TABLE 2.

The most frequently used methods to assess rhizobial competitiveness for nodulation.

Method	Principle	Pros	Cons	Example of latest studies using this method to assess competitiveness
Antibiotic markers Graham, 1969; Josey et al., 1979	Scoring rhizobial infection by plating nodule samples on suitable selected media	• No need for sophisticated equipment; • No need to genetically modify the strains; • Competitiveness of strain not affected.	• Limited number of strains can be evaluated; • Mixed nodules are often missed; • Relies on strain viability and culturability in different antibiotics which becomes very labor intensive.	Bogino et al., 2011; Laguerre et al., 2012; Bourion et al., 2018
Strain-specific fingerprints De Bruijn, 1992; Laguerre et al., 2003	Targeting specific plasmid profiles or genes; afterwards patterns of the resulting PCR products are analyzed	• Suitable as a first step to classify closely related strains in large collections.	• Requires strict standardization of reaction parameters; • Complex comparative analysis of banding patterns; • Does not allow the identification of mixed nodules.	Lardi et al., 2017; Irisarri et al., 2019; Pastor-Bueis et al., 2019
Sequential double staining to detect gusA and celB Sessitsch et al., 1996	Scoring of nodule infection by color detection after enzymatic reactions	• Allows efficient scoring of single or double nodule infections without requiring sophisticated equipment; • Stable insertion of marker genes, ideal for ecological experiments; • Can be used in large-scale assays and in the presence of an unmarked background population.	• Only possible to score two tagged strains simultaneously; • Toxic buffers are needed for the enzymatic reaction to distinguish nodule occupancy; • Not possible to recover viable rhizobia from stained nodules.	Sánchez-Cañizares and Palacios, 2013; Ferguson et al., 2020; Mendoza-Suárez et al., 2020,Westhoek et al., 2021
Fluorescent proteins Stuurman et al., 2000	Detection of dual fluorescence by microscopy	• High resolution even at single cell level; • Viable rhizobia can be recovered from nodules.	• Only a few nodules per plant or a small plant sample size can be assessed due to microscopy complexity.	Checcucci et al., 2016; Regus et al., 2017; Bellabarba et al., 2020
NGS of full genome, core genes or accessory genes	Analysis of partial or full genome to identify individual strains	• Large numbers of rhizobial strains can be assessed simultaneously; • Measures relative strain diversity; • Tracks dynamic changes in strain populations.	• DNA samples are pooled, loosing information of individual strain-nodule relation; • Pre-sequencing of the genomes from the strains is needed; • Complex sequencing data analysis.	Ji et al., 2017; Burghardt et al., 2018; Boivin et al., 2020; Moeskjær et al., 2020
NGS of synthetic DNA fragments Mendoza-Suárez et al., 2020	Introduced unique barcodes (IDs) are detected by NGS to score bacterial populations in individual nodules	• Large numbers of rhizobial strains can be assessed simultaneously; • Strains not previously isolated and genome-sequenced can be identified; • Information at a nodule level; • Easy identification of mixed nodules; • Simultaneous assessment of competitiveness and effectiveness.	• High-throughput cloning methods are needed and bacteria library preparation; • A blue-light transilluminator is needed to detect GFP nodules from tagged strains vs native strains.	Mendoza-Suárez et al., 2020