Biosynthesis of silver nanoparticles (AgNPs) using endophytic bacteria is a safe alternative to the traditional chemical method. The purpose of this research is to biosynthesize AgNPs using endophytic bacterium Bacillus endophyticus strain H3 isolated from onion. The biosynthesized AgNPs with sizes from 4.17 to 26.9 nm were confirmed and characterized by various physicochemical techniques such as Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), UV-visible spectroscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) in addition to an energy dispersive spectrum (EDS) profile. The biosynthesized AgNPs at a concentration of 40 μg/mL had a strong antifungal activity against rice blast pathogen Magnaporthe oryzae with an inhibition rate of 88% in mycelial diameter. Moreover, the biosynthesized AgNPs significantly inhibited spore germination and appressorium formation of M. oryzae. Additionally, microscopic observation showed that mycelia morphology was swollen and abnormal when dealing with AgNPs. Overall, the current study revealed that AgNPs could protect rice plants against fungal infections.

Most endolysins of dsDNA phages are exported by a holin-dependent mechanism, while in some cases endolysins are exported via a holin-independent mechanism. However, it is still unclear whether the same endolysins can be exported by both holin-dependent and holin-independent mechanisms. This study investigated the lysis system of OP2-like phage X2 infecting Xanthomonas oryzae pv. oryzae, causing devastating bacterial leaf blight disease in rice. Based on bioinformatics and protein biochemistry methods, we show that phage X2 employs the classic "holin-endolysin" lysis system. The endolysin acts on the cell envelope and exhibits antibacterial effects in vitro, while the holin facilitates the release of the protein into the periplasm. We also characterized the role of the transmembrane domain (TMD) in the translocation of the endolysin across the inner membrane. We found that the TMD facilitated the translocation of the endolysin via the Sec secretion system. The holin increases the efficiency of protein release, leading to faster and more efficient lysis. Interestingly, in E. coli, the expression of either holin or endolysin with TMDs resulted in the formation of long rod shaped cells. We conclude that the TMD of X2-Lys plays a dual role: One is the transmembrane transport while the other is the inhibition of cell division, resulting in larger cells and thus in a higher number of released viruses per cell.

Rice bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is responsible for a significant reduction in rice production. Due to the small impact on the environment, biogenic nanomaterials are regarded as a new type of antibacterial agent. In this research, three colloids of silver nanoparticles (AgNPs) were synthesized with different biological materials such as Arctium lappa fruit, Solanum melongena leaves, and Taraxacum mongolicum leaves, and called Al-AgNPs, Sm-AgNPs and Tm-AgNPs, respectively. The appearance of brown colloids and the UV-Visible spectroscopy analysis proved the successful synthesis of the three colloids of AgNPs. Moreover, FTIR and XRD analysis revealed the formation of AgNPs structure. The SEM and TEM analysis indicated that the average diameters of the three synthesized spherical AgNPs were 20.18 nm, 21.00 nm, and 40.08 nm, respectively. The three botanical AgNPs had the strongest bacteriostatic against Xoo strain C2 at 20 μg/mL with the inhibition zone of 16.5 mm, 14.5 mm, and 12.4 mm, while bacterial numbers in a liquid broth (measured by OD₆₀₀) decreased by 72.10%, 68.19%, and 65.60%, respectively. Results showed that the three AgNPs could inhibit biofilm formation and swarming motility of Xoo. The ultrastructural observation showed that Al-AgNPs adhered to the surface of bacteria and broke the bacteria. Overall, the three synthetic AgNPs could be used to inhibit the pathogen Xoo of rice bacterial leaf blight.

Phage therapy is a promising biocontrol management on plant diseases caused by bacterial pathogens due to its specificity, efficiency and environmental friendliness. The emergence of natural phage-resistant bacteria hinders the application of phage therapy. Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of the devastating bacterial leaf blight disease of rice. Here, we obtained a spontaneous mutant C2R of an Xoo strain C2 showing strong resistance to the lytic phage X2. Analysis of the C2R genome found that the CDS2289 gene encoding glycosyltransferase acquired a frameshift mutation at the 180th nucleotide site, which also leads to a premature stop mutation at the 142nd amino acid. This mutation confers the inhibition of phage adsorption through the changes in lipopolysaccharide production and structure and bacterial surface morphology. Interestingly, glycosyltransferase-deficient C2R and an insertional mutant k2289 also showed reduced virulence, suggesting the trade-off costs of phage resistance. In summary, this study highlights the role of glycosyltransferase in interactions among pathogenic bacteria, phages and plant hosts, which provide insights into balanced coevolution from environmental perspectives.

Plants grow together with microbes that have both negative and positive impacts on the host, while prokaryotes are in turn also hosts for viruses, co-evolving together in a complex interrelationship. Most research focuses on the interaction of either bacterial pathogens interacting with the plant host, or the impact on viruses on their pathogenic bacterial hosts. Few studies have investigated the co-evolution of bacterial pathogens with their host plants as well as with their bacterial viruses. In this work, we aimed to identify the genes that were associated with both phage sensitivity and host pathogenicity of the bacterium Xanthomonas oryzae pv. oryzae (Xoo), which is the most important bacterial rice pathogen. Using the Tn5 transposon mutation technology, we created a library of Xoo strain C2 comprising 4524 mutants, which were subsequently tested for phage infectability. The phage infection tests showed that less than 1% of the mutants (n = 36) were resistant to phage infection, which was attributed to the Tn5 insertion in 19 genes. Interestingly, three out of 19 genes that conveyed resistance to the phage resulted in reduced pathogenicity to rice seedlings compared to the wild type. We identified three genes involved in both phage infection and bacterial virulence, which were studied by knockout mutants and complementation experiments. All of the three knockout mutants were resistant to infection by phage X2, while the complemented strains restored the susceptibility to the bacterial virus. Surprisingly, the genes are also essential for pathogenicity, which we confirmed by single knockout mutants corresponding to the Tn5 mutants. All three genes are involved in lipopolysaccharide synthesis, thus changing the cell envelope surface molecule composition. Our work shows a possible balance in terms of the connection between bacterial virulence and phage resistance, supporting the deployment of phages for the biocontrol of plant pathogens.

Xanthomonas oryzae pv. oryzae (Xoo) is a significant bacterial pathogen responsible for outbreaks of bacterial leaf blight in rice, posing a major threat to rice cultivation worldwide. Effective management of this pathogen is crucial for ensuring rice yield and food security. In this study, we identified and characterized a novel Xoo phage, ZP3, isolated from diseased rice leaves in Zhejiang, China, which may offer new insights into biocontrol strategies against Xoo and contribute to the development of innovative approaches to combat bacterial leaf blight. Transmission electron microscopy indicated that ZP3 had a short, non-contractile tail. Genome sequencing and bioinformatic analysis showed that ZP3 had a double-stranded DNA genome with a length of 44,713 bp, a G + C content of 52.2%, and 59 predicted genes, which was similar to other OP1-type Xoo phages belonging to the genus Xipdecavirus. ZP3’s endolysin LysZP was further studied for its bacteriolytic action, and the N-terminal transmembrane domain of LysZP is suggested to be a signal–arrest–release sequence that mediates the translocation of LysZP to the periplasm. Our study contributes to the understanding of phage–Xoo interactions and suggests that phage ZP3 and its endolysin LysZP could be developed into biocontrol agents against this phytopathogen.