Quantification of free-living nematode communities using qPCR for monitoring soil biological health in agricultural soils

Nematode communities are widely used indicators of soil biological condition, as shifts in their composition reflect changes in the soil food web and soil ecological function. However, reliance on traditional morphological identification has limited the scalability of nematode community assessments. To our knowledge, this study presents the first validated suite of qPCR assays targeting free-living nematode (FLN) indicator taxa enabling large-scale community assessment and quantification using soil environmental DNA (eDNA). Taxa were selected as indicators using multivariate analyses based on responsiveness to agricultural management and environmental variables across 50 sites in Australia's grain-growing regions over three years. Fifteen qPCR assays were developed targeting key trophic groups, including at the order level Dorylaimida (omnivores) and Mononchida (predators), and at the family level for Aphelenchidae and Aphelenchoididae (fungivores), Cephalobidae, Panagrolaimidae, Mesorhabditidae, and Rhabditidae (bacterivores), and Tylenchidae (plant associates). Assay performance was validated by direct comparison with traditional morphological analyses using split soil samples. Strong correlations were observed between the qPCR assays and the morphological (40 taxa) method for individual taxa, overall FLN community structure, and nematode-based indices (NBIs). Importantly, qPCR-derived communities and NBIs detected the same significant treatment effects across selected environmental variables as morphological data. Some limitations were identified for Tylenchidae, indicating a need for improved assay specificity for this taxonomically complex group. The integration of these FLN qPCR assays alongside existing microbial qPCR tests in an Australian commercial soil DNA service, capable of processing biologically relevant soil samples (500 g), will support large-scale monitoring of soil biological health.