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Soil and root samples were collected from May to October 2010 from cultivated and wild plants during a survey for hop cyst nematodes. Cyst nematodes were detected in roots of common nettle (Urtica dioica L.) with severe plant yellowing in four natural areas of León and Burgos provinces, Spain, where common nettle is grown in organic farming systems as a substrate for pharmaceutical products. Cysts were isolated by flotation and sieving methods. Cysts and juveniles were analyzed by morphological and molecular methods. The cyst nematodes obtained from soil and plant samples from all four locations had uniform morphological and molecular characteristics that differed from those of Heterodera humuli. Cysts (n = 25) had the following characteristics: lemon shaped, yellow to pale brown; cyst wall with ridges forming an irregular zigzag pattern; young cysts covered by subcrystalline layer; vulval cone bifenestrate with circular or subcircular fenestrae; bullae absent; underbridge weak; body length (not including the neck) ranging from 295 to 489 μm (mean of 418 μm); body width ranging from 208 to 375 μm (mean of 310 μm); fenestrate length of 39 to 58 μm (mean of 46.5 μm) and width of 25.2 to 30.9 μm (mean of 25.1 μm); underbridge length from 51 to 90 μm (mean of 69.2 μm); and vulval slit length from 26 to 40 μm (mean of 33 μm). J2 (n = 20) had the following characteristics: body length ranging from 338 to 380 μm (mean of 359.3 μm); stylet length from 21 to 24 μm (mean of 22.1 μm) with knobs rather wide and slightly projecting anteriorly; tail conical with a length of 41 to 52.5 μm (mean of 45.6 μm) and hyaline part of tail ranging from 18 to 25 μm (mean of 23.3 μm); lateral field with four lines. All morphological data and characters were consistent with those of H. ripae (1). DNA from single cysts was extracted to amplify the internal transcribed spacer (ITS) region of rDNA by PCR with forward primer TW81 (5′-GTTTCCGTAGGTGAACCTGC-3′) and reverse primer AB28 (5′-ATATGCTTAAGTTCAGCGGGT-3′) (2). The PCR product was digested by restriction enzymes (AluI, CfoI, HaeI, HinfI, PstI, RsaI, TaqI, and Tru9I) to obtain restriction fragment length polymorphism profiles (2). ITS products cloned and assayed using the ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Salamanca, Spain) were subjected to a database search using BLAST (National Centre for Biotechnology Information) to confirm the identification. These sequences exhibited 99.0% similarity with that of a H. ripae isolate from Germany (GenBank Accession No. AF274407.1). In glasshouse proofs of pathogenicity with these populations of H. ripae, 25 full cysts placed in nylon net bags were inoculated in 9-cm-diameter pots with 10 replicates per plant. After 12 weeks, soil from each pot was dried and cysts extracted. Cysts did not develop on roots of common hop (Humulus lupulus L.) and hemp (Cannabis sativa L.), but in common nettle there was an increase in nematode populations, with all plants severely stunted and yellowing, which confirmed the nematodes' pathogenicity. H. ripae has been previously reported in Russia, Estonia, Latvia, Armenia, Moldova, Ukraine, Bulgaria, Slovakia, Germany, and Belgium (1). To our knowledge, this is the first report of H. ripae in Spain. The identification of H. ripae in nettle fields is important in this region where it could cause large yield reductions if not properly managed. References: (1) S. A. Subbotin et al. Russ. J. Nematol. 5:143, 1997. (2) S. A. Subbotin et al. Nematology 5:515, 2003.

During the 2009 to 2010 corn-growing (Zea mays L. cv. Alexandria) seasons, severely stunted and yellowing corn plants in several commercial fields at Aldearrubia (Salamanca Province) were observed in western Spain. The disease incidence ranged from 80 to 100%. Early symptoms consisted of severely reduced growth of the plants coupled with extensive leaf yellowing. Occurrence of the disease was estimated to cause near complete loss of the crop yields since the corn produced in affected fields was unmarketable. Disease surveys revealed high parasitism in the main and feeder roots and a large soil population of the root-knot nematode Meloidogyne spp. The nematode population was extracted and quantified from soil and root samples according to Barker (1). It was identified as the southern root-knot nematode M. incognita race 1, by female perineal pattern, host-differential test, and multiplex PCR using forward primers H-18S, CF-ITS, I-ITS, and reverse primer HCFI-28S (3,4). ITS products cloned and assayed using the ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Salamanca, Spain) were subjected to a database search using BLAST (National Centre for Biotechnology Information) to confirm the identification. These sequences exhibited 99.0% similarity with that of an M. incognita isolate from France (GenBank Accession No. AF402309.1). M. incognita was found in 80% of soil samples collected from the areas where the disease was observed and 83.5% of root samples with nematode population densities ranging from 26 to 269 eggs and second-stage juveniles (J2s) per 100 cm3 of soil and 234 to 1,634 eggs and J2s per 5 g of fresh roots. In glasshouse proofs of pathogenicity, a mix of 1,500 eggs and J2s of these populations of M. incognita were inoculated in 20-cm-diameter pots with 10 replicates with a single pregerminated seed of corn cv. Alexandria as host plant; another 10 replicates without inoculation were established as control plants. After 6 weeks, all plants inoculated were severely stunted and yellowing; infected roots showed galls on root tips and secondary feeder roots. Galling of root tips that cause stubby root symptoms prevented further root growth into deeper soil layers and induced proliferation of secondary roots, which confirmed the nematodes' pathogenicity. The severe infections in roots of corn plants suggest that parasitism of corn roots by the root-knot nematode must contribute to stunting, yellowing, and decline of corn, reducing yield by restricting access to water and nutrients that are needed for plant growth and development, and can result in the death of younger plants as previously reported (2). To our knowledge, this is the first report of M. incognita infecting corn in Spain. 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) T. P. Heffes et al. Nematropica 22:139, 1992. (3) L. Robertson et al. Crop Prot. 25:440, 2006. (4) C. Zijlstra. Fund. Appl. Nematol. 20:505, 1997.

I(evwor&: Steinernema carpocapsae, beet cyst nematode, sugar beetThe inundative application of Steinernema carpocapsae (DDl36) was shown to increase populations of rhabditid nematodes and decrease plant-parasitic genera in soil (7). The invasion of tomato roots by the root-knot nematode Meloidogyne javanica was markedly decreased by application of S. glaseri (1) and by S. carpocapsae, S. feltiae and S. riobravis (6). The present paper reports on the effect of S. feltiae and Heterorhabditis bacteriophora on the beet cyst nematode, Heterodera schuchtii. and Methods Sugar beet seedlings (Beta vulgaris L.) cv. Dora, transplanted into 8 cm plastic pots (one seedlindpot) containing a sand mixture, were watered daily with Hoagland's nutrient solution and maintained under glasshouse conditions. The entomopathogenic nematodes used were Steinernemafeltiae (UK isolate from Reading University) and a Heterorhabditis bacteriophora isolate from Spain. Both entomopathogenic nematode species were cultured by infecting last instar Galleria mellonella (greater wax moth) with infective juveniles of each species: after 9-10 days the newly produced infective juveniles were harvested from the dead G. mellonella larvae on modified White traps (3). When sugar beet seedlings were 20 cm in height, infective juveniles of both entomopathogenic nematode species were applied daily at a rate of 10 000 per pot over a 10 day period. On the third day after the initial application of the entomopathogenic nematodes, either 1000 or 2000 freshly hatched second stage juveniles of the sugar beet cyst nematode were added per pot. Ten replicates were used in each treatment. The plants were grown on for two months in a glasshouse (photoperiod 15/9 lighudark at a temperature ranging from 25-30°C) when the final populations of the sugar beet cyst nematode were determined by extracting the cysts with the Fenwik can (4). The cysts were separated from the debris by alcohol flotation (7) and were crushed in water in a cyst homogeniser (Eijkelkamp Agrisearch Equipment, Netherlands). Data from the experiment were analysed by ANOVA and the Duncan multiple-range test. IlGsuk2The total number of H. schuchtii eggs was significantly (P = 0.01) decreased by the presence of both entomopathogenic nematode species. Steinernemafeltiae was the most effective entomopathogenic nematode at both levels of H. schachtii inoculum.-100 -

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