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Small Subunit Ribosomal DNA-Based Phylogenetic Analysis of Foliar Nematodes (Aphelenchoides spp.) and Their Quantitative Detection in Complex DNA Backgrounds

First, second, third, fourth, fifth, tenth, and eleventh authors: Laboratory of Nematology, Department of Plant Sciences, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands; sixth author: BLGG AgroXpertus, Binnenhaven 5 6709 PD, 6709 PA Wageningen, The Netherlands; seventh and eighth authors: Flower Bulbs and Nursery Stock-PPO Lisse, Prof. van Slogterenweg 2, 2161 DW Lisse, The Netherlands; and ninth author: Plant Protection Service, Nematology Section, Geertjesweg 15, 6706 EA Wageningen, The Netherlands.

Foliar nematodes, plant-parasitic representatives of the genus Aphelenchoides, constitute a minority in a group dominated by fungivorous species. Distinction between (mostly harmless) fungal feeding Aphelenchoides species and high impact plant parasites such as A. besseyi, A. fragariae, A. ritzemabosi, and A. subtenuis is severely hampered by the scarcity of informative morphological characters, some of which are only observable in specific developmental stages. Poor description of a number of non-plant-parasitic Aphelenchoides species further complicates identification. Based on (nearly) full-length small subunit ribosomal DNA (SSU rDNA) sequences (≈1,700 bp), a phylogenetic tree was generated, and the four target species appeared as distinct, well-supported groups. Notably, this genus does not constitute a monophyletic group: A. besseyi and A. ritzemabosi cluster together and they are phylogenetically isolated from A. fragariae, A. subtenuis, and most other fungivorous species. A phylum-wide SSU rDNA framework was used to identify species-specific DNA motifs. For the molecular detection of four plant-parasitic Aphelenchoides species, polymerase chain reaction primers were developed with high, identical annealing temperatures (63°C). Within the molecular framework presented here, these primers can be used for the rapid screening of plant material and soil for the presence of one or multiple foliar nematode species.