A novel molecular approach for rapid assessment of soil nematode assemblages – variation, validation and potential applications

Abstract

Summary1. Nematode assemblages are commonly used as an indicator of ecosystem health; however, conventional approaches to assemblage analyses are restricted by time-consuming processing and declining availability of expertise. Molecular methods offer a rapid and cost-effective alternative. 2. We have designed a molecular profiling system, using directed terminal-restriction fragment length polymorphism (dT-RFLP), to characterise nematode assemblages by relative abundance of feeding guilds.3. An arable soil was first characterised by cloning and sequencing of small subunit ribosomal DNA, and an enzyme digest selected to discriminate between feeding guilds. This yielded 14 different terminal-restriction fragments (T-RFs) from the sequence set, assigned to five nematode feeding guilds. 4. Robustness of the dT-RFLP methodology was tested. The greatest amount of variation between replicates occurred at the PCR stage, with little variability between replicate digests from the same PCR product or capillary runs. 5. dT-RFLP revealed changes in assemblage composition owing to organic amendments of dairycattle slurry and municipal green compost. The proportion of microbial feeding nematodes was higher in compost and slurry plots than in the no amendment control in the first sampling after organic amendment. Plant feeding nematodes composed a significantly greater proportion of the control assemblage during the growing season and post-harvest. 6. The increased throughput of molecular analysis compared with microscopy increases the feasibility of studies involving large-scale sampling and makes nematode assemblage analysis more attractive as an indicator of soil health for environmental assessment.

Directed terminal restriction analysis tool (DRAT): an aid to enzyme selection for directed terminal‐restriction fragment length polymorphisms

“…As with all new applications, DT-RFLP tools require thorough validation with clones or type strain DNA to confirm suitability. Donn et al (2011) have used DRAT to aid the design and validation of a DT-RFLP approach for the analysis of free-living soil nematodes.…”

“…For instance, the difference between expected and observed T-RF sizes (between 0AE2 and 4AE4 bp, Table 3) whilst within the previously reported range (Kitts 2001;Kaplan & Kitts 2003;Bukovoska´et al 2010) needs to be understood during the full validation stage of any DT-RFLP application. A more complete example including practical validation can be found in Donn et al (2011) where DRAT has been used to aid the design of an approach to separate nematode groups followed by a complete validation of the suggested digest strategies to select an optimum solution both theoretically and practically.…”

“…This process requires careful validation that has, for example, been undertaken with, phytoplasmas (Hodgetts et al 2007) and free-living nematodes (Donn et al 2011). We refer to this as DT-RFLP (for 'Directed T-RFLP') as the design of the PCR primers and selection of restriction enzyme is carefully directed to allow identification of diagnostic T-RFs for specific target species or groups within samples.…”

Congruence of community structure between taxonomic identification and T-RFLP analyses in free-living soil nematodes

“…Each fluorescently labeled band produced through T-RFLP analyses represents a unique operational taxonomic unit (OTU) (Liu et al 1997). Although each OTU lacks a species-level identification by definition, it is possible to assign such a label to them when the technique is conducted using known cultures (Liu et al 1997;Chen et al 2010) or through novel techniques that integrate sequencing of the DNA from specific individuals (Donn et al 2012). T-RFLP methods have proven useful in cases where a previously (Lott et al 2014) or concurrently (Donn et al 2012) characterized reference community of nematodes exists and when novel methodologies are under examination (Donn et al 2008;Edel-Hermann et al 2008;Wiesel et al 2015).…”

“…Indeed the use of molecular techniques to discern community structure has been aggressively explored (Wiesel et al 2015). Polymerase chain reaction (PCR)-based techniques such as denaturing gradient gel electrophoresis (DGGE) (Foucher et al 2004;Okada and Oba 2008), DNA barcoding/sequencing (Vervoort et al 2012), and terminal restriction fragment length polymorphism (T-RFLP) (Donn et al 2008;Donn et al 2012;Lott et al 2014;Wiesel et al 2015) have been commonly used to address questions of community composition in difficult taxonomic groups (Chen et al 2010). Terminal restriction fragment length polymorphism in particular holds much potential as it is a comparatively inexpensive method, the result of which can quickly be compared between different gel runs (Liu et al 1997;Chen et al 2010).…”