding genes (viz. cox1-3, nad1-6, nad4L, atp6 and cob) and two ribosomal RNA genes (rrnL and rrnS), but the atp8 gene was not detected. These mt genomes showed a gene arrangement very different within the Longidoridae species sequenced, with the exception of very closely related species (X. americanum and X. rivesi). The sizes of non-coding regions in the Longidoridae nematodes were very small and were present in a few places in the mt genome. Phylogenetic analysis of all coding genes showed a closer relationship between Longidorus and Paralongidorus and different phylogenetic possibilities for the three Xiphinema species.

A new stem nematode, Ditylenchus oncogenus n. sp. (Nematoda: Tylenchida), parasitizing sowthistle from Adriatic coast dunes in southern Italy.

Morphological and molecular analyses of a stem nematode causing a severe disease on infected sowthistle (Sonchus bulbosus) plants, involving the formation of gall-like structures on infected leaves and stems, have led to the description of a new species named Ditylenchus oncogenus n. sp. Morphologically, the new species is characterized by a medium to large body size (all adults more than 1 mm in length); a delicate stylet (9.0-11.0 µm long) with minute, rounded knobs; a long post-vulval uterine sac (c. 65% of the vulva-anus distance); six incisures at the lateral fields and characteristic D. destructor-pattern of spicules (with pronounced ventral tumulus and anteriorly pointed, less sclerotized, cuticle parts present within the lamina). The results of molecular analysis of rRNA gene sequences, including the D2-D3 expansion regions of 28S rRNA, internal transcribed spacer (ITS) rRNA, partial 18S rRNA gene, the protein-coding mitochondrial gene, cytochrome oxidase c subunit I (COI), and the heat-shock protein 90 (hsp90) gene, support the new species status. The results of a host-suitability test indicated that the new species does not parasitize potato (Solanum tuberosum) tubers and broad bean (Vicia faba) seedlings. Histopathological observations on naturally infected sowthistle tissues revealed that D. oncogenus n. sp. causes floral stem neoplasia and midrib leaf gall formation on the type, and to date only known, host. The galls were characterized by extensive hyperplasia, where several necrotic cells in the neoplasic area were directly damaged by feeding of the nematode, whereas a number of adjacent cells showed typical cytological changes, such as granulated cytoplasm with hypertrophied nuclei and nucleoli.

First Report of the Root-Knot Nematode, Meloidogyne hispanica, Infecting Sunflower in Greece.

Severe plant stunting, chlorosis, and extensive root galling were observed on sunflower (Helianthus annus Pioneer Hi-bred PR64LE19, Dupont) in a commercial field at Agios Athanasios, Drama Province, northeastern Greece at the end of May 2013. Disease symptoms were observed about 1.5 months after planting, and were distributed in patches that covered approximately 2% of the whole cultivated area. Examination of the soil and root samples from selected infected plants revealed the presence of abundant root-knot nematodes. Juveniles, males, and females were extracted by sieving, decanting, and root dissection for identification using morphological traits. Nematode population densities ranging from 100 to 150 J2s per 100 cm3 of soil, and 150 to 3,000 eggs per g of fresh sunflower roots were observed. Identification was confirmed by perineal patterns of females and by sequencing of the D2-D3 expansion segments of 28S ribosomal RNA gene (1,3,4). All identification methods were consistent with typical Meloidogyne hispanica. Morphology of perineal patterns of females and measurements of the second-stage juveniles (J2s) matched those of the original description of M. hispanica (3). Alignment indicated that the D2-D3 sequence (GenBank Accession No. KF501128) was 99% homologous to other sequences of M. hispanica deposited in GenBank from Brazil, Portugal, and Spain (EU443606, EU443608, and GQ375158, respectively), differing in only one nucleotide. Phylogenetic analyses using maximum likelihood of this sequence placed the Meloidogyne sp. in a highly supported (100%) clade that included all M. hispanica sequences available from the GenBank database (4). Root-knot nematodes in general have been reported to cause economic losses in sunflower in Europe (2), but there are no reports of M. hispanica. M. hispanica was first found in Seville Province, southern Spain, infecting rootstocks of Prunus spp. (3). Its distribution has been confirmed worldwide on different agricultural crops. However, to our knowledge, this is the first report of M. hispanica infecting sunflower in Europe and the first report of this species on any crop for Greece. The identification of M. hispanica in sunflower is relevant because it may represent a threat for sunflower production in Greece. Research to develop sunflower varieties resistant to root-knot nematodes should now also consider M. hispanica along with other species of Meloidogyne. References: (1) K. R. Barker. Page 19 in: An Advanced Treatise on Meloidogyne. Vol. II, Methodology. K. R. Barker et al., eds. North Carolina State University Graphics, Raleigh, NC, 1985. (2) M. Di Vito et al. Nematol. Mediterr. 24:109, 1996. (3) H. Hirschmann. J. Nematol. 18:520, 1986. (4) B. B. Landa et al. Plant Dis. 92:1104, 2008.

Pathogenicity and Host-Parasite Relationships of Heterodera cruciferae in Cabbage.

Stunted cabbage ('Lupini') associated with severe soil infestations by a cyst-forming nematode were observed in large patches of open fields in Castellaneta, province of Taranto, southern Italy. Morphological traits based on mature cysts, males, and second-stage juveniles (J2s) and molecular analysis of ribosomal DNA (D2 to D3 expansion segments of 28S and internal transcribed spacer [ITS]1 region) were used to identify the species. ITS1 sequence information supported the identity of Heterodera cruciferae, also showing a high degree of similarity to other species of the Heterodera Goettingiana group, including H. goettingiana, H. carotae, and H. urticae. Nematodes successfully established permanent feeding sites in cabbage roots which caused cellular alterations in the root cortex, endodermis, pericycle, and vascular cylinder by inducing typical multinucleate syncytia. Syncytial cytoplasm was granular and dense, with variously sized vacuoles and hypertrophied nuclei with nucleoli. Cabbage plant growth was also reduced in pathogenicity tests. The relationship between the initial nematode population density in soil and shoot plant weight was well described by the Seinhorst's equation. Tolerance limits with respect to shoot plant weight of cabbage to H. cruciferae was estimated as 1.50 units of eggs plus J2s/cm3 of soil. The minimum relative value (m) for plant height was 0.71 at an initial nematode population density of (Pi) ≥ 64 units of eggs plus J2s/cm3 of soil. The maximum nematode reproduction rate (Pf/Pi) was 4.6 times that of the initial population density of 8 units of eggs plus J2s/cm3 of soil.

Influence of temperature on the life-cycle of Globodera spp.

The potato cyst nematodes (PCN) G. rostochiensis (Woll.) and G. pallida (Stone) are the most economically important pests of potato (Solanum tuberosum L.) in the UK and are widespread in ware potato growing regions in Europe. The new EU directive 2007/33/EC which came into effect July 1, 2010 aims to control their spread and limit further increases in populations. We are investigating the role of temperature in the life cycle of PCN to assess how this effects population multiplication in relation to managing PCN. Hatching and nematode development are stages in the life cycle that are affected by temperature and thus are important life stages that can be examined to determine the impact of temperature on the length of time required for one generation to be completed and the potential for final populations to increase on different potato genetic backgrounds. In some conditions a partial or complete second generation has also been observed within the growing season. Females have been observed on the surface of tubers and "pecking" skin damage can occur which may be a result of a second generation. We are investigating the influence of temperature on the potential for a second generation or the induction of diapause.

First Report of Root-Knot Nematode Meloidogyne hispanica Infecting Grapevines in Southern Spain.

Some commercial vineyards producing the 'Condado de Huelva' wine denomination of origin in Almonte, Bonares, and Rociana (Huelva Province), southern Spain, showed general decline in sandy soils in 2009. Disease surveys revealed severe infections of grapevine rootstock Richter 110 feeder roots and heavy soil infestations by a root-knot nematode (Meloidogyne sp.). Infected plants showed a general decline as the only visible aboveground symptom, but when roots were inspected, moderate to small galls on secondary feeder roots were detected. The Meloidogyne sp. population was extracted and quantified from soil and root samples as previously described (1) and identified by the female perineal pattern, esterase (Est) and malate dehydrogenase (Mdh) phenotypes, and sequencing and maximum parsimony (MP) analysis of the ribosomal DNA region D2-D3 of 28S (2,4). Morphology of the perineal patterns and measurements of the second-stage juveniles (J2s) matched those of the original description of Meloidogyne hispanica (3). Enzyme analysis revealed two slow and a medium Est bands, a strong band, and two additional weaker bands coincident with the S2-M1 and N3 Mdh M. hispanica phenotypes (2,4). D2-D3 sequences of all three populations sampled were 100% homologous (GenBank Accession No. GQ375158). Phylogenetic analyses with MP of those sequences placed the Meloidogyne sp. in a clade (100% support) that included all M. hispanica sequences available from the GenBank database (4). M. hispanica was first found in Seville Province, southern Spain, infecting rootstocks of Prunus spp. Its distribution has been confirmed worldwide on different agricultural crops. Thus, M. hispanica has been reported to be infecting grapevines in South Africa and Australia (4); however, to our knowledge, this is the first report of M. hispanica infecting grapevines in Europe. Our data suggest that M. hispanica may pose a threat for vineyard production in southern Spain since M. hispanica was found in 52.63 and 47.36% of soil and root samples, respectively, from 19 fields in 'Condado de Huelva', with nematode population densities ranging from 2.4 to 129.6 eggs and J2s per 100 cm3 of soil and 1 to 1,797 eggs and J2s per gram of fresh roots. Furthermore, genes that confer resistance to other common root-knot nematodes reported on grapevine in Europe may not protect against M. hispanica. References: (1) K. R. Barker. Nematode extraction and bioassays. Page 19 in: An Advanced Treatise on Meloidogyne. Vol. II, Methodology. K. R. Barker et al., eds. North Carolina State University Graphics, Raleigh, 1985. (2) P. R. Esbenshade and A. C. Triantaphyllou. J. Nematol. 22:10, 1990. (3) H. Hirschmann. J. Nematol. 18:520, 1986. (4) B. B. Landa et al. Plant Dis. 92:1104, 2008.

A New Root-Knot Nematode Parasitizing Sea Rocket from Spanish Mediterranean Coastal Dunes: Meloidogyne dunensis n. sp. (Nematoda: Meloidogynidae).

High infection rates of European sea rocket feeder roots by an unknown root-knot nematode were found in a coastal dune soil at Cullera (Valencia) in central eastern Spain. Morphometry, esterase and malate dehydrogenase electrophoretic phenotypes and phylogenetic trees demonstrated that this nematode species differs clearly from other previously described root-knot nematodes. Studies of host-parasite relationships showed a typical susceptible reaction in naturally infected European sea rocket plants and in artificially inoculated tomato (cv. Roma) and chickpea (cv. UC 27) plants. The species is herein described and illustrated and named as Meloidogyne dunensis n. sp. The new root-knot nematode can be distinguished from other Meloidogyne spp. by: (i) perineal pattern rounded-oval, formed of numerous fine dorsal and ventral cuticle striae and ridges, lateral fields clearly visible; (ii) female excretory pore at the level of stylet knobs, EP/ST ratio 1.6; (iii) second-stage juveniles with hemizonid located 1 to 2 annuli anteriorly to excretory pore and long, narrow, tapering tail; and (iv) males with lateral fields composed of four incisures anteriorly and posteriorly, while six distinct incisures are observed for large part at mid-body. Phylogenetic trees derived from distance and maximum parsimony analyses based on 18S, ITS1-5.8S-ITS2 and D2-D3 of 28S rDNA showed that M. dunensis n. sp. can be differentiated from all described root-knot nematode species, and it is clearly separated from other species with resemblance in morphology, such as M. duytsi, M. maritima, M. mayaguensis and M. minor.