Identification and characterisation of a novel chitosan formulation in its ability to induce resistance against Botrytis cinerea
We tested the water-soluble chitosan-based commercial formulation ChitoPlant, from hereafter termed chitosan, in its capacity to induce resistance against the fungal pathogen B. cinerea . Treatments of chitosan demonstrated that this elicitor successfully triggers resistance in tomato (Fig 1a), Arabidopsis (Fig 1b) and aubergine (Fig S1) against B. cinerea . In tomato, chitosan significantly decreased necrotic lesion size in all concentrations compared with control plants (Fig 1a). The resistance phenotype induced by chitosan had a dose-dependent effect at the two high concentrations (1% and 0.1%), however, the lowest concentration (0.01%) induced a level of resistance in between 0.1% and 1% treatments. In Arabidopsis, chitosan treatment resulted in induced resistance in a concentration-dependent manner, with 1% having the strongest effect (Fig 1b). In aubergine, chitosan treatment resulted in differences in lesion diameter in all concentrations compared to water-treated control plants (Fig S1), however, post-hoc analysis demonstrated that 0.1% was the most effective concentration.
We then tested whether chitosan induces callose deposition in a similar manner to other chitosan formulations (Luna et al., 2011). Plants were treated with increasing concentrations of chitosan one day before aniline blue staining. In both plant species, treatments with chitosan resulted in a direct induction of callose. The lowest concentrations of 0.001% and 0.01% in tomato and Arabidopsis, respectively, triggered the strongest effect (Fig 1c and d).
To determine any antifungal effect of chitosan, different concentrations were tested on B. cinerea hyphal growth in vitro and compared to different concentrations of the fungicide Switch (Syngenta). Whereas all concentrations of Switch arrested pathogen growth, only 0.1% concentration of chitosan or higher had an antifungal effect (Fig S2). However, the lowest concentration of chitosan tested (0.01 %) had no antifungal effect compared to the control. This shows a concentration threshold for chitosan-direct antifungal activity against B. cinerea . Since 0.01% chitosan had no antifungal effect, but reduced B. cinerea lesions and induced callose formation, this concentration was selected for more in-depth analysis.