Background

Plants naturally live with various soil bacteria in the rhizosphere, which refers to a thin layer of soil adhering to the roots. While some rhizobacteria have no observable effects on plants, others are either pathogens that cause detrimental effects or growth-promoting rhizobacteria (PGPR) that promote plant vigor (Mendes et al., 2013; Olanrewaju et al., 2019). The capacity of bacterial pathogens or PGPR to impact plant growth is tightly correlated with their level of bacterial root colonization. Therefore, the investigation of bacterial root colonization is an important stepping stone to understanding the belowground plant-bacteria interactions.

The abundance of bacteria strain can be assessed by visualization of fluorescence signals by modifying them to express a transgenic marker gene encoding the fluorescent protein such as GFP (Rochat et al., 2010; Krzyzanowska et al., 2012; Saad et al., 2018). The abundance of bacteria strain can also be measured by PCR-based amplification of the bacterial genomic DNA (Maciá-Vicente et al., 2009; Mendis et al., 2018). These two methods detect the bacterial strain of interest regardless of the presence or absence of other bacterial species; nevertheless, both methods have their potential limitations. The fluorescence-assisted detection may be limited by technical difficulties during genetic transformation, by the possibility that the introduction of the transgene may interfere with wild-type bacterial behavior, and by the risks posed to environmental safety. On the other side, PCR-based bacterial detection requires that levels of the target DNA templates be above the PCR detection limits, and that the target sequence be specific enough to represent the bacterial strain of interest only.

In addition to the fluorescence-based and the PCR-based methods, root-associated bacteria can also be quantified by in vitro culturing. This increases the detection limits, followed by counting the colony-forming units (CFUs), if the bacterial strain of interest is culturable with an artificial growth medium. With proper experimental setups, the method of counting CFUs is efficient, affordable, reliable, and reproducible for quantification of root-associated bacteria. By using Arabidopsis thaliana and B. megaterium YC4-R4 as a model system for studying plant-bacteria interactions, herein we describe a protocol for bacteria quantification based on counting CFUs from in vitro bacterial cultures. This protocol, which includes an optional genetic fingerprinting step by Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR, has been applied to different bacterial species in studies where plants were either grown in artificial medium or in soil, and under control or stress conditions (Vílchez et al., 2020). In addition to studying root-associated bacteria, this methodology can be applied to studies involving other types of plant organs or other types of bacteria-host systems under optimized conditions.