Valsa pyri is a fatal canker pathogen that causes significant reduction of crop yield in pear orchards. V. pyri invades the trunk phloem, and is difficult to control by chemical treatment. In this work, it was found for the first time that Bacillus subtilis-produced dipicolinic acid (DPA) exhibits antifungal activity against different canker pathogens, including Alteraria alternata, Botryosphaeria dothidea, Rhizoctonia solani, and V. pyri. Growth inhibition of V. pyri was observed at less than 5 mM concentration (pH = 5.6). DPA showed the highest antifungal activity at acidic pH values and in the presence of bivalent metals, such as zinc(II), cobalt(II), and copper(II). Measurement of mRNA expression levels and scanning electron microscope (SEM) observations revealed that DPA causes V. pyri apoptosis via inhibition of chitin biosynthesis and subsequent cell lysis. Interestingly, DPA showed high stability in the pear bark and was able to cross the pear tree bark into the phloem, protecting the internal phases of the pear trunk. In preventive applications, DPA reduced the canker symptoms of V. pyri on Cuigan pear trees by 90%. Taken together, an efficient strategy for the management of V. pyricaused canker disease was developed using a novel antifungal agent, DPA, with strong antifungal activity and particular diffusion properties.

Homologous regulatory factors are widely present in bacteria, but whether homologous regulators synergistically or differentially regulate different biological functions remains mostly unknown. Here, we report that the homologous regulators RpoN1 and RpoN2 of the plant pathogen Xanthomonas campestris pv. campestris (Xcc) play different regulatory roles with respect to virulence traits, flagellar biosynthesis, and basal metabolism. RpoN2 directly regulated Xcc fliC and fliQ to modulate flagellar synthesis in X. campestris, thus affecting the swimming motility of X. campestris.Mutation of rpoN2 resulted in reduced production of biofilms and extracellular polysaccharides in Xcc. These defects may together cause reduced virulence of the rpoN2 mutant against the host plant. Moreover, we demonstrated that RpoN1 could regulate branched-chain fatty acid production and modulate the synthesis of diffusible signal factor family quorum sensing signals. Although RpoN1 and RpoN2 are homologues, the regulatory roles and biological functions of these proteins were not interchangeable. Overall, our report provides new insights into the two different molecular roles that form the basis for the transcriptional specialization of RpoN homologues. K E Y W O R D S flagellar synthesis, sigma factor 54 RpoN, virulence, Xanthomonas campestris pv. campestris S U PP O RTI N G I N FO R M ATI O N Additional supporting information may be found online in the Supporting Information section.

Hypothetical proteins without defined functions are largely distributed in all sequenced bacterial genomes. Understanding their potent functionalities is a basic demand for bacteriologists. Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial leaf blight of rice, is one of the model systems for the study of molecular plant pathology. One-quarter of proteins in the genome of this bacterium are defined as hypothetical proteins, but their roles in Xoo pathogenicity are unknown. Here, we generated in-frame deletions for six hypothetical proteins selected from strain PXO99 and found that one of them (PXO_03177) is required for the full virulence of this strain. PXO_03177 is conserved in Xanthomonas, and is predicted to contain two domains relating to polysaccharide synthesis. However, we found that mutation of this gene did not affect the production or modification of extracellular polysaccharides (EPSs) and lipopolysaccharides (LPSs), two major polysaccharides produced by Xoo relating to its infection. Interestingly, we found that inactivation of PXO_03177 significantly impaired biofilm formation and tolerance to sodium dodecyl sulfate (SDS), both of which are considered to play key roles during Xoo infection in rice leaves. These findings thus enable us to define a function for PXO_03177 in the virulence of Xoo. Furthermore, we also found that the global regulator Clp controls the transcription of PXO_03177 by direct binding to its promoter region, presenting the first cellular regulatory pathway for the modulation of expression of this hypothetical protein gene. Our results provide reference information for PXO_03177 homologues in Xanthomonas.