Soil Biology and Soil Health Partnership Project 3: Molecular approaches for routine soil-borne disease and soil health assessment – establishing the scope

This review is part of a suite of integrated projects (Soil Biology and Soil Health Partnership) specifically aimed at addressing the AHDB and BBRO Soils Programme call – “Management for Soil Biology and Soil Health”. This project is designated Project 3 within WP 1 (Benchmarking and Baselining; see Figure 1). This review specifically aimed to:  Summarise available knowledge on procedures to sample field soils to undertake DNA analysis of the presence and composition of microbial communities and their functions to provide indicators of soil health.  Evaluate standard tools for use in routine sampling and molecular analysis of soil quality so that their value can be demonstrated to growers and agronomists during and beyond the current Soil Biology and Soil Health Research Partnership.  Establish full lists of molecular markers that can be used to quantify: (a) Soil-borne pathogens for use in prediction of crop disease; (b) Indicators of good soil health which can influence crop yield and value. Procedures for sampling soil and extracting DNA from the sample have been reviewed. There are no standardised sampling methods, but it is general practice to take composite samples by mixing multiple cores from the surface to 10-30 cm depth. Corers should be cleaned and flamed between collection of each separate set of composite samples. A sample size of at least 200-500g is recommended. The number and spatial arrangement of samples and sub-samples required depends on the expected distribution of the target. For unknown target distributions, it has been suggested that the area is divided into evenly-sized grids with at least 2 composite samples per grid. An internationally recognised standard (ISO 11063:2012) describes a procedure for direct isolation of DNA from soil, suitable for further analysis using qPCR and high throughput sequencing methods, but this does not include procedures for subsequent DNA purification and is only suitable for small soil samples. Procedures more suitable for direct extraction and purification of total DNA from composite soil samples of 200-500g are described in the Appendices to this report. Various molecular techniques have been used for analysis of soil quality, including methods based on polymerase chain reaction (PCR), microarrays, DNA fingerprinting (DGGE and T-RFLP) and DNA sequencing. Two approaches are considered most suitable for routine analysis of taxonomic or functional markers; quantitative PCR (qPCR) for detection and quantification of specific markers and next generation high throughput sequencing for analysis of whole soil communities. The range of molecular markers that have been used to investigate the taxonomy and function of individual target organisms and communities of organisms in soil are described in full. These include taxon-specific markers, mainly based on selected DNA sequences from within ribosomal (rDNA) or mitochondrial (cytochrome oxidase) DNA loci. Functional markers in genes expressing key enzymes involved in 1 carbon, nitrogen, phosphorus and sulphur cycling are also described. Markers are also listed that have been used to assess soils for presence and activity of other key bioindicators of soil health, including mycorrhizal fungi, nitrogen-fixing microorganisms, plant growth promoting bacteria, biocontrol agents, nematode assemblages and plant pathogens. Some technical challenges remain to be fully overcome in the application of these technologies to ensure a representative and unbiased analysis of soil microbiological communities and their function. These include further standardisation of procedures for sampling, extracting and purifying DNA from soils, improved consistency in the choice of markers to be used in the analyses and the use of appropriate internal controls that ensure accuracy of data interpretation. The high cost of molecular analysis also remains a constraint to its routine application.