Black rot disease is a serious bacterial disease that harms vegetable crops of the Brassica genus (especially cabbage plants) worldwide. The causal agent, Xanthomonas campestris pv. campestris (Xcc), is a seed-borne pathogen that primarily infects seedlings. Previous studies suggest that the bacterial strain, Bacillus amyloliquefaciens PMB05, can intensify the plant immune responses of cabbage against black rot disease and reduce disease occurrence. In plant immunity, several reactions occur during a pathogen attack, but the elevation of calcium ion concentration in plant cells is essential in the induction of plant defense responses. Therefore, this study aims to investigate whether disease control of black rot disease in cabbage plants can be improved by integrating calcium carbonate in the formulation for preparing B. amyloliquefaciens PMB05. Firstly, we found the addition of calcium carbonate in the formulation revealed to have significantly increased the cell and endospore populations of B. amyloliquefaciens PMB05 in the fermentation liquids. To increase the convenience of disease control in the field, these fermentation liquids were converted to powder form for subsequent analysis. Results revealed that the grown seedlings from seeds, mixed with PMB05 powders, significantly intensified plant immune responses and improved black rot disease control. We further compared distinct seed treatments using one PMB05 powder to evaluate its feasibility in field application. The results demonstrated that the disease control efficacy and yield of cabbage were significantly improved in the seed treatment with the powder (SD-160C2) to 56.46% and 5.91%, respectively, at 10 weeks post transplanting. Interestingly, the seed treatment combined with a calcium-containing commercial fertilizer spraying treatment did not increase the control efficacy of black rot disease, but it significantly increased the weight of cabbages after harvest. We concluded that the seed treatment with calcium carbonate-containing Bacillus amyloliquefaciens PMB05 powder is an efficient way to control black rot disease in cabbage.

Sweet pepper is an important vegetable in the world. Bacterial leaf spot, caused by the pathogen Xanthomonas perforans, is a limiting factor that significantly reduces the quality and yield of sweet peppers. The use of chemical fungicides is currently the main disease-control method for bacterial leaf spot disease. It is important to develop an eco-friendly biocontrol method by using antagonistic microorganisms. Bacillus amyloliquefaciens PMB04 has strong antagonistic effects against pathogens and can inhibit the occurrence of diseases. B. amyloliquefaciens PMB04 has the potential for the development of a disease-control product. Primarily, PMB04 contained a strong inhibitory effect against all isolated X. perforans strains. In the inoculation assay, the severity of bacterial leaf spot disease on sweet peppers was reduced by PMB04 bacterial suspensions. To increase the convenience of field applications in future prospects, the development of the PMB04 fermentation liquid was carried out using different ratios of brown sugar and yeast extract in a 30 L fermentation tank. The results exhibited that the fermentation liquid of the 3-1 and 2-1 formulas obtained the highest bacterial population in a 30 L fermentation tank. The fermentation liquid of the 0.5-0.5 formula was the most stable formula for two different conditions in terms of a consistent bacterial population and sporulation. In addition, the 200-fold dilution of the 3-1 and 0.5-0.5 fermentation liquids revealed the best control efficacy on bacterial leaf spot disease of sweet peppers. Additionally, the results of the 0.5-0.5 fermentation liquid (PMB4FL) with different dilution concentrations also demonstrated that the 200- and 500-fold dilutions had the best control efficacy. To understand the effect of commonly used copper-containing fungicides on sweet peppers on the application of microbial agent PMB4FL, the effects of copper hydroxide and tribasic copper sulfate on the growth of X. perforans strains and B. amyloliquefaciens PMB04 were assayed. The results exhibited that the above two fungicides did not have any inhibitory effect on the growth of PMB04 but had a strong inhibitory effect on the X. perforans strain. In the follow-up control experiment, the treatment of copper hydroxide had no synergistic effect with PMB4FL to control bacterial leaf spot disease. We concluded that the use of the PMB4FL fermentation liquid alone on the leaves could effectively control the occurrence of bacterial leaf spots in sweet pepper crops.

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.

Sweet pepper is an important vegetable in the world. Bacterial leaf spot caused by the pathogen Xanthomonas perforans, is a limiting factor that significantly reduces the quality and yield of sweet pepper To control this disease, the use of chemical fungicides is currently the main disease control method. Thus, we want to develop an alternative method by using antagonistic microorganisms. Under this demand, Bacillus amyloliquefaciens PMB04 has strong antagonistic effects against pathogens and can inhibit the occurrence of diseases. B. amyloliquefaciens PMB04 has the potential for the development of a disease control product. Primarily, PMB04 revealed to contain a strong inhibitory effect against all isolated X. perforans strains. In the inoculation assay, the severity of bacterial spot disease on sweet peppers was reduced by PMB04 bacterial suspensions. To increase the convenience of field application for future prospects, the development of PMB04 fermentation liquid was carried out with different ratios of brown sugar and yeast extract in a 30-liter fermentation tank subsequently. Results exhibited that the fermentation liquid of 3-1 formula obtained the highest bacterial population in a 30-liter fermentation tank. The fermentation liquid of 0.5-0.5 formula was the most stable formula under two different conditions in terms of consistent bacterial population and sporulation. In addition, the 200-fold dilution of 3-1 and 0.5-0.5 fermentation liquids revealed best control efficacy on bacterial leaf spot of sweet pepper. Additionally, the results of the 0.5-0.5 fermentation liquid (PMB4FL) with different dilution concentrations also showed that the 200- and 500-fold dilutions had the best control efficacy. To understand the effect of commonly used copper-containing fungicides on sweet peppers on the application of microbial agent PMB4FL, the effects of copper hydroxide and tribasic copper sulfate on the growth of X. perforans strains and B. amyloliquefaciens PMB04 were assayed. The results exhibited that the above two fungicides did not have any inhibitory effect on the growth of PMB04, but had a strong inhibitory effect on the X. perforans strain. In the follow-up control experiment, the treatment of copper hydroxide had no synergistic effect with PMB4FL to control bacterial leaf spot. We concluded that the use of PMB4FL fermentation liquid alone on the leaves can effectively control the occurrence of bacterial leaf spot in sweet pepper crops.

Bacterial fruit blotch (BFB), caused by Acidovorax citrulli, is a major constraint in watermelon (Citrullus lanatus) production worldwide. As a seed-borne disease, reducing the initial inoculum through seed treatment is critical for effective management. This study evaluated the biocontrol potential of Bacillus amyloliquefaciens PMB05 as a seed treatment to enhance resistance against BFB. Seed treatment and spray inoculation assays revealed that the PMB05 fermentation broth provide superior diseased resistance compared with its powder formulation. To optimize seed treatment performance, the addition of 5% maltodextrin improved bacterial adherence on seed surfaces without affecting germination. In our previous research, the co-treatment of sodium metasilicate and PMB05 enhanced plant immunity and resistance to soft rot in cabbage. In this study, we also incorporated this co-treatment to seed coating. This combination markedly strengthened plant immune responses and reduced BFB incidence, achieving a 70.2 % control efficacy. Overall, the co-treatment of PMB05 fermentation broth and sodium metasilicate in seed coating represents a promising, eco-friendly strategy for managing BFB in watermelon production.