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Root-knot nematodes (Meloidogyne spp.) are among the biotic factors that limit tomato production worldwide. The objectives of this study were to assess the distribution and identify Meloidogyne spp. and associated problems from major tomato growing areas of Ethiopia. A total of 212-rhizosphere soil and 123 root samples were sampled from 40 localities during 2012/13 growing season. A total of 646 respondents participated in the questionnaire to assess knowledge and practice of farmers and factors associated with RKN damage on tomato. Out of the 212 soil samples collected, 47.2% were found infested by various Meloidogyne species 8 weeks after the start of the bioassay test. Out of the 123 root samples collected, 65% had root galls. The highest prevalence (100%) of RKN was found on samples collected from Adami Tullu, Babile, Erer Gota, Hurso, Jittu, Tikur Wuha, Tepo Choronke, Zeway and Koka. The highest incidence (100%) of RKN was found from Adami Tullu, Jara Weyo, Babile, Erer Gota, Hurso, Jittu, Tikur Wuha, Tony farm, Tepo Choronke, Zeway and Koka localities based on direct observation of galls on collected root samples. Out of the 646 respondents, 43.3% reported to have RKN damage symptoms when shown the symptoms while 56.7% of them did not report damage. The highest significant effect on the dependent variable RKN damage on tomato roots comes from the previous crop, soil texture, awareness about RKN and source of irrigation water used. The presence of Meloidogyne incognita, M. javanica, M. arenaria and M. hapla on tomato was confirmed using a combination of molecular and biochemical identification tools. Five isolates of Meloidogyne spp. were not properly identified and could be new species. The two tropical species, M. incognita (48.4%) and M. javanica (41.2%) were the most prevalent species. Both species were also co-infesting tomato plants. Meloidogyne hapla was detected for the first time in an open tomato production farmer’s field at ‘Zeway’ locality with 1620 m.a.s.l. elevation. The occurrence of these Meloidogyne species alone, or in mixed populations from samples collected, clearly shows that RKN are widespread in major tomato growing areas of Ethiopia. In the near future, the economic importance of M. arenaria and M. hapla on tomato production in Ethiopian agriculture should be investigated.

Summary Pratylenchus rwandae n. sp., a root-lesion nematode associated with maize (Zea mays) from Rwanda, is described. This new species is characterised by females of medium to large size (469-600 μm) having an offset lip region with three annuli, stylet of 13-14.6 μm long with prominent rounded or anteriorly concave knobs, short to long pharyngeal gland overlap of 10.9-34.7 μm long, variable number of lateral lines (4-14) in different regions of the body, lateral field consisting of smooth bands, oval to slightly rounded spermatheca, vulva located at 75-80% of the total body length, post-vulval uterine sac (PUS) 20.3-26.5 μm long, tail subcylindrical to conoid with variation in tail tip shape from rounded to truncate or indented with generally smooth tip, and male unknown. The results of the phylogenetic analyses based on sequences of the D2-D3 expansion regions of 28S, partial 18S and ITS of rDNA and COI of mitochondrial DNA indicate that P. rwandae n. sp. is a species within the Penetrans group and appears as a sister species to a group comprising P. convallariae, P. dunensis, P. fallax, P. oleae, P. penetrans, P. pinguicaudatus, and three other unidentified species. A comparison of important morphological characters of the closely related Pratylenchus spp. is provided.

An overview is given of all representatives of the genus Typhlopolycystis Karling, 1956: the five known species (T. coeca Karling, 1956; T. mediterranea Brunet, 1965; T. coomansi Schockaert and Karling, 1975; T. schockaerti Karling, 1978 and T. rubra Noldt and Reise, 1985) and eleven new species: T. microphthalma n. sp. from S. France, T. fonsecai n. sp. and T. maikoni n. sp. from Brazil, T. norenburgi n. sp. and T. leasiae n.sp from the Pacific coast of Panama, T. pluvialiae n. sp. from Lanzarote (Canary Islands, Spain), T. riegeri n. sp. from Tanzania, T. tahitienis n. sp. from French Polynesia, T. australiensis n. sp. from South Australia, T. sarda n. sp. from Sardinia (Italy), the S. of France and from Lanzarote, and T. errata n. sp. from Sardinia (Italy). We consider T. limicola Schilke, 1970 as a species inquirenda of which no type material exists. Finally, some comments are given on the distribution of the genus of which representatives have been found in almost all places that have been sampled world wide.

Comprehensive morphological and molecular analyses revealed that published ITS sequences of the economically important plant-parasitic nematodePratylenchus goodeyiare actually sequences from distantly free-living bacterivorous ‘cephalobids’. We demonstrated that this incorrect labelling resulted in a cascade of erroneous interpretations, as shown by the reports of ‘P. goodeyi’ on banana in China and on cotton in India. This clearly illustrates the risk of mislabelled sequences in public databases. Other mislabelledPratylenchuscases are discussed to illustrate that this is not an isolated case. Herein,P. lentisn. syn. is considered a junior synonym ofP. pratensiswhileP. flakkensiswas for the first time linked to DNA sequences using topotype material. As taxonomic expertise is decreasing and sequence-based identification is growing rapidly, the highlighted problem may yet increase and a strong link between morphology and DNA sequences will be of crucial importance in order to prevent, or at least minimise, sequence-based misidentifications.

Background: Intravenous (IV) administration of IPI and NIVO has low activity in patients (pts) with recurrent glioblastoma (rGB). IT administration of IPI and NIVO was safe in cohort C1 and C2 of the GlITIpNi phase I clinical trial (Schwarze et al. ASCO 2020, abstract #2534). In C3 and C4, IT administration of IPI and NIVO was followed by repeated IC and IV administrations of NIVO. Method(s): Pts with resectable rGB were recruited in C4; pts with non-resectable rGB in C3 (biopsy only). IT administration (in the brain tissue lining the resection cavity) of IPI (5 mg) plus NIVO (10 mg), was followed by IC-NIVO (through an Ommaya reservoir) at a dose of 1, 5 or 10 mg Q2w plus IV-NIVO (10mg) Q2w (max 12x). Cerebrospinal fluid (CSF) was collected prior to each drug administration for biochemical/cytological analysis, and concentration measurements of IPI and NIVO. NGS (RNA/DNA) was performed on resected tissue. Result(s): 32 pts (23 male; med age 55y [38-77]; IDH1 R132H mutation [n=2], 1p/19q LOH [n=1]) diagnosed with rGB were enrolled (C3 [n=17], C4 [n=15]). After a median FU of 37w (1-82w), study treatment is ongoing in 2 pts. All pts received the predefined IT dose of IPI and NIVO. Median number of IC/IV NIVO administrations in C3 and C4 were respectively 5 [1-12] and 4 [1-12]. Most frequent AEs were fatigue (n=27), headache (n=17), fever (n=9), and seizures (n=5). Bacterial colonization of the Ommaya reservoir requiring removal and antibiotic therapy occurred in 4 pts. There were no G5 AEs. In C3, 1 pt has an ongoing PR after 83w (best response was PD in all other 15 evaluable pts); 11 pts have died (due to PD; median OS is 38w [95% CI 9-61]). In C4, 3 pts remain relapse-free after 30, 31 and 32w; 5 pts have died (all due to PD; median OS not reached). In 5 (C3) and 6 (C4) pts, elevated protein levels as well as lymphocytic pleocytosis were documented. There was no evidence for accumulation of NIVO in the CSF during therapy. Cytokine, cfDNA analysis of CSF and NGS on tumor tissue are ongoing. Conclusion(s): IT administration of NIVO and IPI followed by IC/IV administration of NIVO was feasible and sufficiently safe to warrant further investigation in pts with rGB. Clinical trial identification: NCT03233152. Legal entity responsible for the study: Department of Medical Oncology, Universitair Ziekenhuis Brussel. Funding(s): Stichting Tegen Kanker. Disclosure: J.K. Schwarze: Travel/Accommodation/Expenses: Merck Sharp & Dohme; Travel/Accommodation/Expenses: Amgen. G. Awada: Research grant/Funding (institution): Pfizer; Travel/Accommodation/Expenses: Merck Sharp & Dohme; Advisory/Consultancy, Research grant/Funding (institution), Travel/Accommodation/Expenses: Novartis; Travel/Accommodation/Expenses: Astellas. B. Neyns: Research grant/Funding (institution): Roche; Advisory/Consultancy: Bristol Myers Squibb; Advisory/Consultancy, Research grant/Funding (institution): Novartis; Advisory/Consultancy, Travel/Accommodation/Expenses: Merck Sharp & Dohme; Advisory/Consultancy: AstraZeneca; Advisory/Consultancy: EtheRNA; Advisory/Consultancy: CryoStorage; Research grant/Funding (institution): Pfizer; Research grant/Funding (institution): Merck-Serono. All other authors have declared no conflicts of interest.Copyright © 2020 European Society for Medical Oncology

Amphimictic populations of root-lesion nematodes with numerous males and females having three lip annuli, a functional spermatheca and non-areolated lateral field occur on sword fern (Nephrolepis exaltata) in Florida. Identified for decades asPratylenchus penetrans, they appeared to be a morphologically separated species on the basis of a longer stylet (17.8-18.3 μm) thanP. penetrans(15-17 μm) and different lip pattern inen faceview (rectangularvsdumb-bell inP. penetrans). Morphologically similar amphimictic root-lesion nematodes have also been detected on flax lily in Costa Rica. Subsequent morphological observations indicated that these amphimictic root-lesion nematodes from fern and flax lily are closely related to the parthenogenetic speciesP. bolivianus, which has areolated lateral fields. In spite of the reproductive and morphological dissimilarities between these populations, their separation into different species was not supported by the results of molecular analyses of their DNA sequences. The populations used in these analyses included those that are amphimictic from Florida and Costa Rica and others that are parthenogenetic from the type locality in Bolivia, and geographically distant localities in Chile, China, Colombia and Europe. Phylogenetic analyses of the ITS and D2-D3 expansion segments of the 28S rRNA gene indicated that they belong to the same species,P. bolivianus, which consists of two morphotypes,P. bolivianus(am) amphimictic andP. bolivianus(pm) parthenogenetic, herein described and illustrated. Contradictory results were obtained by the analyses using a portion of thehsp90gene. The phylogenetic study, which included sequences of other root-lesion nematodes, a topotype and geographical distant populations ofP. zeae, revealed thatP. bolivianusandP. zeaeformed highly supported clades in the majority consensus trees. PCR with species-specific primers for rapid diagnostics ofP. bolivianusandP. zeaewere developed and tested.

Root-lesion nematodes of the genus Pratylenchus are an important pest parasitizing a wide range of vascular plants including several economically important crops. However, morphological diagnosis of the more than 100 species is problematic due to the low number of diagnostic features, high morphological plasticity and incomplete taxonomic descriptions. In order to employ barcoding based diagnostics, a link between morphology and species specific sequences has to be established. In this study, we reconstructed a multi-gene phylogeny of the Penetrans group using nuclear ribosomal and mitochondrial gene sequences. A combination of this phylogenetic framework with molecular species delineation analysis, population genetics, morphometric information and sequences from type location material allowed us to establish the species boundaries within the Penetrans group and as such clarify long-standing controversies about the taxonomic status of P. penetrans, P. fallax and P. convallariae. Our study also reveals a remarkable amount of cryptic biodiversity within the genus Pratylenchus confirming that identification on morphology alone can be inconclusive in this taxonomically confusing genus.

Surveys conducted in peanut production areas of China revealed peanut pod rot in several fields in Shandong and Hebei Provinces, China. A large quantity of an unknown stem nematode was isolated from the hulls and seeds of peanuts, herein described as Ditylenchus arachis n. sp. The new species is characterized by a combination of the following features: lateral lip sectors distinctly projected, stylet delicate, 8·4–10 μm in length, six lines in the lateral field, tail elongate–conoid, bursa covering about 68–86% of tail length. Pathogenicity tests showed that D. arachis n. sp. could infect peanut (Arachis hypogaea), but not sweet potato (Ipomoea batatas) or potato (Solanum tuberosum). Morphologically, D. arachis n. sp. appears closest to D. africanus, D. myceliophagus and D. destructor, but can be differentiated based upon a combination of morphological characteristics, host preference and molecular sequence data. The results of the phylogenetic analysis, based on 18S rDNA, the D2–D3 expansion region of 28S rDNA, and the ITS1–5·8S–ITS2 region, confirmed its status as a new species. A sister relationship with D. destructor was appointed, rather than with its ecologically very similar congener D. africanus.

Composed mostly of fungivorous species, the genus Aphelenchoides also comprises 14 plant-parasitic species. The most common and devastating, A. besseyi, A. fragariae, A. ritzemabosi and A. subtenuis have been reported on more than 900 plant species. The combination of low inter-specific and high intra-specific morphological variability makes morphology-based identification extremely difficult within this genus, and has led to molecular tools being employed to ensure accurate diagnoses. rDNA markers are widely used for the identification of nematodes while the Cytochrome Oxidase I gene (COI) remains relatively unexplored despite its role as the standard barcode for almost all animal groups. To explore its suitability as a diagnostic tool, we studied a fragment of the mtCOI region of the four main plant-parasitic Aphelenchoides within a phylogenetic framework. We generated 69 mtCOI and 123 rDNA sequences of diverse Aphelenchoides taxa; 67 belong to the main plant-parasitic species including the first mtCOI sequence of A. fragariae and the first mtCOI and 28S sequences of A. subtenuis. mtCOI had a similar success rate for PCR amplification. Phylogenetic trees based on the three studied markers are largely in agreement with one another, validating their use for Aphelenchoides diagnosis; additionally, we were able to locate several misidentified sequences of plant-parasitic Aphelenchoides in existing databases. The concatenated analysis from the three markers resulted in a more robust insight into the phylogeny and evolution of Aphelenchoides, revealing that plant-parasitism has evolved independently at least three times within this genus, presumably from fungal-feeding ancestors.

Root-knot nematodes, Meloidogyne spp., cause huge economic losses worldwide. Currently, three Meloidogyne spp. are present on the quarantine A2 list of EPPO, Meloidogyne chitwoodi, Meloidogyne fallax and Meloidogyne enterolobii. As a quarantine organism, Meloidogyne fallax has been detected in England and Northern Ireland on sport turf in 2011, and in England on leek in 2013. However, its presence in Ireland has probably been overlooked since 1965, when Mr. John F. Moore and Dr. Mary T. Franklin had detected a new Meloidogyne species for that time. While the relevant data was recorded and a preliminary manuscript describing the species was prepared but never submitted for publication, and together with the original slides, pictures and drawings, it was restudied recently. We compared the population of Irish Meloidogyne sp. to other similar Meloidogyne spp. Careful observation and comparison shows that it belongs to Meloidogyne fallax. The characters found to be common for Irish Meloidogyne sp. and Meloidogyne fallax are female stylet length (14.6 μm) with oval to rounded basal knobs, oval shaped perineal pattern with moderately high dorsal arch, slender stylet in males (18.5 μm) with set off and rounded basal knobs, slightly set off male head with one post-labial annule and incomplete transverse incisures, and second-stage juveniles with large and rounded stylet basal knobs, and a gradually tapering tail (46.9 μm) with a broadly rounded tip and a clearly delimitated smooth hyaline part sometimes marked by constrictions (12.9 μm). The host test and gall formation also correspond to Meloidogyne fallax. The identification could not be additionally supported by molecular analysis, as we were unable to extract DNA from the old permanent slides. Nevertheless, our study reveals that the Meloidogyne species detected in Ireland in 1965 belongs to Meloidogyne fallax.

Root-knot nematodes, Meloidogyne spp., cause huge economic losses worldwide. Currently, three Meloidogyne spp. are present on the quarantine A2 list of EPPO, Meloidogyne chitwoodi, Meloidogyne fallax and Meloidogyne enterolobii. As a quarantine organism, Meloidogyne fallax has been detected in England and Northern Ireland on sport turf in 2011, and in England on leek in 2013. However, its presence in Ireland has probably been overlooked since 1965, when Mr. John F. Moore and Dr. Mary T. Franklin had detected a new Meloidogyne species for that time. While the relevant data was recorded and a preliminary manuscript describing the species was prepared but never submitted for publication, and together with the original slides, pictures and drawings, it was restudied recently. We compared the population of Irish Meloidogyne sp. to other similar Meloidogyne spp. Careful observation and comparison shows that it belongs to Meloidogyne fallax. The characters found to be common for Irish Meloidogyne sp. and Meloidogyne fallax are female stylet length (14.6 μm) with oval to rounded basal knobs, oval shaped perineal pattern with moderately high dorsal arch, slender stylet in males (18.5 μm) with set off and rounded basal knobs, slightly set off male head with one post-labial annule and incomplete transverse incisures, and second-stage juveniles with large and rounded stylet basal knobs, and a gradually tapering tail (46.9 μm) with a broadly rounded tip and a clearly delimitated smooth hyaline part sometimes marked by constrictions (12.9 μm). The host test and gall formation also correspond to Meloidogyne fallax. The identification could not be additionally supported by molecular analysis, as we were unable to extract DNA from the old permanent slides. Nevertheless, our study reveals that the Meloidogyne species detected in Ireland in 1965 belongs to Meloidogyne fallax.

The polyphagous parthenogenetic root-knot nematodes of the genus Meloidogyne are considered to be the most significant nematode pest in sub-tropical and tropical agriculture. Despite the crucial need for correct diagnosis, identification of these pathogens remains problematic. The traditionally used diagnostic strategies, including morphometrics, host-range tests, biochemical and molecular techniques, now appear to be unreliable due to the recently-suggested hybrid origin of root-knot nematodes. In order to determine a suitable barcode region for these pathogens nine quickly-evolving mitochondrial coding genes were screened. Resulting haplotype networks revealed closely related lineages indicating a recent speciation, an anthropogenic-aided distribution through agricultural practices, and evidence for reticulate evolution within M. arenaria. Nonetheless, nucleotide polymorphisms harbor enough variation to distinguish these closely-related lineages. Furthermore, completeness of lineage sorting was verified by screening 80 populations from widespread geographical origins and variable hosts. Importantly, our results indicate that mitochondrial haplotypes are strongly linked and consistent with traditional esterase isozyme patterns, suggesting that different parthenogenetic lineages can be reliably identified using mitochondrial haplotypes. The study indicates that the barcode region Nad5 can reliably identify the major lineages of tropical root-knot nematodes.

With 556 species described to date, Kalyptorhynchia includes about one-third of all species of rhabdocoel flatworms. In this study, we present the first molecular phylogenetic analysis of this taxon. The final analysis comprises 110 species. These represent 11 of the 17 known families. The largest family (241 known species), Polycystididae, is represented by nine of 10 subfamilies and 33 of the 58 genera. Sequence data from 18S rDNA and 28S rDNA were analysed using maximum likelihood and Bayesian approaches. Of the two taxa traditionally recognised within Kalyptorhynchia, Eukalyptorhynchia and Schizorhynchia, only Schizorhynchia is monophyletic. All eukalyptorhynch families, except Cicerinidae, are monophyletic. On the other hand, two of the three schizorhynch families included are not monophyletic. Within Polycystididae, the traditional taxonomy was not reflected in our phylogenetic analyses and most subfamilies are polyphyletic. These results suggest that current classification, mostly based on characters of the genital system, suffers from homoplasy. Where possible, a revised classification, taking into account these new findings, is given.

Five new species of the new taxon Brunetorhynchus n. gen. are described: B. deconincki n. sp., B. microstylis n. sp. and B. complicatus n. sp. are from the Mediterranean, B. canariensis n. sp. is from the Canary Island Lanzarote, B. cannoni n. sp. is from the Australian East coast and one species from the Galapagos, formerly described as Limipolycystis spec., is transferred to the new genus as B. dubius n. sp.. As in Limipolycystis, these species have a single stylet, (accessory stylet type II), an accessory secretion vesicle (type II) and a prostate vesicle (type III) in the male atrium, although the latter vesicle is absent in some species. Unlike the species of Limipolycystis, where the seminal receptacle is a sclerotized tubule, the species of the new taxon have a pear-shaped seminal receptacle on the oviduct.

Five new species of the new taxon Brunetorhynchus n. gen. are described: B. deconincki n. sp., B. microstylis n. sp. and B. complicatus n. sp. are from the Mediterranean, B. canariensis n. sp. is from the Canary Island Lanzarote, B. cannoni n. sp. is from the Australian East coast and one species from the Galapagos, formerly described as Limipolycystis spec., is transferred to the new genus as B. dubius n. sp.. As in Limipolycystis , these species have a single stylet, (accessory stylet type II), an accessory secretion vesicle (type II) and a prostate vesicle (type III) in the male atrium, although the latter vesicle is absent in some species. Unlike the species of Limipolycystis, where the seminal receptacle is a sclerotized tubule, the species of the new taxon have a pear-shaped seminal receptacle on the oviduct.

The Macrostomorpha-an early branching and species-rich clade of free-living flatworms-is attracting interest because it contains Macrostomum lignano, a versatile model organism increasingly used in evolutionary, developmental, and molecular biology. We elucidate the macrostomorphan molecular phylogeny inferred from both nuclear (18S and 28S rDNA) and mitochondrial (16S rDNA and COI) marker genes from 40 representatives. Although our phylogeny does not recover the Macrostomorpha as a statistically supported monophyletic grouping, it (i) confirms many taxa previously proposed based on morphological evidence, (ii) permits the first placement of many families and genera, and (iii) reveals a number of unexpected placements. Specifically, Myozona and Bradynectes are outside the three classic families (Macrostomidae, Microstomidae and Dolichomacrostomidae) and the asexually fissioning Myomacrostomum belongs to a new subfamily, the Myozonariinae nov. subfam. (Dolichomacrostomidae), rather than diverging early. While this represents the first evidence for asexuality among the Dolichomacrostomidae, we show that fissioning also occurs in another Myozonariinae, Myozonaria fissipara nov. sp. Together with the placement of the (also fissioning) Microstomidae, namely as the sister taxon of Dolichomacrostomidae, this suggests that fissioning is not basal within the Macrostomorpha, but rather restricted to the new taxon Dolichomicrostomida (Dolichomacrostomidae+Microstomidae). Furthermore, our phylogeny allows new insights into the evolution of the reproductive system, as ancestral state reconstructions reveal convergent evolution of gonads, and male and female genitalia. Finally, the convergent evolution of sperm storage organs in the female genitalia appears to be linked to the widespread occurrence of hypodermic insemination among the Macrostomorpha.