When a plant is infected by a pathogen, endogenous immune responses are initiated. When the initiation of these defense responses is induced by a pathogen-associated molecular pattern (PAMP) of a pathogen, it is called PAMP-triggered immunity (PTI). Previous studies have shown that Bacillus amyloliquefaciens PMB05 can enhance PTI signals and improve disease control of bacterial soft rot and wilt in Arabidopsis thaliana. In the context of controlling bacterial wilt disease, the involvement of a mitogen-activated protein kinase (MAPK) signaling pathway has been established. Nevertheless, it remains unclear whether this pathway is also required for B. amyloliquefaciens PMB05 in controlling bacterial soft rot. In this study, A. thaliana ecotype Columbia (Col-0) and its mutants on a MAPK pathway-related pathway were used as a model and established that the ability of B. amyloliquefaciens PMB05 to control soft rot requires the participation of the MAPK pathway. Moreover, the enhancement of disease resistance by PMB05 is highly correlated with the activation of reactive oxygen species generation and stomata closure, rather than callose deposition. The spray inoculation method was used to illustrate that PMB05 can enhance stomatal closure, thereby restricting invasion by the soft rot bacterium. This control mechanism has also been demonstrated to require the activation of the MAPK pathway. This study demonstrates that B. amyloliquefaciens PMB05 can accelerate stomata closure via the activation of the MAPK pathway during PTI, thereby reducing pathogen invasion and achieving disease resistance against bacterial soft rot.

PAMP-triggered immunity (PTI) is the first layer of plant defense response that occurs on the plant plasma membrane. Recently, the application of a rhizobacterium, Bacillus amyloliquefaciens strain PMB05, has been demonstrated to enhance flg22_Pst- or harpin-triggered PTI response such as callose deposition. This PTI intensification by PMB05 further contributes to plant disease resistance to different bacterial diseases. Under the demand for rapid and large-scale screening, it has become critical to establish a non-staining technology to identify microbial strains that can enhance PTI responses. Firstly, we confirmed that the expression of the GSL5 gene, which is required for callose synthesis, can be enhanced by PMB05 during PTI activation triggered by flg22 or PopW (a harpin from Ralstonia solanacearum). The promoter region of the GSL5 gene was further cloned and fused to the coding sequence of gfp. The constructed fragments were used to generate transgenic Arabidopsis plants through a plant transformation vector. The transgenic lines of AtGSL5-GFP were obtained. The analysis was performed by infiltrating flg22_Pst or PopW in one homozygous line, and the results exhibited that the green fluorescent signals were observed until after 8 h. In addition, the PopW-induced fluorescent signal was significantly enhanced in the co-treatment with PMB05 at 4 h after inoculation. Furthermore, by using AtGSL5-GFP to analyze 13 Bacillus spp. strains, the regulation of PopW-induced fluorescent signal was observed. And, the regulation of these fluorescent signals was similar to that performed by callose staining. More importantly, the Bacillus strains that enhance PopW-induced fluorescent signals would be more effective in reducing the occurrence of bacterial wilt. Taken together, the technique by using AtGSL5-GFP would be a promising platform to screen plant immunity-intensifying microbes to control bacterial wilt.

Cabbage is an important vegetable crop worldwide. In Taiwan, during cabbage production, bacterial soft rot caused by Pectobacterium carotovorum subsp. carotovorum often leads to significant yield losses. Aligning with the Sustainable Development Goals, there is a high demand for sustainable disease control strategies. Silicates are considered to be effective elicitors in activating plant defense responses and are reported to improve resistance to certain plant diseases. Bacillus amyloliquefaciens PMB05 fermentation liquid has been shown to enhance plant immunity and control many bacterial diseases. The supplementation of silicates to the PMB05 fermentation liquid may further improve its efficacy to control bacterial soft rot in cabbage. This study evaluated the effects of sodium metasilicate and potassium silicate on PMB05-mediated plant immune responses and disease control. Initial assays confirmed that treatment with B. amyloliquefaciens PMB05 suspension significantly increased HrpN-triggered reactive oxygen species (ROS) generation and callose deposition; moreover, PMB05 treatment alone reduced bacterial soft rot severity by 39.7%. When combined with B. amyloliquefaceins PMB05 fermentation liquid, sodium metasilicate at 2000 μM further enhanced ROS generation and callose deposition by 100% and 133%, respectively, compared to the treatment of PMB05 alone (p < 0.05). In contrast, potassium silicate exhibited inconsistent effects on ROS production, with both 500 and 1000 µM concentrations significantly reducing ROS generation by 26% and 38%, respectively, while none of the tested concentrations affected callose deposition (p < 0.05). Lastly, disease severity assessments in cabbage inoculated with P. carotovorum subsp. carotovorum PCCSB1 revealed that B. amyloliquefaciens PMB05 fermentation liquid was able to reduce bacterial soft rot symptoms by 60.3%. Supplementation with 1500 and 2000 µM sodium metasilicate further decreased disease severity by 77.9% and 76.4%, respectively (p < 0.05). Although the supplementation of potassium silicate also significantly reduced disease severity compared to P. carotovorum subsp. carotovorum PCCSB1 alone, it was less effective than PMB05 fermentation alone. Overall, these results demonstrate that sodium metasilicate enhances the biocontrol activity of B. amyloliquefaciens PMB05 by further intensifying plant immune responses. This approach may broaden the large-scale use of B. amyloliquefaciens PMB05 fermentation liquid for sustainable soft rot management in cabbage, although the stability and cost-effectiveness of sodium metasilicate under field conditions still require validation.