| There are numerous important agricultural pests in Lepidoptera noctuidae,such as Helicoverpa armigera,Spodoptera frugiperda,Spodoptera exigua and Spodoptera litura.These pests are widely distributed in the field and have miscellaneous feeding habits.they can course a devastating impact on agricultural production.Bt formulation based on Bacillus thuringiensis and Transgenic Bt-modified crops have been developed,and have already achieved remarkable results in controlling Lepidopteran pests.However,long-term use has also led to resistance evolution problems in some pests.The successful application of Bt gave us a sufficient proof that the use of entomopathogenic bacteria for pest control has great application potential,and the evolution of pest resistance to Bt has prompted plant protection researchers to seek new entomopathogenic bacteria instead of Bt for sustainable biological control of pests.The lack of efficient insecticidal strains is an important factor restricting the development of entomopathogenic bacteria.In our previous studies,an unreported insect pathogenic bacterium(Enterobacter cancerogenus)had been isolated from the dead body of H.armigera larvae,which is named HaEc1.The insecticidal toxicity of HaEcl to different field populations of H.armigera and various lepidopteran pests was detected by bioassay.FISH technology was used to locate the process of HaEcl invasion into the midgut and fat body of H.armigera and S.frugiperda larvae,and evaluate its application potential in pest control.The results can provide bacterial resources and theoretical basis for microbial control of H.armigera and a variety of Lepidoptera pests.The main results are listed as follows.1.Detection of virulence of Enterobacter cancerogenus to field populations of cotton bollworm and other lepidopteran pestsThis paper detected the virulence for pathogenic bacterium HaEcl to various field populations of H.armigera and larvae of different lepidopteran insects(S.frugiperda,S.litura,Plutella xylostella and S.exigua)with bioassay.The results showed that HaEcl showed high virulence to different field populations of cotton bollworm.The LC50 was lower than 1 × 109cfu/mL.Although there were significant differences in the susceptibility of larvae from different field populations to HaEc1.HaEcl showed high toxicity to larvae of S.frugiperda,S.litura,P.xylostella,and S.exigua,LC50 was lower than 0.6×109 cfu/ml.When the concentration of HaEcl was 0.89×109cfu/mL.the corrected mortality of S.exigua and S.frugiperda was close to 90%,and that of larvae of S.litura was about 75%,and that of P.xylostella was the lowest,is about 50%.The above results showed that the HaEcl had higher insecticidal toxicity to different field populations of H.armigera and four Lepidoptera pests.it has the potential to be developed as a broad-spectrum bacterial insecticide against Lepidoptera pests.2.FISH detection of the process of E.cancerogenus invasing into the larval tissues of Noctuidae pestsIn this paper,FISH technique was used to detect the invasion process of HaEcl strain into the midgut and adipose tissue of H.armigera and S.frugiperda.The results showed that obvious bacterial fluorescence could be detected in midgut and adipose tissue of H.armigera larvae after feeding on HaEcl strain for 2-12 hours.After feeding HaEc1 for 2 hours,the extent of bacterial infection in midgut cells was the deepest,and the bacterial signal surrounded the nucleus tightly,which indicated that the bacteria might invade the cytoplasm.At this time,the fluorescent signals of the bacteria detected in the fat body cells were arranged in a regular circle,which implied that the bacteria might take the fat droplets in the fat body cells as the nutrition source.It also showed that HaEcl could cross the intestinal wall cells and enter the hemolymph rapidly,and the fatbody cells distributed in the blood cavity were used as targets.Over time,the bacteria continued to invade,leading to a gradual decrease in the number of midgut cells,while the fat body cells became more and more loose and eventually disintegrated.After S.frugiperda feeding on HaEcl,the invasion process and pathogenicity of the larvae were similar to that of the cotton bollworm.However,due to the late ingestion of HaEc1 by the sixth instar larvae after starvation,the bacterial invasion process was detected 4-12 hours after the larvae fed on HaEcl strain.Compared with the midgut cells of H.armigera,the midgut cells of the S.frugiperda larvae are smaller but more closely arranged.HaEcl also rapidly invades the midgut cells and adipocytes in the blood lumen,causing abnormalities in the midgut and fatbody tissues.The above results indicate that HaEcl entered the intestinal cavity of Lepidoptera Noctuidae larvae through the mouth,could rapidly infect the midgut cells,penetrate the intestinal wall and enter the body cavity,and plunder the nutrition inside the fat body cells.It is consistent with its effective oral insecticidal activity and has the potential to be developed as a bacterial insecticide. |
