Biological Control of Distrbution Grapholita molesta Thtrough Immunological Features

Abstract

The oriental fruit moth, Grapholita molesta (Busck) is a key pest of tree fruit of Europe, Asia, America, Africa, Australia, and New Zealand, which makes a big damage to apple trees, pear tree and the stone fruit of the peach, plum, apricot, nectarine, cherry and so on. The hidden damage of larvae makes their prevention and control difficult and traditional chemical method is rarely effective. Meanwhile with the increasing demand for food safety and awareness of environmental protection, biological control method to this pest has attracted more and more people's attentions. Beauveria bassiana is one of the most widely studied and used entomopathogenic fungi, which can also infect and kill the oriental fruit moth as a biological control agent. The entomopathogenic fungi with a wide range host and they are harmless to the environment, human and animal. Using entomopathogenic fungi to control pests has many advantages and it has been an important part in biological control of pests. However, entomopathogenic fungi as a better alternate control method still with some disadvantages, such as easy to be affected by environmental conditions. In order to make good use of entomopathogenic fungi in the future, it is necessary to deeply understand their living conditions and infection mechanism to insects. Entomopathogenic fungi can invades the insects from the body wall through contact directly, but also can through the digestive tract, stomata and wounds and other ways into the insect body. But insects have evolved a strong innate immune system to protect themselves from infection by the pathogens and adverse conditions. When insects are infected by entomopathogenic fungi, their innate immune system will firstly be activated. And the insects will resist the infection by their immune response, which will lead to the reduction of infection efficiency and the control effect. So, it is necessary to study the immune response of insects introduced by entomopathogenic fungi, and it has been a study hotspot in pest control. The immune ability of insects to entomopathogenic fungi directly affects the infection efficiency, which is an area of active research in the field of entomopathogenic fungi currently. In this paper, the optimal concentration and infection mode of B. bassiana on G. molesta larvae were selected under laboratory conditions. Secondly, the transcriptome of G. molesta after infected by B. bassiana were analyzed，and the immune-related genes of G. molesta larva against B. bassiana were screened out. Thirdly, the peptidoglycan recognition proteins (GmPGRP-SC), β-1,3-glucan recognition proteins (GmBGRP) and serine protease inhibitor genes (GmSerpin-2 and GmSerpin-3) involved G. molesta larva in immune response were identified by molecular methods. We carried out phylogenetic analysis based on amino acid sequences to identify homologous protein in G. molesta belonging to the same clade as that encoding the target proteins. The spatio-temporal expression patterns of the selected target genes in the larva of G. molesta after infection with B. bassiana were assessed by qPCR. Moreover, we used nanoparticles mediation the GmSerpin-2 and GmSerpin-3 genes, which effectively increasing the interference efficiency of RNAi. Finally, we have a good grasp of the occurrence rule of oriental fruit moth in Xinxiang area by investigation of the damage of adult and larva in peach orchard. Meanwhile, the field efficacy test of five different biological pesticides showed that Bt, Spinetoram, Methoxyfenozide, Celastrus angulatus MaXim and B. bassiana all have better control effect on oriental fruit moth. But the control effect of Bt and B. bassiana are relatively lower than other three agents. We need to do more future research to improve their effectiveness. This study laid a foundation for the exploration of new biological control targets of G. molesta IPM system and the enhancement of B. bassiana for effectively biological control. The main results and conclusions are as follows: Laboratory Evaluation of the effect of B. bassiana on the vital activity of G. molesta: G. molesta infected by B. bassiana through cuticular infection had higher corrected mortality and better weight inhibition than that of digestive tract infection. Meanwhile, B. bassiana at concentration of 1×107 conidia/mL may have potential to be used as control measure against G. molesta in fruit orchards. Transcriptome analysis of G. molesta larvae 24 h after infected by B. bassiana: The second-generation high-throughput transcriptome sequencing technology were used, the transcriptomic profile of G. molesta larva infected by B. bassiana after 24 h were analyzed, the differentially expressed genes (DEGs) were screened and annotated. Totally, 1,755 DEGs were obtained, with 965 up-regulated and 790 down-regulated genes. The fold change (log2 ratio) of the gene expression ranged from -14.224 to 10.718. We focused on the up-regulated genes in G. molesta infected by B. bassiana, and 14 genes related to immune response of G. molesta induced by B. bassiana were selected and quantified by qRT-PCR method, and 10 genes were significantly up-regulated. Finally, 1,755 DEGs were enriched in 1,965 GO terms, among which 107 GO terms were significantly enriched, including 62 Biological processes, 7 Cellular components and 38 Molecular functions. These results indicated the G. molesta has changed its physiological and biochemical state as defense responses against B. bassiana. Characterization and functional analysis of Immune recognition gene GmPGRP-SC, GmBGRP, GmSerpin-2 and GmSerpin-3: The complete sequence of GmPGRP-SC gene, GmBGRP, GmSerpin-2 and GmSerpin-3 are obtained and characterized. They have been submitted to NBCI GenBank respectively, and the GenBank accession number is MW773839, ON055286, OQ359960 and OQ35996. The gene sequences, structures, physical and chemical properties were analyzed. To explore the spatio-temporal expression patterns of each gene, the qPCR data form different tissues and developmental stages were analyzed, besides we analyzed the specific functions of each gene. In order to better understand the molecular interaction mechanism between B. bassiana and G. molesta, the effect of B. bassiana on the regulation of immune-related gene expression were analyzed. Finally, we successfully silenced the target gene using RNAi technology, and the effects of target gene silencing on larvae ability to resist fungal infection were analyzed, which laid a good foundation for further improving the control effect of entomopathogenic fungi. In addition, we analyzed the effects of GmBGRP gene silencing on Toll immune signaling pathway related gene expression and PPO enzyme activity. And effectively improve the interference efficiency of GmSerpin-2 and GmSerpin-3 genes mediated by nanoparticles M2L and NP5. Investigation on the occurrence and dynamics of G. molesta in peach orchard and evaluation on the control of five different biological pesticides: According to the survey results in the Xinxiang area, overwintered larva began to pupate in late March, the pupal stage was 10-20 days, and first-generation adult appeared in early April, which reaching a high level around 4, May. Then, the number of G. molesta began to increase continuously with the rise of temperature. The occurrence of male adult G. molesta in Xinxiang has no obvious rule, and with serious overlap generations. There is no obvious boundary between each generation, especially between the 2~3 generation and 3~4 generation. Larvae of oriental fruit moth had boring into the peach shoots from April 18. Meanwhile the larvae have transferring harm habit to peach shoot. The middle May is the key time for controlling of G. molesta in Xinxiang area. The damaged peach shoots can be cut off manually and taken out of the orchard for centralized destruction in order to reduce the occurrence of overwintering generation. The damaged shoots rate by G. molesta in peach orchard was higher than that in nectarine orchard. And in the same peach orchard, the damaged shoots are also different due to different planting densities. The shoots damage is higher with planting density of 4×4 meters than that with planting density of 3×3 meters. The five biological agents all had adverse effects on the growth of peach trees. The results of this study indicating that all the five biological agents were safe for the growth of peach trees. And the 5 biological control agents all have certain control effects on G. molesta. On day 7 in treatment groups, the control effec of the five agents was 56.3% ~ 86.4%. Its control effect was 63.1% ~ 80.1% on day 14 of treatment. Among them, 60 g/L pinetoram suspension 2000 times liquid and 240 g/L Methoxyfenozide suspension 5 000 times liquid have better control effectiveness with 86.4% and 74.6% control effect on day 7 in the treatment groups. Moreover, the control effect of these two agents was still more than 78% on day 14. The control effects of 0.2% Celastrus angulatus MaXim and 10 billion spores B. bassiana were 68.25% and 66.8% respectively on day 7 in treatment groups, and the control effects of these two agents were 61.27% and 67.1% on day 14 in treatment groups which indicating that the two agents had relatively good quick and lasting effect on the control of G. molesta. In summary, all these results lay a foundation for better understanding of the immune mechanism between entomopathogenic fungi and insects, and effectively improve the control effect of entomopathogenic fungi, which lay a solid foundation for the biological control of G. molesta pests. Moreover, the study of immune-related genes, in order to find more suitable target genes, which can also be applied to the development of other biological control agents and improve their control effect. The results providing strong support for better biological control through immune characters of oriental fruit moth.