First Report of Heterodera ripae on Common Nettle in Spain.

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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.