Xenorhabdus nematophila HB310 secreted the insecticidal protein toxin complex (Tc). The chi60 and chi70 chitinase genes are located on the gene cluster encoding Tc toxins. To clarify the insecticidal activity of chitinases and their relationship with Tc toxins, the insecticidal activity of the chitinases was assessed on Helicoverpa armigera. Then, the chi60 and chi70 genes of X. nematophila HB310 were knocked out by the pJQ200SK suicide plasmid knockout system. The insecticidal activity of Tc toxin from the wild-type strain (WT) and mutant strains was carried out. The results demonstrate that Chi60 and Chi70 had an obvious growth inhibition effect against the second instar larvae of H. armigera with growth-inhibiting rates of 81.99% and 90.51%, respectively. Chi70 had a synergistic effect with the insecticidal toxicity of Tc toxins, but Chi60 had no synergistic effect with Tc toxins. After feeding Chi60 and Chi70, the peritrophic membrane of H. armigera became inelastic, was easily broken and leaked blue dextran. The Δchi60, Δchi70 and Δchi60-chi70 mutant strains were successfully screened. The toxicity of Tc toxins from the WT, Δchi60, Δchi70 and Δchi60-chi70 was 196.11 μg/mL, 757.25 μg/mL, 885.74 μg/mL and 20,049.83 μg/mL, respectively. The insecticidal activity of Tc toxins from Δchi60 and Δchi70 was 3.861 and 4.517 times lower than that of Tc toxins from the WT, respectively, while the insecticidal activity of Tc toxins from the Δchi60-chi70 mutant strain almost disappeared. These results indicate that the presence of chi60 and chi70 is indispensable for the toxicity of Tc toxins.

Foxtail millet (Setaria italica), a vital cereal crop in China, serves as both a staple food and forage source but is threatened by rust disease caused by Uromyces setariae-italicae (Usi), leading to severe yield and quality losses. The NAM, ATAF1/2, and CUC2 (NAC) transcription factor family represents one of the largest plant-specific regulatory gene families, playing pivotal roles in development and stress responses. However, the functional relevance of NAC genes in foxtail millet’s defense against this pathogen remains unexplored. Here, we systematically analyzed 33 SiNAC genes from the foxtail millet genome. Phylogenetic analysis classified these genes into 11 subgroups, while chromosomal mapping localized them to nine chromosomes unevenly. Promoter analysis identified stress- and plant hormone-related cis-elements, suggesting functional diversity. Expression profiling analysis showed that most SiNAC genes exhibit tissue-specific expression patterns. Quantitative real-time PCR (qRT-PCR) results indicated that 30 genes responded to Usi infection, with 17 showing a strong association with rust resistance. Three resistance-associated genes demonstrated transactivation activity and nuclear localization, indicating their regulatory function in defense responses. This study provides both mechanistic insights into SiNAC-mediated rust resistance and potential targets for molecular breeding in foxtail millet.

MAPK plays an important role in plant growth and development regulation, biotic and abiotic stress responses, and hormone signal transduction. In order to elucidate the biological function of the SiMPK genes in foxtail millet, we identified the SiMPK family members in the genome and analyzed the distribution, structure, evolution, and its expression characteristics in response to different stresses. In this study, the SiMPK gene family members were identified in the genome-wide level using the amino acid sequences of conserved domains and specific TXY motifs of MAPK proteins between foxtail millet and rice. The protein physicochemical property, phylogenetic evolution, chromosome distribution, gene structure, protein conserved motif, promoter cis-acting regulatory elements, and collinearity were analyzed. The relative expression patterns of SiMPK genes in the different tissue, under the biotic stresses of Uromyces setariae-italicae Yoshino and Ostrinia furnacalis and with different hormone treatments were analyzed by qRT-PCR. The results showed that a total of 15 SiMPK genes were identified, and the encoded proteins contained 220-611 amino acids, the relative molecular weight ranged from 25.77 kD to 69.63 kD, and the isoelectric point ranged from 5.46 to 9.34. Phylogenetic analysis showed that SiMPK genes were divided into four groups. Group A, B, and C contained TEY motifs, and group D contained TDY motifs. SiMPK genes were distributed on chromosomes 1, 3, 4, 5, 8, and 9, and contained 3-11 exons. All SiMPK proteins contained motif 1 and motif 2. A number of cis-acting elements related to stress, hormones and plant growth and development were predicted in the promoter regions of the SiMPK genes. Most genes had obvious tissue expression specificity. Except for SiMPK21-2 and SiMPK6, the other members had obvious responses to 1 to 3 kinds of stresses, such as Uromyces setariae-italicae Yoshino infection, Ostrinia furnacalis feeding, and SA and MeJA treatments. The results laid a theoretical foundation for further research on the function of SiMPK genes in the biotic stresses of disease and pest in foxtail millet.

Foxtail millet blast, caused by Pyricularia oryzae, is a widespread airborne disease that severely threatens the sustainable production and industrial development of foxtail millet. Developing eco-friendly and non-toxic management strategies has been widely acknowledged as a core prerequisite for establishing sustainable disease control systems. In this study, a bacterial strain NR2065B9 with prominent in vitro antagonistic activity against P. oryzae was isolated from healthy foxtail millet rhizosphere soil. Taxonomic identification via morphological characterization and 16S rRNA gene sequence homology analysis confirmed that this strain belonged to the species Pseudomonas silesiensis. Dual culture assays revealed that strain NR2065B9 exerted a significant inhibitory effect on the mycelial growth of P. oryzae. Detached-leaf inoculation assays demonstrated that strain NR2065B9 could remarkably restrict the expansion of blast lesions on foxtail millet leaf tissues. Field trials confirmed that NR2065B9 application could reduce foxtail millet blast severity compared with the controls. Collectively, these findings indicated that the antagonistic strain NR2065B9 possessed great application potential in the biocontrol of foxtail millet blast, and could be further exploited as a promising microbial resource for the development of biocontrol agents against this disease.