In the present study, the determination and computer-assisted characterization of ribosomal DNA (rDNA) sequence data were performed to improve classification and diagnostics of nematodes of the genus Ditylenchus. Dot-plot analyses of the ITS1-5AE8S-ITS2 segments revealed the presence of short repeated DNA motifs localized in the ITS1 region of D. destructor, whilst no similar DNA repeats were identified in other members of the genus. Additionally, three-domain secondary structure models were inferred for ITS2 transcripts, providing a valuable resource for complex phylogenetic studies. Finally, alignment of the ITS-rDNA sequence data and subsequent oligonucleotide combinations trials led to the development of taxon-specific amplified regions. Together, this is the first report in which the three most common taxa of crop-parasitic nematodes of the genus Ditylenchus, namely D. dipsaci sensu stricto, Ditylenchus sp. B and D. destructor, have been simultaneously detected using a multiplex polymerase chain reaction (PCR)-based approach. This novel DNA technology is particularly valuable for the precise molecular diagnostics applicable to phytosanitary control and marker-assisted selection (MAS)-based plant breeding.

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Abstract

The use of DNA-based analyses in molecular plant nematology research has dramatically increased over recent decades. Therefore, the development and adaptation of simple, robust, and cost-effective DNA purification procedures are required to address these contemporary challenges. The solid-phase-based approach developed by Flinders Technology Associates (FTA) has been shown to be a powerful technology for the preparation of DNA from different biological materials, including blood, saliva, plant tissues, and various human and plant microbial pathogens. In this work, we demonstrate, for the first time, that this FTA-based technology is a valuable, low-cost, and time-saving approach for the sampling, long-term archiving, and molecular analysis of plant-parasitic nematodes. Despite the complex structure and anatomical organization of the multicellular bodies of nematodes, we report the successful and reliable DNA-based analysis of nematode high-copy and low-copy genes using the FTA technology. This was achieved by applying nematodes to the FTA cards either in the form of a suspension of individuals, as intact or pestle-crushed nematodes, or by the direct mechanical printing of nematode-infested plant tissues. We further demonstrate that the FTA method is also suitable for the so-called "one-nematode-assay", in which the target DNA is typically analyzed from a single individual nematode. More surprisingly, a time-course experiment showed that nematode DNA can be detected specifically in the FTA-captured samples many years after initial sampling occurs. Collectively, our data clearly demonstrate the applicability and the robustness of this FTA-based approach for molecular research and diagnostics concerning phytonematodes; this research includes economically important species such as the stem nematode (Ditylenchus dipsaci), the sugar beet nematode (Heterodera schachtii), and the Northern root-knot nematode (Meloidogyne hapla).

The plant-parasitic nematodes are the most important pests around the world. Many species of the plant-parasitic nematodes cause high losses of crop yield and many of them have a quarantine status. The stem and bulb nematode Ditylenchus dipsaci (Kühn 1857) Filipjev 1836 is a migratory endoparasite nematode of over five hundred vascular plant species. The stem nematode D. dipsaci is prevalent in a wide range of climatic conditions, where moisture regimes enable nematode infection, multiplication and dispersal. The main method of D. dipsaci control is crop rotation, but the presence of morphologically indistinguishable host races with different host preferences makes rotation difficult (Wendt et al. 1993). The biological races exhibit different degrees of reproductive isolation, such as partial or complete reproductive incompatibility (Erikson 1974). Each biological race is able to complete its life cycle only on a specific plant host. Among the most important plant hosts of D. dipsaci in Central Europe are bulb vegetables (onion, garlic and leek), carrot, alfalfa, clover, sugar beet, chicory, potatoes, strawberry, ornamental bulb plants (Narcissus spp.), and also many common weeds. Symptoms on host plants are not always specific as for appearance. Early infested plants and low infested seeds show no symptoms. Nematodes cause swellings and distortion of aerial plant parts and necrosis or rotting of stem bases, bulbs, tubers and rhizomes. The Conversion of sequence-characterized amplified region (SCAR) bands into high-throughput DNA markers based on RAPD technique for detection of the stem nematode ABSTRACTDitylenchus dipsaci, the stem nematode, is a migratory endoparasite of over 500 species of angiosperms. The main method of D. dipsaci control is crop rotation, but the presence of morphologically indistinguishable host races with different host preferences makes rotation generally ineffective. Therefore, a sensitive, rapid, reliable, as well as cost effective technique is needed for identification of D. dipsaci in biological samples. This study describes the development of species-specific pairs of PCR oligonucleotides for detection and identification of the D. dipsaci stem nematode in various plant hosts. Designed DIT-2 primer pair specifically amplified a fragment of 325 bp, while DIT-5 primer pair always produced a fragment of 245 bp in all D. dipsaci isolates. Two developed SCAR primer pairs were further tested using template DNA extracted from a collection of twelve healthy plant hosts; no amplification was however observed. The developed PCR protocol has proved to be quite sensitive and able to specifically detect D. dipsaci in artificially infested plant tissues.