Background

Most Pseudomonas strains secrete exopolysaccharides, such as alginate, which is an important matrix molecule for biofilm formation (Hentzer et al., 2001; Nivens et al., 2001). Biofilm formed by the human pathogen P. aeruginosa plays important roles in its virulence and antibiotic resistance, and contributes to acute or chronic infections (Donlan and Costerton, 2002).

To date, various methods have been reported for biofilm characterization and quantification. Originally, biofilms were detected in microtiter plates made of polystyrene or polypropylene (O'Toole and Kolter, 1998; Merritt et al., 2005). During the growth of P. aeruginosa on a surface, the expression of genes involved in extracellular polysaccharide synthesis is induced (Davies et al., 1993; Davies et al., 1995), which promotes the adherence of cells to the surface. Crystal violet specifically stains the bacterial cells, and has been developed as a widely used dye for bacterial biofilm (George et al., 1998). Some recent studies have analyzed biofilm structure in a flow chamber coupled with confocal laser scanning microscopy (Sternberg and Tolker-Nielsen, 2006; Chua et al., 2016).

Biofilms formed by P. syringae strains have also been found in plant tissues (Osman et al., 1986; Fakhr et al., 1999; Preston et al., 2001). Alginate produced by P. syringae is an important polymer for P. syringae biofilm formation and contributes to its virulence and fitness, indicating its importance in plant-pathogen interaction (Preston et al., 2001; Engl et al., 2014). The formation of P. syringae and P. fluorescens biofilms can also be measured using crystal violet staining in microwell plates (Carezzano et al., 2017; Zhu et al., 2018; Patange et al., 2019).

In this study, we modified and applied the current method of P. aeruginosa biofilm measurement to P. syringae (Kong et al., 2015; Zhao et al., 2016; Shao et al., 2018). We present an economic, rapid and visual biofilm detection protocol that combines the use of borosilicate glass tubes and crystal violet staining methods, which have been efficiently used in our recent studies (Wang et al., 2018; Wang et al., 2019; Xie et al., 2019) for visualizing the biofilm of the model plant pathogen P. syringae.