The Adaptability Of Cydia Pomonella Granulovirus Response To Strong UV Index Sunlight In Xinjiang Relative To The Biological Control Of The Codling Moth

Cydia pomonella granulovirus(Cp GV) is an important biological agent for codling moth control due to its strong specificity and high pathogenicity to the host while being safe to non-target organisms. The granulovirus is vulnerable to ultra violet(UV) radiation. However a native Cp GV-ZY isolate was found in northwest of China with a strong UV index and surviving in a dry climate. Therefore, there should be some adaptability in this native isolate. The mechanism may be a new one in the research of adaptations of microorganisms facing an extreme environment. This also advances basic research on microbial pesticide development and screening out new granuloviruses that adapt to areas with dry climate and a strong UV index.Collecting the codling moth from the field and rearing it in the lab was the first necessity to provide an abundant source of study specimens. Then the quality of this lab population was assessed by comparing it with field populations. The gene granulin coding the granulin protein that was the main protein of the granule was cloned and a virus micro-detection method using RT-q PCR was used. The proliferation of granulovirus in the larvae of the codling moth and the oriental fruit moth was compared. An UVB irradiation mode was established for accurate research of the relationship between UV and inactivity of the granulovirus. Electron microscopy observation, sunlight irradiation, effect of temperature on inactivation of Cp GV and disintegration of the granule from two granulovirus isolates were conducted. Restriction enzyme tests were also conducted for analysis of differences between the two isolates. RNA-seq was completed for finding differently expressed genes and then these gene expressions including the granulin gene were tested by RT-q PCR. Finally, two inorganic oxides were evaluated for their UV protecting ability, and an insecticidal active material, cantharidin, was tested for determining whether it can be used for biological control of codling moth.1. Quality assessment of Cydia pomonella lab population and proliferation dynamics of Cp GV in two hostsThe laboratory population was generally similar to other laboratory strains although parameters such as survival rate and larval duration were low when compared with field populations of C. pomonella. This shows that some improvement might be carried out in the future for maintaining the lab population. The real-time absolute quantitative PCR reaction was performed using the standard curve method that can be used for detection of granulin gene with 104~1011copies/μL. Cp GV replicated quickly in the host of C. pomonella in comparison with Grapholitha molesta. The proliferation of granulovirus reached plateau on day 3 post-infection in C. pomonella. The LC50 and LC90 for G. molesta were 1000-fold and 7300-fold higher than that for C. pomonella. Research on the proliferation dynamics of granulovirus is a basis for study of granulovirus-host interaction and application of native Cp GV isolate.2. Relationship between granule size and Cp GV’s UV toleranceThe LT50 of granulovirus was 3.75 h under UVB irradiation in the lab. The activity of the granulovirus was significantly affected under ≥60℃ for 2 h, or ≥4h in 40℃. UV was the main factor influencing granulovirus activity under sunlight conditions for a short time(2h). The Cp GV-ZY with big granules was damaged slowly while the Cp GV-M with small granules was cracked quickly under alkaline solution(p H 9). Bioassays showed that the weak alkaline was not the factor that significantly influenced the activity of the granulovirus, but UVB is the main factor. What’s more, only the Cp GV-M isolate was significantly inactivated(72%) when treated with a weak alkaline and UVB. These results show that Cp GV-M is weaker than Cp GV-ZY when undergoing UV irradiation, and there is a positive correlation between granule size and UV tolerance.The expression of the granulin of Cp GV-ZY was fast at the beginning of infection, but it was slower than Cp GV-M when proliferation achieves the plateau period. Totally, no significant difference was found in proliferation between the two granuloviruses, although the total number of granules of Cp GV-ZY was a litter higher than that for Cp GV-M in dead larvae. These results show that production of big granules is not at the cost of a reduction of granule number.3. Restriction enzyme map analysis of viral genomeSome differences were found between the two Cp GV isolates at the genome level. Cp GV-ZY has a sub molecular fragment of Sal I and Eco RI, 7.5 kbp, 14.7kbp, respectively. After comparison with other Cp GV isolates reported in the literature, we speculate that mutations are happening in two enzyme sites, which may be an identification characteristic for Cp GV-ZY.4. Transcriptome analysis of two granulovirus isolates in the midgut of hostThe total RNA of the midgut of larvae infected with the two different Cp GV isolates was sequenced. The results show the reads of granulovirus mapped the Cp GV genome at a relative low level(2.69%~2.85%) in comparison with total reads. Only two differentially expressed genes were found statistically significant and both were up-regulated in Cp GV-ZY compared with Cp GV-M. Gene of orf20 is specific to Cp GV and its function was not found by GO and KEGG annotations. As a result, further work should be focused on its function. Another gene orf91 is a homolog gene that encodes ODV-E25. The gene is a core gene and expressed in the late period, and the protein is located at the envelope of BV and ODV. Semi quantitative PCR showed the gene was highly expressed in Cp GV-M on day 2 post-infection, which is consistent with the proliferation dynamic of Cp GV-M. Due to only one point being chosen for transcriptome analysis, other differentially expressed genes should be paid attention to across the whole infection period.5. Application of UV protectants for granulovirusTwo inorganic oxides Zn O and Ti O2 were tested to determine whether they can protect Cp GV from UV damage. Bioassays showed the mortalities were improved 55.8% and 46.5% in comparison with the control. Semi-field tests indicated both compounds are effective as UV protectants at low concentrations(Zn O with 15mg/m L and Ti O2 with 10mg/m L). This work will advance the research on improvement of granulovirus persistence on the field after its application.6. The toxicology and biochemical characterization of cantharidin on codling mothCantharidin, a natural specific toxin and insect defensive substance, has the two poison effects of touch-kill and stomach poison with LC50 0.125 mg/m L and 0.057 mg/m L, respectively. Neonate larvae exposed to LC10 of cantharidin showed increased glutathione S-transferase activity and significantly reduced the carboxylesterase and cytochrome P450-dependent mixed-function oxidase activities. Field trials demonstrated cantharidin has a significant effect on both the first and second generations of larvae. It may be considered a valuable tool for the control of codling moth in organic agriculture.In summary, a C. pomonella lab population was established and assessed, and the proliferation dynamic of Cp GV in the host was clarified. Granule size was directly related to the virus’ UV tolerance, which was first proved by morphological observation. We also found the native Cp GV isolate that has big granules was not at the cost of reduction of proliferation. Two mutated enzyme sites in the genome of Cp GV-ZY and two up-regulated differentially expressed genes were found, which might be related to the adaptability of native granulovirus response to strong UV index sunlight, but it should be verified in further research. Moreover, relative to the biological control of codling moth, application of UV protectants for granulovirus was studied and insect-derived cantharidin was validated for the pest management.