Markerless gene deletion in Ralstonia solanacearum based on its natural transformation competence.

Ralstonia solanacearum species complex (RSSC) is a group of Gram-negative bacterial pathogen capable of infecting numerous plants and crops, causing severe vascular wilt diseases. Functional analysis of the genes associated with bacterial virulence is critical for elucidating the molecular mechanisms that govern the bacterial pathogenicity. To this end, an efficient gene deletion method would be of great help. In this study, we set to develop an efficient and simple markerless gene deletion method by exploiting its natural transformation competence and the FLP/FRT recombination system. We found that natural transformation using PCR products provided much higher transformation frequency than the plasmid-based triparental mating and electroporation. We thus generated the gene deletion fusion PCR fragments by incorporating the upstream and downstream DNA fragments of the target gene and an antibiotic resistance gene flanked by FRT sites, and delivered the PCR products into R. solanacearum cells through natural transformation. Using this method, we knocked out the epsB and phcA genes, which are associated with exopolysaccharide (EPS) biosynthesis and regulation, respectively, in several R. solanacearum strains isolated from different host plants at a frequency from 5 (1E-08) to 45 (1E-08). To remove the antibiotic marker gene, the plasmid expressing the FLP enzyme was introduced into the above knockout mutants, which enabled removal of the marker gene. The effective combination of natural transformation and the FLP/FRT recombination system thus offers a simple and efficient method for functional study of putative virulence genes and for elucidation of R. solanacearum pathogenic mechanisms.

Burkholderia gladioli CGB10: A Novel Strain Biocontrolling the Sugarcane Smut Disease.

In this study, we isolated an endophytic Burkholderia gladioli strain, named CGB10, from sugarcane leaves. B. gladioli CGB10 displayed strong inhibitory activity against filamentous growth of fungal pathogens, one of which is Sporisorium scitamineum that causes sugarcane smut, a major disease affecting the quality and production of sugarcane in tropical and subtropical regions. CGB10 could effectively suppress sugarcane smut under field conditions, without itself causing any obvious damage or disease, thus underscoring a great potential as a biocontrol agent (BCA) for the management of sugarcane smut. A toxoflavin biosynthesis and transport gene cluster potentially responsible for such antifungal activity was identified in the CGB10 genome. Additionally, a quorum-sensing gene cluster was identified too and compared with two close Burkholderia species, thus supporting an overall connection to the regulation of toxoflavin synthesis therein. Overall, this work describes the in vitro and field Sporisorium scitamineum biocontrol by a new B. gladioli strain, and reports genes and molecular mechanisms potentially involved.

Pseudomonas sp. ST4 produces variety of active compounds to interfere fungal sexual mating and hyphal growth.

Sexual mating of compatible sporida is essential for Sporisorium scitamineum to form dikaryotic mycelia and then cause infection on sugarcane. Our previous work identified a Pseudomonas sp. ST4 from a soil sample, which showed a promising biocontrol potential by inhibiting the mating of S. scitamineum sporida and hyphal growth. In this study, we set to isolate the active compounds from Pseudomonas sp. ST4 through solid fermentation. High-performance liquid chromatography (HPLC) separation coupling with bioassay showed that Pseudomonas sp. ST4 produced a range of antimicrobial compounds. Two of the major components were purified following acetate extraction, silica gel and HPLC separation. Nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS) analysis identified these active compounds are 4-hydroxybenzaldehyde and indole-3-carbaldehyde respectively. Further analysis showed that the former compound only inhibited the hyphal growth of the fungus at a concentration of 3 mM, while the latter interfered the fungal sexual mating at a concentration of 0.6 mM and affected hyphal growth at a concentration of 2 mM. Treatment of corn plants with 3 mM indole-3-carbaldehyde significantly inhibited corn smut infection, with a control rate up to 94%. Further analysis of the structure and activity relationship revealed that indole has a much stronger inhibitory activity against the fungal sexual mating than indole-3-carbaldehyde. The results from this study provide new agents for control and prevention of the sugarcane smut disease, and the active compounds could also be used to probe the molecular mechanisms of fungal sexual mating.

Biocontrol of Sugarcane Smut Disease by Interference of Fungal Sexual Mating and Hyphal Growth Using a Bacterial Isolate.

Sugarcane smut is a fungal disease caused by Sporisorium scitamineum, which can cause severe economic losses in sugarcane industry. The infection depends on the mating of bipolar sporida to form a dikaryon and develops into hyphae to penetrate the meristematic tissue of sugarcane. In this study, we set to isolate bacterial strains capable of blocking the fungal mating and evaluate their potential in control of sugarcane smut disease. A bacterial isolate ST4 from rhizosphere displayed potent inhibitory activity against the mating of S. scitamineum bipolar sporida and was selected for further study. Phylogenetic analyses and biochemical characterization showed that the isolate was most similar to Pseudomonas guariconensis. Methanol extracts from minimum and potato dextrose agar (PDA) agar medium, on which strain ST4 has grown, showed strong inhibitory activity on the sexual mating of S. scitamineum sporida, without killing the haploid cells MAT-1 or MAT-2. Further analysis showed that only glucose, but not sucrose, maltose, and fructose, could support strain ST4 to produce antagonistic chemicals. Consistent with the above findings, greenhouse trials showed that addition of 2% glucose to the bacterial inoculum significantly increased the strain ST4 biocontrol efficiency against sugarcane smut disease by 77% than the inoculum without glucose. The results from this study depict a new strategy to screen for biocontrol agents for control and prevention of the sugarcane smut disease.