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.

The effect of temperature on the cotoxicity coefficient (CTC) value was used to evaluate mixture efficacy of different temperature coefficient chemicals from 15 to 35°C by exposing third-instar Apolygus lucorum (Meyer-Dür) to dip-treated asparagus bean pods. The results indicated the joint toxicity of same temperature coefficient insecticide (TCI) types were unaffected by temperature. This means that even when temperatures change, the mixture ratios of the highest CTC values remained the same, and the effect of temperature on the joint toxicity of same TCI types was only on the CTC values. However, the effect of temperature was variable when considering the joint toxicity of different TCI types. The effect of temperature on the joint toxicity of both strong positive and strong negative TCI types was clear, and the highest CTC values of mixture ratios changed with temperature regularly. When comparing the influence of temperature between strong/slight positive/negative insecticides, the results indicated a greater influence of the strong TCI. Paradoxically, the highest CTC value of the imidacloprid and methomyl mixture did not change with temperature changes consistently, even with the variance of imidacloprid ratios, a strong TCI. These results will guide pest managers in choosing the most effective insecticide mixtures for A. lucorum control under given environmental conditions.

Background: Genome sequence analysis (GenBank access No.: FN667742.1) shows that Xenorhabdus nematophila ATCC19061 contains one gene (Xn-cbp) encoding chitin binding protein (Xn-CBP). Objective: The present work aims to clarify the characteristics and function of Xn-CBP from X. nematophila HB310. Methods: In this study, the Xn-cbp gene was cloned and expressed in Escherichia coli BL21 (DE3). Substrate binding assays were performed to explain the ability of Xn-CBP combined with the polysaccharide. The insecticidal toxicity of Xn-CBP against the second-instar larvae of Helicoverpa armigera was determined by feeding method. Besides, the antifungal activity of Xn-CBP against Coniothyrium diplodiella, Verticillium dahlia, and Fusarium oxysporum was tested by spore germination assay and hyphal extension assay. Results: Xn-CBP encoded 199 amino acids with a calculated mass of 28 kDa, which contained a signal peptide and a chitin binding domain. The Bmax and Kd values of Xn-CBP to colloidal chitin were 2.46 and 4.08, respectively. Xn-CBP had insecticidal activity against the H. armigera with a growth inhibition rate of 84.08%. Xn-CBP had the highest spore germination inhibitory effect on C. diplodiella with the inhibition rate of 83.11%. The hyphal growth inhibition rate of Xn-CBP to F. oxysporum, 41.52%, was higher than the other two fungi. Conclusion: The Xn-CBP had the highest binding ability to colloidal chitin and it showed insecticidal activity and antifungal activity. The present study laid a foundation for further exploitation and utilization of X. nematophila.

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.