First Report of Plant-Parasitic Nematode Meloidoderita salina in the Netherlands.

After the description of the root-parasitic nematode Meloidoderita salina from a tidal salt marsh in France (1), an additional sampling was carried out to search for the presence of this unusual nematode in a tidal salt marsh area close to Sint-Annaland, Zeeland Province, the Netherlands. In August and October 2012, a total of 25 soil and root samples were collected from the halophytic plants Atriplex portulacoides L. (so far the only known host for this nematode species), A. littoralis L., A. prostrata Boucher ex DC., Limonium vulgare Mill., Salicornia europaea L., Aster tripolium L., and Plantago maritima L. All these halophytes grow in a cohesive muddy soil type within the salt marsh, except A. littoralis and A. prostrata, which grow in the litter tidal zones on the edges of this area. Nematodes from roots and soil were extracted by centrifugal flotation (2) and Oostenbrink's cotton-wool filter methods (4), respectively. Additionally, roots were used for direct observation of females and young cystoids with a dissecting microscope. Finally, all stages were compared morphologically with available type material (1). Root and soil samples demonstrated that only nematodes isolated from A. portulacoides, A. littoralis, and A. prostrata contained all life stages of the genus Meloidoderita, while on the roots of L. vulgare, S. europaea, A. tripolium, and P. maritima, no Meloidoderita was observed. The soil samples included males, cystoids, and second-stage juveniles (J2) in low densities (<20 nematodes/100 ml), while swollen females and young cystoids were observed on root samples. All stages (n = 10 per life stage) fit morphologically with the recently described M. salina. Females were swollen with an oval to pear shaped body with a small posterior protuberance, irregular and twisted neck, oval and backwardly sloping stylet knobs, a prominent secretory-excretory (S-E) pore with cuticular lobes, and a swollen uterus with a thick hyaline wall. Males were without stylet, strongly sclerotized S-E duct, and tail tapering to rounded terminus ending in one or two ventrally terminal mucron. J2s had a well-developed stylet and rounded knobs set off from shaft and conical tail slightly curved ventrally and tapering to a finely pointed terminus with a finger-like projection. Cystoids showing the unique sub-cuticular hexagonal beaded pattern (1). J2s were also used for molecular analysis. DNA was extracted by incubating individual J2s in a lysis buffer as described in (3). Two primer combinations were used to amplify the small subunit ribosomal DNA (SSU rDNA) from a 100-times-diluted crude lysate (two overlapping fragments, [3]). The resulting (nearly) full-length SSU-rDNA sequences (GenBank KF751617 and KF751618) showed >99% identity with M. salina sequences from nematodes collected in the aforementioned tidal salt marsh in France (FJ969126 and FJ969127). To our knowledge, this is the first report of M. salina in the Netherlands. Moreover, this is the first record of M. salina parasitizing A. littoralis and A. prostrata. Although these Atriplex species are used for human consumption, the effect of M. salina on the host is unknown so far. References: (1) S. Ashrafi et al. Zookeys 249:1, 2012. (2) W. A. Coolen. Pages 317-329 in: Root-knot nematodes (Meloidogyne species). Systematics, biology and control. Academic Press, New York, 1979. (3) Holterman et al. Mol. Biol. Evol. 23:1792, 2006. (4) M. Oostenbrink. Pages 85-102 in: Nematology. University of North Carolina Press, Chapel Hill, 1960.

Small subunit rDNA-based phylogeny of the Tylenchida sheds light on relationships among some high-impact plant-parasitic nematodes and the evolution of plant feeding.

Cyst (Heteroderidae), root knot (Meloidogyne spp.), and lesion (Pratylenchus spp.) nematodes all belong to a single nematode order, Tylenchida. However, the relationships between and within these economically highly relevant groups, and their relatedness to other parasitic Tylenchida is unclear. We constructed a phylogeny of 116 Tylenchida taxa based on full length small subunit ribosomal DNA (small subunit [SSU] rDNA) sequences. Ancestral state reconstruction points at a gradual development of simple to more complex forms of plant parasitism. Good resolution was observed in distal clades that include cyst, root knot, and lesion nematodes, and monophyly of most families was confirmed. Our data suggest that root knot nematodes have evolved from an ancestral member of the genus Pratylenchus, but it remains unclear which species is closest to this branching point. Contrary to the notoriously polyphagous distal representatives, basal members of the genus Meloidogyne (and probably, their common ancestor) have narrow host ranges. Our analysis also shows that mitotic parthenogeny has arisen at least two times independently among root knot nematodes. In many cases resolution till species was observed, suggesting that SSU rDNA sequences have a potential for DNA barcode-based species identification with, due to the overall conserved nature of this gene, limited intra-species variation.

First Report of Root-Knot Nematode Meloidogyne enterolobii on Tomato and Cucumber in Switzerland.

Severe stunting and extensive root galling were observed on tomato rootstock (Solanum lycopersicum L. cv. Maxifort) resistant to Meloidogyne incognita (Kofoid & White, 1919) Chitwood, 1949, M. javanica (Treub, 1885), and M. arenaria (Neal, 1889) Chitwood, 1949 and cucumber (Cucumis sativus L. cv. Loustik) from two commercial greenhouses in the cantons Aargau and Lucerne in northern Switzerland. Examination of the roots of infected plants revealed the presence of root-knot nematodes in large numbers. Juveniles, males, and females were isolated, and the species was determined on the basis of morphological characteristics, including the female perineal pattern. Identification was confirmed by female esterase (Est) and malate dehydrogenase (MdH) electrophoresis (20 each for Est and MdH). All methods of identification were consistent with M. enterolobii Yang & Eisenback, 1983 (4). For further confirmation, type material of M. enterolobii (from the original host Enterolobium contortisiliquum (Vell.) Morong) from China (4) was used. Furthermore, comparison of the sequence data from 12 individuals of each of the two Swiss populations and the type material of a 310-bp fragment of cytochrome oxidase I (COI), a 723-bp fragment covering the internal transcribed spacer (ITS) region 1, 5.8s, ITS2, and part of the 26s, the mtDNA 63-bp repeat region, and a 780-bp fragment of the intergenic spacer region (1-3) showed 100% homology and confirmed the identification as M. enterolobii. The species M. enterolobii is of great importance because it is able to reproduce on resistant tobacco, pepper, watermelon, and tomato (4). To our knowledge, this is the first report of M. enterolobii in Switzerland. References: (1) M. A. M. Adam et al. Plant Pathol. 56:190, 2007. (2) V. C. Blok et al. Nematology 4:773, 2002. (3) T. C. Vrain et al. Fundam. Appl. Nematol. 15:565, 1992. (4) B. Yang and J. D. Eisenback. J. Nematol. 15:381, 1983.

First Report of the Root-Knot Nematode Meloidogyne ethiopica on Tomato in Slovenia.

The root-knot nematode Meloidogyne ethiopica Whitehead originally described from Tanzania is also distributed in South Africa, Zimbabwe, and Ethiopia (3). Although this species is a relatively unknown root-knot nematode, M. ethiopica parasitizes several economical important crops, such as tomato, cowpea, bean, cabbage, pepper, pumpkin, tobacco, lettuce, and soybean (4). This species can be morphologically confused with M. incognita (Kofoid & White), and therefore, is probably easily overlooked (1). Recently, characteristic esterase isozyme patterns were described for this species, which provide a more reliable identification (2). In October 2003, Lycopersicum esculentum cv. Belle plants with large root-galls were observed in a greenhouse at Dornberk, Slovenia, which were identified as an unknown root-knot nematode within the M. incognita group. Subsequent sampling revealed infestation of all tomato plants within this greenhouse with obvious aboveground symptoms of stunting and wilting. Additional morphological and isozyme studies identified this root-knot nematode as M. ethiopica. The identification was based on female, male, and second-stage juvenile morphology and malate dehydrogenase and esterase isozyme phenotypes from young egg-laying females isolated from 20 tomato plants and compared with a culture of M. ethiopica from Africa. To our knowledge, this is not only the first report of M. ethiopica in Slovenia, but also the first report of this species in Europe. References: (1) A. M. Golden. Fundam. Appl. Nematol. 15:189, 1992. (2) W. Mandefro and K. Dagne. Afr. J. Plant Prot. 10:39, 2000. (3) A. G. Whitehead. Trans. Zool. Soc. Lond. 31:263, 1968. (4) A. G. Whitehead. Nematologica 15:315, 1969.

First Report of the Dagger Nematode Xiphinema rivesi, a Member of the X. americanum Group, from Slovenia.

The dagger nematode, Xiphinema rivesi Dalmasso, is a virus-transmitting plant-parasitic nematode described from France (2) and also reported from Germany, Portugal, and Spain. While this species is only known from a few local sites within Europe, it is widespread in North America and also present in Peru and Pakistan (3). X. rivesi is able to transmit at least four North American nepoviruses, including Cherry rasp leaf virus, Tobacco ringspot virus, Tomato ringspot virus, and Peach rosette mosaic virus (1,4). In early spring 2002, nematodes of the X. americanum group were detected in soil samples taken near the roots of peach plants from an orchard in Bilje near Nova Gorica, Slovenia. Resampling in June and September at the same site and at an adjacent, approximately 30-year-old peach orchard confirmed the presence of the nematode in relatively high numbers (5 per 100 ml of soil). Adult females were isolated and sent for further identification to the Plant Protection Service in Wageningen, the Netherlands. The nematode was morphologically identified as X. rivesi and confirmed at the rDNA-level with internal transcribed spacer restriction fragment length polymorphism analysis. Because both sampling sites where the nematode was found are close to the Vipava River in an area of extensive stone and pome fruit production, it is likely that the nematode is also present in other orchards in this region. The origin of this quarantine nematode is unknown as there is no direct link with Slovenian orchards and import of plant material from abroad in the last decades. To our knowledge, this is the first report of X. rivesi in Slovenia and the fourth report of this species in Europe. References: (1) D. J. F. Brown et al. Phytopathology 84:646, 1994. (2) A. Dalmasso. Mem. Mus. Hist. Nat. Nouv. Série A., Zool. 61:33, 1969. (3) Data Sheets on Quarantine Pests: Xiphinema americanum sensu lato. Page 629 in: Quarantine Pest for Europe, CABI, Wallingford, UK, 1997. (4) L. W. Stobbs et al. Plant Dis. 80:105, 1996.

Species identification of cyst and root-knot nematodes from potato by electrophoresis of individual females.

To carry out rapid and reliable species identification of Globodera and Meloidogyne specimens, two electrophoretic methods were modified and adapted for an automated electrophoresis system (PhastSystem, Pharmacia). Proteins of individual Globodera cysts were identified using isoelectric focusing and a sensitive silver stain. Proteins of young single Meloidogyne females were separated using polyacrylamide gradient gels and stained for the isozymes esterase and malate dehydrogenase.