ABSTRACT
Chondrina Reichenbach, 1828 is a highly diverse genus of terrestrial molluscs currently including 44 species with about 28 subspecific taxa. It is distributed through North Africa, central and southern Europe, from Portugal in the West to the Caucasus and Asia Minor in the East. Approximately 70% of the species are endemic to the Iberian Peninsula constituting its main center of speciation with 34 species. This genus includes many microendemic taxa, some of them not yet described, confined to limestone habitats (being strictly rock-dwelling species). They are distributed on rocky outcrops up to 2000 m.a.s.l. It is a genus of conical-fusiform snails that differ mainly in shell characters and in the number and position of teeth in their aperture. So far, molecular studies on Chondrina have been based exclusively on the mitochondrial Cytochrome Oxidase subunit I region (COI). These studies gave a first view of the phylogeny of the genus but many inner nodes were not statistically supported. The main objective of the study is to obtain a better understanding of the phylogeny and systematics of the genus Chondrina on the Iberian Peninsula, using multilocus molecular analysis. Partial sequences of the COI and 16S rRNA genes, as well as of the nuclear Internal Transcribed Spacer 1 (ITS1-5.8S) and Internal Transcribed Spacer 2 (5.8S-ITS2-28S) were obtained from individuals of all the extant Chondrina species known from the Iberian Peninsula. In addition to this, the newly obtained COI sequences were combined with those previously published in the GenBank. Phylogenetic relationships were inferred using maximum likelihood and Bayesian methods. The reconstructed phylogenies showed high values of support for more recent branches and basal nodes. Moreover, molecular species delimitation allowed to better definethe studied species and check the presence of new taxa.
Subject(s)
Snails , Animals , Bayes Theorem , Europe , Humans , Phylogeny , RNA, Ribosomal, 16S/genetics , Snails/geneticsABSTRACT
In 2014, glasshouse-grown wasabi (Eutrema japonica) grown in a compost based media displayed symptoms of poor growth and wilting. Visual assessment of the roots showed that 25% of the symptomatic plants sampled had raised black lesions on the roots affecting between 5 and 20% of the total root area. To isolate the causal agent, affected material (approximately 5 mm3) was surface disinfested in sodium hypochlorite (2%) for 30 s, rinsed twice in sterile water and plated on to water agar medium amended with penicillin G (0.2 g/liter) and streptomycin sulfate (0.8 g/liter). Plates were incubated at 20ºC until fungal colonies were visible. After three days, colonies of Rhizoctonia solani were identified based on the presence of septate hyphae with right-angle branching, a pure culture was obtained through hyphal tip transfer onto a new plate of PDA. DNA was extracted from a 7-day old plate of the isolate (WAS1) as described previously (Woodhall et al., 2013). The AG of WAS1 was determined as AG2-1 using a subgroup specific real-time PCR assay (Budge et al., 2009b) and confirmed by DNA sequencing as described previously (Lekuona Gomez et al., 2015). The sequence was 100% identical (587/587bp) to a previously identified AG2-1 isolate 1971 (GenBank accession FJ435126) (Budge et al., (2009a). Pathogenicity of the isolate was confirmed by inoculating three healthy one-year-old wasabi plants grown in loam based compost (John Innes No.3) each with four 5 mm fully colonised PDA plugs of isolate WAS1 placed at approx. 40 mm depth in the soil. Four sterile PDA plugs were place in each of three control plants. All six plants were placed in a greenhouse at 21°C, 18h:6h light: dark and watered as required. After 21 days, multiple black root lesions typically 3-5mm in length were observed on the roots of all inoculated plants. No lesions were observed on the control plants. From three lesions per plant, isolations were attempted as described above. Rhizoctonia solani was recovered from all isolations and the resulting cultures all tested positive for AG2-1 using the real-time PCR assay. Isolations were attempted from the roots of healthy control plants but Rhizoctonia was not recovered. Here we demonstrate that R. solani AG2-1 is associated with root necrosis of Eutrema japonica. Rhizoctonia solani AG2-1 has been reported previously in various Brassica crops in the UK (Budge et al., 2009a) and on Matthiola incana (Lekuona Gómez et al., 2015). It has also been reported causing disease in potatoes and as widely present in UK field soils (Woodhall et al., 2013). Although R. solani AG1 and AG4 of R. solani have been reported to infect Eutrema japonica in Japan (Takeuchi et al., 2003; 2008), this is the first finding that identifies AG2-1 as the causal agent. The potential presence of AG2-1 in soil and/or as plant debris should be considered prior to planting susceptible hosts.
