An Improved Phenotyping Method for Evaluation of Yam (Dioscorea spp.) Resistance to Nematodes Belonging to the Genera Meloidogyne and Scutellonema.

Phenotyping yam (Dioscorea spp.) germplasm for resistance to parasitic nematodes is hampered by the lack of an efficient screening method. In this study, we developed a new method using rooted yam vine cuttings and yam plantlets generated from semi-autotrophic hydroponics (SAHs) propagation for phenotyping yam genotypes for nematode resistance. The method was evaluated using 26 genotypes of D. rotundata for their reaction to Scutellonema bradys and four root-knot nematode species, Meloidogyne arenaria, M. enterolobii, M. incognita, and M. javanica. Yam plantlets established in nursery bags filled with steam-sterilized soil were used for screening against single nematode species. Plants were inoculated four weeks after planting and assessed for nematode damage eight weeks later. A severity rating scale was used to classify genotypes as resistant, tolerant, or susceptible determine based on the nematode feeding damage on tubers and the rate of nematode multiplication in the roots of inoculated plants. The results demonstrated putative resistance and tolerance against S. bradys in 58% of the genotypes and 88%, 65%, 65%, and 58% against M. arenaria, M. javanica, M. incognita, and M. enterolobii, respectively. The method is rapid, flexible, and seasonally independent, permitting year-round screening under controlled conditions. This method increases the throughput and speed of phenotyping and improves the selection process.

Exploring the use of metabarcoding to reveal eukaryotic associations with mononchids nematodes.

Nematodes play a vital ecological role in soil and marine ecosystems, but there is limited information about their dietary diversity and feeding habits. Due to methodological challenges, the available information is based on inference rather than confirmed observations. The lack of correct dietary requirements also hampers rearing experiments. To achieve insight into the prey of mononchid nematodes, this study employed high-throughput Illumina paired-end sequencing using universal eukaryotic species 18S primers on 10 pooled mononchid nematode species, namely Mylonchulus brachyuris, M. brevicaudatus, Mylonchulus sp., Clarkus parvus, Prionchulus sp. M. hawaiiensis, M. sigmaturellus, M. vulvapapillatus, Anatonchus sp. and Miconchus sp. The results indicate that mononchids are associated with a remarkable diversity of eukaryotes, including fungi, algae, and protists. While the metabarcoding approach, first introduced here for mononchids, proved to be a simple and rapid method, it has several limitations and crucial methodological challenges that should be addressed in future studies. Ultimately, such methods should be able to evaluate the dietary complexity of nematodes and provide a valuable avenue for unraveling the dietary requirements of previously unculturable nematodes. This can contribute to the methodology of understanding their feeding habits and contributions to ecosystem dynamics.

A New Root-Knot Nematode species, Meloidogyne karsseni n. sp. (Nematoda: Meloidogynidae), From Mexico and a Taxonomic Update on M. paranaensis From Guatemala.

A new root-knot nematode (RKN) species, Meloidogyne karsseni n. sp., associated with sweet pepper from Mexico, and a population of M. paranaensis from Guatemala, are described using data from morphological, biochemical (isozyme enzymes), molecular, and phylogenetic analyses. Meloidogyne karsseni n. sp. can be morphologically diagnosed using the combined features of the second-stage juveniles, viz. body length (345 to 422 µm), a conical rounded head region, a post-labial annule lacking transverse striation, a thin stylet 11 to 12 µm long, rounded to oval and backwardly sloping knobs, dorsal gland orifice (DGO) at 5.2 to 6.0 µm from the knobs, a hemizonid just above the secretory-excretory (SE) pore, a tapering tail with finely rounded terminus and one or two very weak constrictions at hyaline tail tip; the female characters viz. oval-to-rounded perineal pattern with coarse striation on lateral sides around the anus, low dorsal arch with finer striations, and distinctly visible lateral lines; and the male characteristics viz. a rounded and continuous head, a post-labial annule without transverse striations, a robust stylet 20 to 24 µm long, rounded-to-oval and slightly backwardly sloping knobs, and a DGO at 2.4 to 2.9 µm from the knobs. In all the studied males of M. paranaensis, a characteristic sclerotization around the duct of SE-pore was also observed for the first time. Sequences of 18S, D2-D3 of 28S, and ITS of rDNA, and cox1 of mtDNA were generated for the two species, and in the phylogenetic trees based on these genes, both species appeared in the tropical RKN species complex clade.

Pellioditis pelhamensis n. sp. (Nematoda: Rhabditidae) and Pellioditis pellio (Schneider, 1866), earthworm associates from different subclades within Pellioditis (syn. Phasmarhabditis Andrássy, 1976).

Recently, much attention has been focused on a group of rhabditid nematodes called Phasmarhabditis, a junior synonym of Pellioditis, as a promising source of biocontrol agents for invasive slugs. Pellioditis pelhamensis n. sp. was first isolated from earthworms near Pelham Bay Park in Bronx, New York, USA, in 1990 and has been found to be pathogenic to slugs as well as some earthworms. It has also been used in several comparative developmental studies. Here, we provide a description of this species, as well as a redescription of a similar earthworm-associated nematode, Pellioditis pellio Schneider, 1866, re-isolated from the type locality. Although P. pelhamensis n. sp. and P. pellio are morphologically similar, they are reproductively isolated. Molecular phylogenetic analysis places both species in a clade that includes all species previously described as Phasmarhabditis which are associated with gastropods. Phasmarhabditis Andrássy, 1976 is therefore a junior synonym of Pellioditis Dougherty, 1953. Also, Pellioditis bohemica Nermut', Puza, Mekete & Mrácek, 2017, described to be a facultative parasite of slugs, is found to be a junior synonym of Pellioditis pellio (Schneider, 1866), adding to evidence that P. pellio is associated with both slugs and earthworms. The earthworm-associated species P. pelhamensis n. sp. and P. pellio represent different subclades within Pellioditis, suggesting that Pellioditis species in general have a broader host range than just slugs. Because of this, caution is warranted in using these species as biological control agents until more is understood about their ecology.

Characterization of Hoplolaimus seinhorsti and Hoplolaimus pararobustus (Tylenchina: Hoplolaimidae) from banana, with phylogeny and species delineation in the genus Hoplolaimus.

The morphological and molecular characterisations of two lance nematode species isolated from the rhizosphere of banana, Hoplolaimus seinhorsti and H. pararobustus, are provided based on an integrative study that includes light and scanning electron microscopy, phylogenetic analysis and two tree-based molecular species delimitation methods (GMYC and bPTP). Nineteen new sequences were obtained, including 5 partial 18S rRNA, 6 D2-D3 of 28S rRNA, 1 ITS rRNA and 7 COI mtDNA (the first COI sequences of H. seinhorsti and H. pararobustus), and an updated morphological character comparison of 37 Hoplolaimus species is presented. The tree-based molecular species-delimitation approaches employed gave markedly differing results, and also showed remarkable discrepancies among the investigated genes, although the bPTP output was found to agree well with established morphological species delimitations. Both species-delimitation approaches did, however, provide the same output for the COI mtDNA sequences, and the COI mtDNA gene sequence was also found to correspond better to established morphological species. It is therefore recommended by this paper as representing the most suitable barcode marker for Hoplolaimus species identification. This integrative study also resulted in the corrective reassignment of 17 gene sequences that were previously unidentified or incorrectly classified, as well as concluding that H. pararobustus consists of two cryptic species.

All quiet on the western front? The evolutionary history of monogeneans (Dactylogyridae: Cichlidogyrus, Onchobdella) infecting a West and Central African tribe of cichlid fishes (Chromidotilapiini).

Owing to the largely unexplored diversity of metazoan parasites, their speciation mechanisms and the circumstances under which such speciation occurs - in allopatry or sympatry - remain vastly understudied. Cichlids and their monogenean flatworm parasites have previously served as a study system for macroevolutionary processes, e.g., for the role of East African host radiations on parasite communities. Here, we investigate the diversity and evolution of the poorly explored monogeneans infecting a West and Central African lineage of cichlid fishes: Chromidotilapiini, which is the most species-rich tribe of cichlids in this region. We screened gills of 149 host specimens (27 species) from natural history collections and measured systematically informative characters of the sclerotised attachment and reproductive organs of the parasites. Ten monogenean species (Dactylogyridae: Cichlidogyrus and Onchobdella) were found, eight of which are newly described and one redescribed herein. The phylogenetic positions of chromidotilapiines-infecting species of Cichlidogyrus were inferred through a parsimony analysis of the morphological characters. Furthermore, we employed machine learning algorithms to detect morphological features associated with the main lineages of Cichlidogyrus. Although the results of these experimental algorithms remain inconclusive, the parsimony analysis indicates that West and Central African lineages of Cichlidogyrus and Onchobdella are monophyletic, unlike the paraphyletic host lineages. Several instances of host sharing suggest occurrences of intra-host speciation (sympatry) and host switching (allopatry). Some morphological variation was recorded that may also indicate the presence of species complexes. We conclude that collection material can provide important insights on parasite evolution despite the lack of well-preserved DNA material.

Title: À l'Ouest, rien de nouveau ? L'histoire évolutive des monogènes (Dactylogyridae : Cichlidogyrus, Onchobdella) infectant une tribu de poissons cichlidés (Chromidotilapiini) d'Afrique occidentale et centrale. Abstract: En raison de la nature largement inexplorée de la diversité des parasites métazoaires, leurs mécanismes de spéciation et les circonstances dans lesquelles cette spéciation se produit­allopatrie ou sympatrie­restent très peu étudiés. Les cichlidés et leurs parasites Plathelminthes monogènes ont déjà servi de modèle pour l'étude des processus macro-évolutifs, par exemple pour le rôle des radiations d'hôtes de l'Afrique de l'Est sur les communautés de parasites. Ici, nous étudions la diversité et l'évolution des monogènes peu étudiées qui infestent une lignée de poissons cichlidés d'Afrique occidentale et centrale : les Chromidotilapiini, qui est la tribu de cichlidés la plus riche en espèces dans cette région. Nous avons examiné les branchies de 149 spécimens hôtes (27 espèces) provenant de musées d'histoire naturelle et mesuré systématiquement les caractères informatifs des pièces sclérifiées du hapteur et des organes copulateurs des parasites. Dix espèces de monogènes (Dactylogyridae : Cichlidogyrus et Onchobdella) ont été trouvées ; huit sont nouvelles pour la science et une est redécrite. Les positions phylogénétiques des espèces de Cichlidogyrus infectant les chromidotilapiines ont été déduites par une analyse de parcimonie des caractères morphologiques. En outre, nous avons utilisé des algorithmes d'apprentissage automatique pour détecter les caractéristiques morphologiques associées aux principales lignées de Cichlidogyrus. Bien que les résultats de ces algorithmes expérimentaux restent peu concluants, l'analyse de parcimonie indique que les lignées de Cichlidogyrus et d'Onchobdella de l'Afrique de l'Ouest et Central sont monophylétiques, contrairement aux lignées d'hôtes qui sont paraphylétiques. Plusieurs cas de partage d'hôtes suggèrent des occurrences de spéciation synxénique (sympatrie) et de changement d'hôte (allopatrie). Certaines variations morphologiques ont été enregistrées et peuvent également indiquer la présence de complexes d'espèces. Nous concluons donc que le matériel de collection peut fournir des informations importantes sur l'évolution des parasites malgré le manque d'ADN exploitable.

First Reports and Morphological and Molecular Characterization of Pratylenchus delattrei and Quinisulcius capitatus Associated with Chickpea in Ethiopia.

Chickpea (Cicer arietinum L.) is classed among the most important leguminous crops of high economic value in Ethiopia. Two plant-parasitic nematode species, Pratylenchus delattrei and Quinisulcius capitatus, were recovered from chickpea-growing areas in Ethiopia and characterized using molecular and morphological data, including the first scanning electron microscopy data for P. delattrei. New sequences of D2-D3 of 28S, ITS rDNA and mtDNA COI genes have been obtained from these species, providing the first COI sequences for P. delattrei and Q. capitatus, with both species being found for the first time on chickpea in Ethiopia. Furthermore, Pratylenchus delattrei was recovered in Ethiopia for the first time. The information obtained about these nematodes will be crucial to developing effective nematode management plans for future chickpea production.

Paratylenchus Ilicis n. Sp. (Nematoda: Paratylenchinae) Associated with Holly from the Netherlands and New Taxonomical and Phylogenetic Support for the Synonymization of Cacopaurus with Paratylenchus.

Paratylenchus ilicis n. sp. was found associated with holly in the Netherlands and was described based on morphology, morphometrics, rRNA and mitochondrial cox1 genes, phylogenetic relationships with other Paratylenchus species, host information and geographical distribution. This species can be morphologically diagnosed based on its light brown, slightly obese to obese females with tubercles on cuticle, lateral bands widening into an ovoid field around vulva level, stylet length of 70 µm to 100 µm, outstretched to reflexed ovary, rounded sperm-filled spermatheca, vagina opening into a thick-walled rounded space, absence of vulval flaps and vulva at 89% to 95% of body length, very short tail in all life stages, and a characteristic finger-like tail tip in juveniles (J2). The new species is morphologically closest to Cacopaurus pestis but differs based on the absence of a scutellum-like differentiation in the lateral field, the ovaries that can be outstretched or reflexed, and the finger-like tail tip in J2. Furthermore, both species were found to be molecularly distant from one another, found in different habitats, and are associated with different hosts. The high morphological similarity between Cacopaurus and Paratylenchus and our phylogenetic analyses, revealing that the former is embedded within different Paratylenchus clades and thus polyphyletic, provide new evidences for the synonymization of Cacopaurus with Paratylenchus.

Remarks on phylogeny and molecular variations of criconematid species (Nematoda: Criconematidae) with case studies from Vietnam.

The family Criconematidae is a remarkable group of nematodes, containing roughly 600 nominal root-ectoparasitic species, of which many species are known to be significant agricultural pests. Strikingly, our phylogenetic analyses based on 18S, D2-D3 of 28S rRNA, and COI mtDNA sequences of criconematid species, supported by tree topology tests (SH and AU tests), revealed that almost all studied genera, including Criconema, Ogma, Crossonema, Discocriconema, Hemicriconemoides, Criconemoides, Mesocriconema, and Lobocriconema, are not monophyletic groups, a finding that is partly contrary to those of previous studies on these groups. Our results suggest that key morphological characters used in the classification of Criconematidae are the consequence of convergent evolution. It is clear from our studies that the species status of at least 40 sequences of criconematid species from GenBank must be either revised or reconsidered, with analyses based on a polyphasic approach that includes different tree- and distance-based molecular species-delimitation methods (bPTP, GMYC, ABGD1, and ABGD2). Our studies found the ABGD2 output of the automatic barcode method to agree remarkably well with established species delimitations, while in general, the four species-delimitation results corresponding to three barcode regions forwarded significantly more putative species compared to those originally considered. This study also characterised for the first time the populations of Criconemoides myungsugae and Discocriconemella hensungica associated with Vietnamese ginseng, one of the most precious and rare ginseng varieties in the world. Although these populations are morphologically in agreement with the original descriptions of C. myungsugae and D. hengsungica, their molecular data display notable variations compared to the sequences deposited in GenBank. These species demonstrate clearly the immense molecular variations that can be observed in several species of the family Criconematidae.

First Report of the Root-Knot Nematode Meloidogyne enterolobii Parasitizing Plantain (Musa spp., AAB) in Nigeria.

Plantain (Musa spp., AAB), an important staple food in Africa with West Africa accounting for 32% of global production, is prone to numerous pests and diseases of which plant-parasitic nematodes are a key concern. This includes root-knot nematodes (RKN; Meloidogyne spp.), which infect the roots, causing them to become galled, deformed and swollen. The nematode Meloidogyne enterolobii is considered a global threat to production of many important agricultural crops due to its extremely virulent and aggressive nature (Philbrick et al. 2020). In 2019, during a survey to identify the diversity of nematodes associated with Musa spp. in Nigeria, RKN females (n = 13) were isolated from a heavily galled root (50-75% galling) from a single plantain cv. Agbagba (Musa spp., AAB) plant in Onne, Rivers State, Nigeria (4°43'08.8"N 7°10'37.5"E). Genomic DNA was extracted from three females and processed individually using worm lysis buffer and proteinase K (Bert et al. 2008). The females were identified as M. enterolobii based on Nad5 mtDNA (Janssen et al. 2016), with GenBank accession no. ON010028, ON010027, ON010026, and were 100% homologous to the M. enterolobii sequences MW965454, KU372358 and KU372359 (Supplementary Figure S1). The sampled plant did not show any specific above-ground symptoms but swellings were apparent on the secondary and tertiary roots, which were associated with RKN females that were embedded in the root tissue. All the life stages were found clustered together in the root cortex, where they created necrotic brown-black lesions. A mean value of 2,604 ± 820 (mean ± standard deviation) males, eggs and second-stage juveniles (J2) were extracted from 5 g of root sub-samples (n = 6) using the Hussey and Barker (1973) NaOCl method. On average 39 females were hand-picked (n = 6) from 5 g fresh root. Pure cultures were established from single egg masses and maintained on RKN-susceptible tomato plants (Solanum lycopersicum cv. Marmande). To conduct Koch's postulates, two-month old plantlets of plantain cv. Agbagba (n = 5) were inoculated with 8000 J2s and eggs (initial population) of M. enterolobii pure cultures in 8 L pots in a screenhouse in Nigeria. Non-inoculated plantlets were included as negative controls. The nematode reproduction factor (RF = final density / initial population) and root damage symptoms were assessed 90 days post-inoculation. All the inoculated plantlets had similar galling symptoms and extensive necrosis as was observed in the field (Supplementary Figure S2), with an average RF = 25.9. No symptoms were observed on control plants. Adult females (n = 2) removed from the roots were identified as M. enterolobii based on Nad5 mtDNA (ON532789, ON532790) confirming that plantain cv. Agbagba is a host of M. enterolobii. In Nigeria, M. enterolobii has been reported to be associated with four plant species belonging to four plant families: Euphorbiaceae (Oyetunde et al. 2022), Cucurbitaceae (Bello et al. 2020), Dioscoreaceae (Kolombia et al. 2016), and Solanaceae (dos Santos et al. 2019). To our knowledge, this is the first report of M. enterolobii on a member of the Musaceae in Nigeria and globally the first report on plantain (Musa spp., AAB). The impact of M. enterolobii on plantain productivity has yet to be determined but given the RF value obtained in the pathogenicity test, plantain is a suitable host. This calls for a comprehensive RKN diversity study to evaluate the geographic spread of M. enterolobii on this important staple food crop in West Africa.

Genomic Reconstruction of the Introduction and Diversification of Golden Potato Cyst Nematode Populations in Indonesia.

Potato cyst nematodes (PCNs), the umbrella term for Globodera rostochiensis and G. pallida, coevolved with their Solanaceous hosts in the Andean Mountain region. From there, PCN proliferated worldwide to virtually all potato production areas. PCN is a major factor limiting the potato production in Indonesia. In our survey, only G. rostochiensis was found. Fourteen field populations were collected on Java and Sumatra, and unique variants were called by mapping resequencing data on a G. rostochiensis reference genome. A phylogenetic tree based on 1.4 million unique variants showed a genotypic separation between the outgroup, a Scottish Ro1 population, and all Indonesian populations. This separation was comparable in size with the genotypic distinction between the Javanese and the Sumatran PCN populations. Next, variants within PCN effector gene families SPRYSEC, 1106, 4D06, and venom allergen-like protein (VAL) that all interfere with the host innate immune system were compared. Distinct selective pressures acted on these effector families; while SPRYSECs (4,341 single-nucleotide polymorphisms [SNPs]/insertions or deletions of bases [indels]) behaved like neutral genes, the phylogenetic trees of 1106, 4D06, and VAL proteins (235, 790, and 150 SNPs/indels, respectively) showed deviating topologies. Our data suggest that PCN was introduced on Java not too long after the introduction of potato in the middle of the eighteenth century. Soon thereafter, the pathogen established on Sumatra and started to diversify independently. This scenario was corroborated by diversification patterns of the effector families 1106, 4D06, and VAL. Our data demonstrate how genome resequencing data from a nonindigenous pathogen can be used to reconstruct the introduction and diversification process.

An Integrated Approach for Synonymization of Rotylenchus rhomboides with R. goodeyi (Nematoda: Hoplolaimidae) Reveals High Intraspecific Mitogenomic Variation.

Rotylenchus is a widely distributed, economically important plant-parasitic nematode group whose species-level identification relies largely on limited morphological characters, including character-based tabular keys and molecular data of ribosomal and mitochondrial genes. In this study, a combined morphological and molecular analysis of three populations of Rotylenchus goodeyi from Belgium, Poland, and the Netherlands revealed important character variations of this species, leading to synonymization of R. rhomboides with R. goodeyi and a high nucleotide variation within cox1 gene sequences in these populations. Additional Illumina sequencing of DNA from individuals of the Dutch population revealed two variants of mitogenomes, each approximately 23 Kb in size, differing by approximately 9% and containing 11 protein-coding genes, 2 ribosomal RNA genes, and as many as 29 transfer RNA genes. In addition to the first representative whole-genome shotgun sequence datasets of the genus Rotylenchus, this study also provides the full-length mitogenome and the ribosomal DNA sequences of R. goodeyi.

'Candidatus Xiphinematincola pachtaicus' gen. nov., sp. nov., an endosymbiotic bacterium associated with nematode species of the genus Xiphinema (Nematoda, Longidoridae).

An intracellular bacterium, strain IAST, was observed to infect several species of the plant-parasitic nematode genus Xiphinema (Xiphinema astaregiense, Xiphinema incertum, Xiphinema madeirense, Xiphinema pachtaicum, Xiphinema parapachydermum and Xiphinema vallense). The bacterium could not be recovered on axenic medium. The 16S rRNA gene sequence of IAST was found to be new, being related to the family Burkholderiaceae, class Betaproteobacteria. Fungal endosymbionts Mycoavidus cysteinexigens B1-EBT (92.9 % sequence identity) and 'Candidatus Glomeribacter gigasporarum' BEG34 (89.8 % identity) are the closest taxa and form a separate phylogenetic clade inside Burkholderiaceae. Other genes (atpD, lepA and recA) also separated this species from its closest relatives using a multilocus sequence analysis approach. These genes were obtained using a partial genome of this bacterium. The localization of the bacterium (via light and fluorescence in situ hybridization microscopy) is in the X. pachtaicum females clustered around the developing oocytes, primarily found embedded inside the epithelial wall cells of the ovaries, from where they are dispersed in the intestine. Transmission electron microscopy (TEM) observations supported the presence of bacteria inside the nematode body, where they occupy ovaries and occur inside the intestinal epithelium. Ultrastructural analysis of the bacterium showed cells that appear as mostly irregular, slightly curved rods with rounded ends, 0.8-1.2 µm wide and 2.5-6.0 µm long, possessing a typical Gram-negative cell wall. The peptidoglycan layer is, however, evident only occasionally and not detectable by TEM in most cells. Another irregularly occurring shell surrounding the endosymbiont cells or the cell clusters was also revealed, probably originating from the host cell membrane. Flagella or spore-like cells do not occur and the nucleoid is diffusely distributed throughout the cell. This endosymbiont is transmitted vertically through nematode generations. These results support the proposal of IAST as a new species, although its obligate intracellular and obligate endosymbiont nature prevented isolation of a definitive type strain. Strain IAST is therefore proposed as representing 'Candidatus Xiphinematincola pachtaicus' gen. nov., sp. nov.

Rotylenchus wimbii n. sp. (Nematoda: Hoplolaimidae) associated with finger millet in Kenya.

Rotylenchus wimbii n. sp. was found associated with finger millet in Kenya and is described based on light microscopy, scanning electron microscopy, and molecular information. Sequence analysis was performed on ITS, 18S, and D2-D3 of 28S of ribosomal DNA and COI of mitochondrial DNA. This new species is characterized by a moderate female body size of 0.6 to 0.8 mm, a continuous hemispherical lip region with four annuli, 3 to 4 irregular blocks on the basal lip annule, absence of longitudinal cuticular striations in anterior region, four lateral lines forming three equal bands which are areolated mainly at pharynx level, a robust stylet of 23 to 27 µm of which 45 to 53% is cone part, and with rounded to sometimes indented knobs, a secretory-excretory pore around level of pharyngo-intestinal junction, didelphic-amphidelphic reproductive system, vulva without distinct epiptygma, indistinct to empty spermatheca, tail usually truncated with 5 to 9 annuli, phasmids located at 7 to 17 annuli anterior to anus, and absence of males. Molecular phylogenies, in combination with species delimitation, supported the distinctiveness of Rotylenchus wimbii n. sp. and revealed some mislabeled Rotylenchus brevicaudatus sequences in GenBank.

Integrative Taxonomy and Molecular Phylogeny of the Plant-Parasitic Nematode Genus Paratylenchus (Nematoda: Paratylenchinae): Linking Species with Molecular Barcodes.

Pin nematodes of the genus Paratylenchus are obligate ectoparasites of a wide variety of plants that are distributed worldwide. In this study, individual morphologically vouchered nematode specimens of fourteen Paratylenchus species, including P. aculentus, P. elachistus, P. goodeyi, P. holdemani, P. idalimus, P. microdorus, P. nanus, P. neoamblycephalus, P. straeleni and P. veruculatus, are unequivocally linked to the D2-D3 of 28S, ITS, 18S rRNA and COI gene sequences. Combined with scanning electron microscopy and a molecular analysis of an additional nine known and thirteen unknown species originating from diverse geographic regions, a total of 92 D2-D3 of 28S, 41 ITS, 57 18S rRNA and 111 COI new gene sequences are presented. Paratylenchus elachistus, P. holdemani and P. neoamblycephalus are recorded for the first time in Belgium and P. idalimus for the first time in Europe. Paratylenchus is an excellent example of an incredibly diverse yet morphologically minimalistic plant-parasitic genus, and this study provides an integrated analysis of all available data, including coalescence-based molecular species delimitation, resulting in an updated Paratylenchus phylogeny and the corrective reassignment of 18 D2-D3 of 28S, 3 ITS, 3 18S rRNA and 25 COI gene sequences that were previously unidentified or incorrectly classified.

Structure, development, and evolutive patterns of spermatozoa in rhabditid nematodes (Nematoda: Rhabditida).

Spermatogenesis of five rhabditid nematodes was studied using transmission electron microscopy and is described herein. Structure and development of nematode sperm in all available representatives of the extensive order Rhabditida have been analysed and the main characteristics of each infraorder are discussed. The ancestral sperm of the order Rhabditida was reconstructed using maximum likelihood and Bayesian methods based on 44 ultrastructural sperm characters. The hypothetical ancestral spermatogenesis of the order Rhabditida agrees with the previously suggested "rhabditid" pattern and appears to be conserved throughout the order Rhabditida. Despite the enormous variation of rhabditid nematodes, few groups deviate from the ancestral pattern. This conserved pattern can be informative within the phylum Nematoda at order level, but poses limitations when used in taxonomic and phylogenetic analysis within Rhabditida.

Plant-parasitic nematodes associated with sugarcane in Kilimanjaro, Tanzania.

Morphological and molecular analyses of plant-parasitic nematodes (PPN) from 12 sugarcane plantation sites of Tanganyika Planting Company (TPC) Limited in Kilimanjaro region of Tanzania revealed the presence of six PPN genera, i.e. Helicotylenchus, Hemicycliophora, Pratylenchus, Rotylenchulus, Scutellonema, and Tylenchorhynchus. The genera with the highest densities and present in virtually all samples were Pratylenchus and Rotylenchulus, and the most important species appeared to be R. parvus, P. zeae, T. crassicaudatus, and T. ventrosignatus. A total sequences of 11 partial ITS, 15 D2-D3 of 28S, and 6 partial 18S of rRNA gene, and 7 partial COI gene of mtDNA of these species were obtained in this study. Morphology and molecular data comparisons between the Tanzanian R. parvus and the South African R. parvus indicated that R. parvus is a cryptic species complex. Based on the results of morphological and molecular analyses of T. crassicaudatus and T. agri from China, Haiti, Indonesia, Iran, Niger and the USA, T. agri syn. n. is proposed as a junior synonym of T. crassicaudatus.Morphological and molecular analyses of plant-parasitic nematodes (PPN) from 12 sugarcane plantation sites of Tanganyika Planting Company (TPC) Limited in Kilimanjaro region of Tanzania revealed the presence of six PPN genera, i.e. Helicotylenchus, Hemicycliophora, Pratylenchus, Rotylenchulus, Scutellonema, and Tylenchorhynchus. The genera with the highest densities and present in virtually all samples were Pratylenchus and Rotylenchulus, and the most important species appeared to be R. parvus, P. zeae, T. crassicaudatus, and T. ventrosignatus. A total sequences of 11 partial ITS, 15 D2-D3 of 28S, and 6 partial 18S of rRNA gene, and 7 partial COI gene of mtDNA of these species were obtained in this study. Morphology and molecular data comparisons between the Tanzanian R. parvus and the South African R. parvus indicated that R. parvus is a cryptic species complex. Based on the results of morphological and molecular analyses of T. crassicaudatus and T. agri from China, Haiti, Indonesia, Iran, Niger and the USA, T. agri syn. n. is proposed as a junior synonym of T. crassicaudatus.

First report of Rotylenchulus reniformis infecting turmeric in Vietnam and consequent damage.

Turmeric (Curcuma longa L.) is one of the common medicinal crops of high economical value in Vietnam. A survey in the Central Highlands of Vietnam revealed a turmeric growing area showing serious disease symptoms, including stunting of the plant, yellowing or darkening of the leaf margins and tips, and underdeveloped dry and rotten rhizomes. An inspection for plant pathogens in soil samples from this area revealed a high density and frequency of Rotylenchus reniformis, with a significant relationship between the density of R. reniformis, rhizome weight, and level of plant damage. This study provides the first report of R. reniformis found in parasitic association with turmeric in Vietnam with the support of molecular data and examines its resulting damage.Turmeric (Curcuma longa L.) is one of the common medicinal crops of high economical value in Vietnam. A survey in the Central Highlands of Vietnam revealed a turmeric growing area showing serious disease symptoms, including stunting of the plant, yellowing or darkening of the leaf margins and tips, and underdeveloped dry and rotten rhizomes. An inspection for plant pathogens in soil samples from this area revealed a high density and frequency of Rotylenchus reniformis, with a significant relationship between the density of R. reniformis, rhizome weight, and level of plant damage. This study provides the first report of R. reniformis found in parasitic association with turmeric in Vietnam with the support of molecular data and examines its resulting damage.

Effects of synthetic fertilizer and farm compost on soil nematode community in long-term crop rotation plots: A morphological and metabarcoding approach.

Soil biodiversity plays a key regulation role in the ecosystem services that underpin regenerative sustainable agriculture. It can be impacted by agricultural management techniques, both positively (through measures such as compost application) and negatively (through, for example, application of synthetic nitrogen). As one of the most numerous members of the soil biota, nematodes are well established as indicators for the soil food web. However, compost application also includes the addition of nematodes present in compost and their subsequent survival in soil is unknown. Nematode communities within the compost applied to soil, and nematode communities in the soil of a multi-year rotational cropping field trial in Melle (Belgium) were studied using morphological and metabarcoding techniques. Compost (C) and nitrogen fertilizer (NF) treated plots were compared. Three replicate plots were investigated for each of the following treatments: C application only; C and NF application; NF only; no C and no NF (control). Plots were sampled six times between 2015-2017, before and after C or NF were added each spring and after crop harvest (except for 2017). NF treatment resulted in a significant decrease of fungal feeding and predatory nematodes, while herbivorous nematodes were positively affected. Remarkably, we did not find compost addition to exert any noticeable effects on the soil nematode community. The morphological and metabarcoding data resulted in different results of the nematode community composition. However, trends and patterns in the two data sets were congruent when observed with NMDS plots and using the nematode maturity index. Metabarcoding of individual compost nematode taxa demonstrated that nematodes originating from compost did not persist in soil.

Morphological and molecular characterization of Heterodera dunensis n. sp. (Nematoda: Heteroderidae) from Gran Canaria, Canary Islands.

Heterodera dunensis n. sp. from the coastal dunes of Gran Canaria, Canary Islands, is described. This new species belongs to the Schachtii group of Heterodera with ambifenestrate fenestration, presence of prominent bullae, and a strong underbridge of cysts. It is characterized by vermiform second-stage juveniles having a slightly offset, dome-shaped labial region with three annuli, four lateral lines, a relatively long stylet (27-31 µm), short tail (35-45 µm), and 46 to 51% of tail as hyaline portion. Males were not found in the type population. Phylogenetic trees inferred from D2-D3 of 28S, partial ITS, and 18S of ribosomal DNA and COI of mitochondrial DNA sequences indicate a position in the 'Schachtii clade'.