Interactive effects of Pseudomonas entomophila strain 23S and Clavibacter michiganensis subsp. michiganensis on proteome and anti-Cmm compound production.

Pseudomonas entomophila strain 23S is an effective biocontrol bacterium for tomato bacterial canker caused by Clavibacter michiganensis subsp. michiganensis (Cmm); it produces an inhibitory compound affecting the growth of Cmm. In this study, the interactions between pure cultures of P. entomophila 23S and Cmm were investigated. First, the population dynamics of each bacterium during the interaction was determined using the selective media. Second, the amount of anti-Cmm compound produced by P. entomophila 23S in the presence of Cmm was quantified using HPLC. Lastly, a label-free shotgun proteomics study of P. entomophila 23S, Cmm, and a co-culture was conducted to understand the effects of the interaction of each bacterium at the proteomic level. Compared with the pure culture grown, the total number of proteins decreased in the interaction for both bacteria. P. entomophila 23S secreted stress-related proteins, such as chaperonins, peptidases, ABC-transporters and elongation factors. The bacterium also produced more proteins related with purine, pyrimidine, carbon and nitrogen metabolisms in the presence of Cmm. The population enumeration study revealed that the Cmm population declined dramatically during the interaction, while the population of P. entomophila 23S maintained. The quantification of anti-Cmm compound indicated that P. entomophila 23S produced significantly higher amount of anti-Cmm compound when it was cultured with Cmm. Overall, the study suggested that P. entomophila 23S, although is cidal to Cmm, was also negatively affected by the presence of Cmm, while trying to adapt to the stress condition, and that such an environment favored increased production of the anti-Cmm compound by P. entomophila 23S. SIGNIFICANCE: Pseudomonas entomophila strain 23S is an effective biocontrol bacterium for tomato bacterial canker caused by Clavibacter michiganensis subsp. michiganensis (Cmm); it produces an inhibitory compound affecting the growth of Cmm. In this study, secreted proteome of pure cultures of P. entomophila 23S and Cmm, and also of a co-culture was first time identified. Furthermore, the study found that P. entomophila strain 23S produced significantly higher amount of anti-Cmm compound when the bacterium was grown together with Cmm. Co-culture enhancing anti-Cmm compound production by P. entomophila 23S is useful information, particularly from a commercial point of view of biocontrol application, and for scale-up of anti-Cmm compound production.

Plant Associated Rhizobacteria for Biocontrol and Plant Growth Enhancement.

Crop disease remains a major problem to global food production. Excess use of pesticides through chemical disease control measures is a serious problem for sustainable agriculture as we struggle for higher crop productivity. The use of plant growth promoting rhizobacteria (PGPR) is a proven environment friendly way of controlling plant disease and increasing crop yield. PGPR suppress diseases by directly synthesizing pathogen-antagonizing compounds, as well as by triggering plant immune responses. It is possible to identify and develop PGPR that both suppress plant disease and more directly stimulate plant growth, bringing dual benefit. A number of PGPR have been registered for commercial use under greenhouse and field conditions and a large number of strains have been identified and proved as effective biocontrol agents (BCAs) under environmentally controlled conditions. However, there are still a number of challenges before registration, large-scale application, and adoption of PGPR for the pest and disease management. Successful BCAs provide strong theoretical and practical support for application of PGPR in greenhouse production, which ensures the feasibility and efficacy of PGPR for commercial horticulture production. This could be pave the way for widespread use of BCAs in agriculture, including under field conditions, to assist with both disease management and climate change conditions.

Biocontrol Rhizobacterium Pseudomonas sp. 23S Induces Systemic Resistance in Tomato (Solanum lycopersicum L.) Against Bacterial Canker Clavibacter michiganensis subsp. michiganensis.

Tomato bacterial canker disease, caused by Clavibacter michiganensis subsp. michiganensis (Cmm) is a destructive disease and has been a serious concern for tomato industries worldwide. Previously, a rhizosphere isolated strain of Pseudomonas sp. 23S showed antagonistic activity toward Cmm in vitro. This Pseudomonas sp. 23S was characterized to explore the potential of this bacterium for its use in agriculture. Pseudomonas sp. 23S possesses ability to solubilize inorganic phosphorus, and to produce siderophores, indole acetic acid, and hydrogen cyanide. The strain also showed antagonistic activity against Pseudomonas syringae pv. tomato DC 3000. A plant assay indicated that Pseudomonas sp. 23S could promote growth of tomato seedlings. The potential of treating tomato plants with Pseudomonas sp. 23S to reduce the severity of tomato bacterial canker by inducing systemic resistance (ISR) was investigated using well characterized marker genes such as PR1a [salicylic acid (SA)], PI2 [jasmonic acid (JA)], and ACO [ethylene (ET)]. Two-week-old tomato plants were treated with Pseudomonas sp. 23S by soil drench, and Cmm was inoculated into the stem by needle injection on 3, 5, or 7 days post drench. The results indicated that plants treated with Pseudomonas sp. 23S, 5 days prior to Cmm inoculation significantly delayed the progression of the disease. These plants, after 3 weeks from the date of Cmm inoculation, had significantly higher dry shoot and root weight, higher levels of carbon, nitrogen, phosphorus, and potassium in the leaf tissue, and the number of Cmm population in the stem was significantly lower for the plants treated with Pseudomonas sp. 23S. From the real-time quantitative PCR (qRT-PCR) analysis, the treatment with Pseudomonas sp. 23S alone was found to trigger a significant increase in the level of PR1a transcripts in tomato plants. When the plants were treated with Pseudomonas sp. 23S and inoculated with Cmm, the level of PR1a and ACO transcripts were increased, and this response was faster and greater as compared to plants inoculated with Cmm but not treated with Pseudomonas sp. 23S. Overall, the results suggested the involvement of SA signaling pathways for ISR induced by Pseudomonas sp. 23S.