Synergistic Mechanism Of Natural Product Citral Incorporated Wtih Guar Gum Film On Rice Brown Planthopper

Brown planthopper,Nilaparvata lugens（St?l）（Hemiptera:Delphacidae）is an important pest that seriously affects the safe production of rice in China.The development of high-efficiency,green and safe prevention and pest control technology is in line with national food security and the strategic demand of“double reduction of pesticides and fertilizer”.Therefore,based on the theory of plant protective film,a natural film-formingubstance with excellent performance were screened out,and by adding active ingredients and optimizing processing technology,a natural product guar gum incorporated with citral film agent（GC film）with synergistic effect on rice brown planthopper was created.Whereafter,the properties of GC film were characterized by scanning electron microscope（SEM）,atomic force microscope（AFM）and fourier transform infrared spectroscopy（FTIR）to clarify its physical and chemical properties.Then,the action mode of GC film on N.lugens and its synergistic mechanism were proved by electrical penetration graph（EPG）and Transcriptomics.In the end,field experiment and indoor experiment were conducted to determine the field control effect of natural product GC film on N.lugens and its safety on rice.The aim is to provide new technology for green prevention and control of N.lugens.The main results are as follows:1.A natural product GC film,which has significant synergistic effect on N.lugens was developed.Taking film-forming property,dilution,adhesion and film-forming effect as screening indexes,43 kinds of film-formingperties substances,such as guar gum,chitosan and sodium alginate,were evaluated.The results showed that natural product guar gum is a good film-forming property with excellent adhesion,so it was selected as the main film-forming material of the film.Taking the mortality of the third instar nymph of N.lugens as an index,13 kinds of plant-derived active substances,such as citral,mandelic acid and tea saponin,were screened out to incorporated wtih guar gum.The results showed that the natural product citral incorporated wtih guar gum had the best effect,and the mortality of N.lugens reached 68.89%after 36 hours treated.The activity of GC film agent with five ratios（w/w:3:1,2:1,1:1,1:2,1:3）against N.lugens was performed with rice stem dipping method.The results showed that the most effective ratio of citral to guar gum was 1:1 with the median lethal concentration(LC₅₀)of 4.30 mg/m L.Therefore,the formula of GC film is 20%citral,20%guar gum,10%Tween-20 and 50%distilled water.2.The physicochemical properties,structural characteristics and the release rule of citral,the main component of the natural product,were expounded.The 100 times dilution multiples of GC film emulsion achieved the minimum p H and luminousness values,and the maximum viscosity values.FTIR、SEM、AFM and other techniques were used to analyze the microstructure of the films,and it was found that citral was enveloped by guar gum in GC film,and the surface of GC film was rougher,with deeper gullies and fewer nodes than guar gum film.Under the conditions of 25℃and50℃,the release rates of citral a and citral b increased gradually with time in 1～30days,but the release rates of citral a and citral b in GC film were lower than those of the control citral essential oil.On the 30th day,the release rates of citral a and citral b in GC film at 25℃were 28.00%and 37.08%respectively,while the release rates of control group were 36.81%and 51.22%respectively.Therefore,GC film can effectively delay the volatilization of citral and increase its stability.3.It was proved that GC film had significant synergistic effect on N.lugens and inhibited its P450 enzyme activity.Co-toxicity coefficient（CTC）method showed that GC film had significant synergistic effect on N.lugens,and its CTC value reached1571.24.The results of feeding choice test showed that the number of N.lugens selected100 dilution multiples of GC film treated rice for feeding was only 16.99%,which was significantly lower than that of the control（83.01%）.Olfactory reaction analysis were consistent with this result.EPG analysis showed that GC film influenced the feeding behavior of N.lugens by disturbing the sense of smell,and the average duration of nonpenetration（np）waveforms was 50.27 min after treated by GC film,longer than that of the control.According to toxicity bioassay,the corrected mortality rate of N.lugens treated by 100 times dilution multiples of GC film emulsion was 69.68%.And after treated for 36 h,the total amount of citral detected in N.lugens was 0.021 mg/kg,and the its P450 enzyme activity treated by different concentration of GC film emulsion was significantly lower than that of the control.Therefore,the GC film can achieve synergistic effect by influencing the feeding behavior of N.lugens and inhibiting its P450 enzyme activity.4.Screening out the crucial candidate genes for synergistic effect of GC film on N.lugens.Total RNA was extracted from the third-instar nymphs of N.lugens that feed on the rcie sprayed with 100 dilution multiples concentrations of GC film-forming emulsion for 48 h.The results of Illumina transcriptome sequencing showed that the expression of 963 genes of N.lugens treated with GC film was significantly different from that of control,including 679 significantly up-regulated genes and 284significantly down-regulated genes.GO enrichment analysis showed that the differential expression was highly enriched in the items of“Localization”and“Oxidation-reduction process”in biological process.Odorant binding protein genes（OBPs）797,and gustatory receptor genes（GRs）/odorant receptor genes（ORs）13110 were significance down differential expression and significance up differential expression,respectively.Meanwhile,there are 19 differential expression genes related to P450,and 7 genes are significantly down-regulated.Among significantly down-regulated genes,gene 19665 is most prominent down-regulated gene,with 0.47 differential expression multiple.The results of real-time quantitative PCR（q RT-PCR）were consistent with those of transcriptome sequencing.According to results described above,we reasoned that,gene797 and gene 19665 are crucial candidate genes for synergistic effect of GC film on N.lugens.The schematic diagram of the action mechanism of GC film on N.lugens was constructed:GC film affected expression of OBPs gene and GRs/ORs,resulting in interference in the recognition and localization of N.lugens.It also affected the expression of detoxification enzyme P450 related genes,through affecting expression of P450 related genes to inhibit the activity of P450 enzyme,which led to the poisoning death of N.lugens.5.It was confirmed that GC film had good control effect on N.lugens and was safe for rice growth.Using spray method,the effects of four different dilution times（800,400,200 and 100 times）of GC film on the control of N.lugens and the growth of rice were studied.The indoor testing results showed that GC film had a good control effect on nymph and adult of N.lugens,for72.90%and 68.77%corrected death efficiency respectively,after treated for 48 h at the 100 dilution multiples concentrations of GC film-forming emulsion.The field control testing results showed that the control effect on N.lugens was still 43.67%after spraying the GC film for 9 days,and there was no significant difference in the control effect between the GC film treatment and that of 10%ethofenprox SC.Futher,the influencings of spraying GC film on chlorophyll content,net photosynthetic rate,stomatal conductance,intercellular CO₂ concentration,transpiration rate and other indexes of rice leaves were measured.It was found that there were no significant difference in growth indexes of rice between spraying GC film in four diluted solution（800,400,200 and 100 times）and that of control even treated after 20?d.