Inoculum concentration and moisture chamber incubation period for inoculation of Xanthomonas citri subsp. citri, the causal agent of citrus canker

Xanthomonas citri subsp. citri is a plant bacterium that causes citrus canker, an important disease in several citrus-growing areas. Due to its relevance, this pathosystem has been frequently investigated, requiring suitable protocols of inoculation for symptom reproduction. This study aimed to evaluate the effect of the concentration of X. citri and the duration of the moisture chamber period on the incidence and severity of citrus canker symptoms under greenhouse conditions. Inoculum concentrations ranging from 10⁰ to 10⁸ cfu/mL were evaluated using both infiltration and spray-inoculation methods. The effect of the moisture chamber on the development of citrus canker lesions was assessed for 24 or 48 h before and/or after inoculation. The minimum concentrations required to infect 100% of the inoculated leaves using infiltration and spray-inoculation were 10⁴ and 10⁷ cfu/mL, respectively. Intermediate severity levels, e.g., 3.2 to 11.4 and 2.5 to 6.7 lesions/cm², were observed using 10³ – 10⁴ cfu/mL for infiltration and 10⁶ – 10⁷ cfu/mL for spray-inoculation. This severity level is suitable when the number of lesions in a given treatment needs to be determined. Conversely, lower or higher inoculum concentrations resulted in severities either extremely low, with no or very few lesions, or excessively high, with coalesced lesions forming a large necrotic area, respectively. A moisture chamber period of 24 h after spray-inoculation was sufficient for reproducing citrus canker lesions under greenhouse conditions. Suitable inoculum concentrations and the use of a moisture chamber after spray-inoculation of X. citri are critical for the successful reproduction of citrus canker symptoms.