Study On The Cotoxicity And Innate Immunity Mechanism Of Trans-Anethole And Metarhizium Flavoride To Pieris Rapae

Pieris rapae is one of the most widely distributed and resistant pests in cruciferous plants.Metarhizium flavoride is an important biocontrol fungus that can infect agricultural pests such as Lepidoptera Pieridae in the field,and has a wide range of application prospects in biological control.Trans-Anethole is the main ingredient in fennel essential oil,and studies have shown that Trans-Anethole has insecticidal potential and is a good substitute for botanical insecticide.In order to clarify the compatibility of Trans-Anethole with a highly virulence entomopathogenic fungi of M.flavoride Mf82 and their cotoxicity to P.rapae Linne（Lepidoptera:Pieridae）（alone and in combinations）,we evaluated the effects of Trans-Anethole(LC₅₀)and different concentrations conidial suspensions of M.flavoride Mf82 mixed in different ratios on the growth of M.flavoride and determined their co-toxicity against 2nd instar larvae of P.rapae in the laboratory.In the long evolutionary process,insects have gradually formed unique and efficient innate immune response to various stresses,and the insect immune system include cellular immunity and humoral immunity.In this paper,we determined the changes hemocyte counts and phenoloxidase activity in the innate immunity of M.flavoride and Trans-Anethole after acting on P.rapae,which will help us to further understand the pathogenic mechanism of biological insecticides acting on the host,and laid the foundation for the future development of biological pesticides and application technology.The main results are as follows:1.M.flavoride and Trans-Anethole showed good insecticidal activities against P.rapae.After 5 days of treatment,the corrected mortality rate of M.flavoride was91.89%at 1.5×10⁷conidia/m L.After 48 hours of treatment,the corrected mortality rates of 5.00 mg/m L and 7.50 mg/m L of Trans-Anethole against P.rapae were92.68%and 97.56%,respectively.2.The mixture of the two has a certain promotion effect on the growth of M.flavoride,the treatment ratios of 5∶5,4∶6 and 3∶7 significantly promoted the growth of mycelia,and the promotion effect was significant as the proportion of Trans-Anethole in the mixture increased,the conidia germination rate is also higher than that after the two are mixed at 2∶8 and 1∶9.3.The mortality of the mixture varied with the mixing ratios,the mixture of the two in mixing ratios of 5∶5,4∶6 and 3∶7 treatments have the higher mortality than that of the two in mixing ratios of 2∶8 and 1∶9 treatments,the co-toxicity coefficients were 287,108 and 83,respectively,indicating synergistic and additive effects.The LT₅₀values combined with mixing ratios of 5:5,4∶6 and 3∶7 were also shorter than that of M.flavoride alone.4.The hemocyte counts of P.rapae is directly proportional to the concentration of M.flavoride.The LC₅₀of trans-Anethole was mixed with different concentrations of M.flavoride conidial suspensions to treat the test insects.There were differences in the number of hemocyte between the treatment groups at different time periods,the hemocyte counts under the mixed treatment of fungus and insecticide increased with the increase of the proportion of Trans-Anethole in the mixture,the number of hemocyte in the mixed treatment was 5∶5﹥4∶6﹥3∶7.After 12 hours of treatment,the cellular immunity began to be activated and the number of hemocyte increased in each treatment group,after 24 hours of treatment,the number of hemocyte decreased but remained at a high level.During the whole infection process,the hemocyte counts increased first and then decreased,and the cellular immunity was activated first and then inhibited.The difference of P.rapae hemocyte counts were affected by the concentration of fungus and the dose of trans-anisole.5.M.flavoride infected P.rapae,the phenoloxidase activity increased with the increase of the strain concentrations,which were significantly higher than that of the control groups.The phenoloxidase activity first increased and then decreased.The fungus inhibited the phenoloxidase activity in P.rapae.The phenoloxidase activity in the hemolymph were significantly higher than that of M.flavoride and the control groups alone after combined treatment with fungus and insecticide for different times.The phenoloxidase activity of LC₅₀of Trans-Anethole mixed with M.flavoride was5∶5﹥4∶6﹥3∶7.The phenoloxidase activity of each mixed treatment group showed a trend of first increasing and then decreasing.The combination of fungus and insecticide further inhibited the immune response of P.rapae.In summary,this study initially explored the insecticidal activity of M.flavoride and Trans-Anethole against the 2nd instar larvae of P.rapae,and the LC₅₀of Trans-Anethole can accelerated the infection of M.flavoride to P.rapae,shorten the infection time.Preferentially select the mixing ratios with good compatibility and high co-toxicity coefficient,that is,Trans-Anethole(LC₅₀)and M.flavoride Mf82 are combined with mixing ratios of 5∶5,4∶6 and 3∶7.Inhibition of innate immunity of P.rapae by combination of M.flavoride and Trans-Anethole.M.flavoride integrated with Trans-Anethole can be a potential alternative to the chemicals and as an effective component of IPM program against P.rapae.