A ribosomal DNA-based framework for the detection and quantification of stress-sensitive nematode families in terrestrial habitats

Indigenous communities of soil-resident nematodes have a high potential for soil health assessment as nematodes are diverse, abundant, trophically heterogeneous and easily extractable from soil. The conserved morphology of nematodes is the main operational reason for their under-exploitation as soil health indicators, and a user-friendly biosensor system should preferably be based on nonmorphological traits. More than 80% of the most environmental stress-sensitive nematode families belong to the orders Mononchida and Dorylaimida. The phylogenetic resolution offered by full-length small subunit ribosomal DNA (SSU rDNA) sequences within these two orders is highly different. Notwithstanding several discrepancies between morphology and SSU rDNA-based systematics, Mononchida families (indicated here as M1¿M5) are relatively well-supported and, consequently, family-specific DNA sequences signatures could be defined. Apart from Nygolaimidae and Longidoridae, the resolution among Dorylaimida families was poor. Therefore, a part of the more variable large subunit rDNA (¿ 1000 bp from the 5'-end) was sequenced for 72 Dorylaimida species. Sequence analysis revealed a subclade division among Dorylaimida (here defined as D1¿D9, PP1¿PP3) that shows only distant similarity with `classical¿ Dorylaimid systematics. Most subclades were trophically homogeneous, and ¿ in most cases ¿ specific morphological characteristics could be pinpointed that support the proposed division. To illustrate the practicability of the proposed molecular framework, we designed primers for the detection of individual subclades within the order Mononchida in a complex DNA background (viz. in terrestrial or freshwater nematode communities) and tested them in quantitative assays (real-time polymerase chain reaction). Our results constitute proof-of-principle for the concept of DNA sequence signatures-based monitoring of stress sensitive nematode families in environmental sample

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

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 other, most 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, PCR 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 specie

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

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 other, most 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, PCR 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 specie