| Beauveria bassiana is an important entomopathogenic fungus,which has a good biocontrol effect on Lepidoptera,Coleoptera,Homoptera and Hymenoptera and other pests.The interaction mechanism between insects and fungi has always been a hot spot in recent years,but the researches on the mechanism of fungi and their metabolites on insect blood cells are still unclear.In this study,Bombyx mori,Beauveria bassiana and its secondary metabolite Beauverin were used as the research objects.And the effects of Beauveria bassiana strains on the pathogenicity and body length of silkworms were studied by high virulence strain screening,laboratory virulence determination,and bioassay;the effects of Beauveria bassiana and Beauvericin on the number,morphology,activity,nodules and encapsulation of silkworm blood cells were studied by hemacytometer,scanning electron microscopy,transmission electron microscopy and trypan blue staining.The results of this study provide an important theoretical basis for the interaction mechanism between Beauveria bassiana and insects.The main findings are as follows:1.The effect of Beauveria bassiana on the growth and development of silkwormThe TST,FDB and BF strains of Beauveria bassiana were used to infect silkworms.The three strains have no significant virulence to silkworm eggs(P>0.05),but have strong virulence to silkworm larvae.The cumulative mortality of TST strains infecting silkworm larvae was the highest,96.67±5.77%;the lethal time of TST strains infecting silkworm larvae was shortest,3.08d;the time for its mycelium to cover the silkworm epidermis was short relatively,5.29 d.Compared with the control group,the TST strain can significantly inhibit the growth of the body weight and length of the silkworm larvae(P<0.001).The median lethal concentration of the TST strain to the silkworm larvae is 2.25×106spores/ml.2.The effect of Beauveria bassiana on silkworm blood cellsThe TST strain of Beauveria bassiana was used to infect the silkworm.Compared with the control,the number of total circulating blood cell in the silkworm infected with the TST strain of 1×107 spores/ml was significantly reduced at 0h,3h,and 24 h after treated(P<0.05).The number of granulocytes was the lowest at 3h after treated,and the number of plasma blood cells was the lowest at 6h after treated.The number of nodules reached the maximum at 6h.Optical microscope observation showed that the infection of TST strain,blood cells protruded pseudopod-like protrusions to identify and phagocytize invading hyphae in the low-concentration groups(1×105 spores/ml),while a large number of blood cells formed nodules and melaninized cysts to remove pathogen in the high-concentration group(1×107spores/ml).Scanning electron microscope observations showed that the infection of TST strain caused cracks,nicks,tumor-like protrusions and perforations appeared on the surface of the silkworm blood cells,and the cell membrane was destroyed.Transmission electron microscope observation showed that the infection of TST strain caused the cell membrane,nucleus and mitochondria,endoplasmic reticulum and various organelles of silkworm blood cells were destroyed,and vacuoles appeared in the cells.Observation by trypan blue staining showed that the survival rate of blood cells in vivo was the lowest at0.5h after treated,only 28.25%,after the TST strain infecting the silkworm;the survival rate of blood cells in vitro was the lowest at 36 h after treated,only 21.24%,after the TST strain infecting the silkworm.The results showed that the number of blood cells was changed,the morphology of blood cells was destructed,and the activity of blood cells was inhibited after B.bassiana strain TST infecting the silkworm larvae.3.The effect of Beauvericin on the blood cells of silkwormThe lethality experiment showed that Beauvericin(BEA)has a strong virulence to silkworm,with a median lethal concentration(LC50)was 362.36μM.After injected 350μM BEA,the total number of circulating blood cells in silkworm decreased after 12 h,and reached the lowest at 72h;the number of granulocyte in silkworm began to decrease at 24 h,and significantly decreased at 72h(P<0.05);the number of plasma blood cells in silkworm was highest at 12 h,and then decreased,it was the lowest at72 h.A large number of blood cells formed nodules after 48 h treated by 350μM BEA under an optical microscope.Different concentrations of BEA can reduce the survival rate of silkworm in vivo and in vitro cultured blood cells,and there is a dose-dependent relationship,and the survival rate at 400μM was the lowest.BEA could enhance the encapsulation of silkworm blood cells on QFF gel particles.The blackening ratio was significantly higher than that of the control group after 1.5h and 3h injection of BEA(P<0.05),and the blackening ratio of QFF gel particles increased with time,reaching 55.33% at 24 h,which was significantly higher than other times Point(P<0.05).In summary,this study took Beauveria bassiana and BEA as the research objects,and explored their effects on silkworm blood cells.Transmission electron microscopy was used to observe the destruction of inside blood cells caused by Beauveria bassiana TST infecting the silkworm,and to explore the pathogenicity of BEA to the lepidopteran silkworm.The research results will provide a theoretical basis for the interaction mechanism between Beauveria bassiana and insects. |
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