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.

Background: Entomopathogenic nematodes (EPNs) have long been used for controlling soil-dwelling insects. Steinernema carpocapsae HB310, previously showed a high virulence against many pests including Agrotis ipsilon Hufnagel (Lepidoptera: Noctuidae). Due to the lack of durable formulations, up until now, S. carpocapsae HB310 has thus far been prevented from use in large-scale farming. The present study aimed to get a better EPNs capsule formulation suitable for long-term storage and effective application. Results: An improved EPNs capsule formulation, herein named: Capsule-C was prepared by the following composition: Solution I: 18% glycerol, 0.075% formaldehyde, 1% sodium alginate, 0.2% xanthan gum, 0.5% potassium sorbate, 9% glucose, 2% fructose, 2% sucrose, and the remainder was distilled water. The nematodes suspension was added to the alginate mixture in 2 × 10⁴ IJs/mL; Solution II: 18% glycerol, 0.075% formaldehyde, 0.5% calcium chloride, 0.5% potassium sorbate, with the remainder being distilled water. After storage for 180 days at 16 °C and 100% RH, the survival rate of nematodes in Capsule-C was 75.68 ± 0.48% and the nematodes caused 82.33 ± 1.45% mortality in the 5th instar larvae of Galleria mellonella. A. ipsilon larvae preferred to chew and ingest Capsule-C due to the addition of the glucose compound. The feeding rate of A. ipsilon larvae on Capsule-C reached to 100% within 24 h and the larval mortality of A. ipsilon was 90.48 ± 6.35%. Conclusion: EPNs-containing capsules were as effective as sprayed EPNs in water solution at killing A. ipsilon. These results will provide ideas to acquire a stable and efficient EPNs capsule formulation and further promote the application of environmental friendly biological pesticides.