The Mechanism Of Action Of Isoxadifen-ethyl Hydrolysate Alleviate The Fenoxaprop-P-ethyl Phytotoxicity Of Rice

As the planting area of direct-seeded rice fields has expanded year by year,the difficulty of weed control has gradually increased.Fenoxaprop-P-ethyl can effectively controls Digitaria sanguinalis(L.)Scop,Echinochloa crusgalli(L.)Beauv and Leptochloa chinensis(L.)Nees in paddy fields,but it can cause phytotoxicity to rice easily.Isoxadifen-ethyl hydrolysate can significantly improve the tolerance of rice to fenoxaprop-P-ethyl,and avoid the damage to rice when fenoxaprop-P-ethyl is used for weeding in paddy fields.However,the mechanism of action is still unclear.The whole plant bioassay was used to systematically analyzed the safen of fenoxaprop-P-ethyl to rice under the action of isoxadifen-ethyl hydrolysate.The dynamic effect of isoxadifen-ethyl hydrolysate on the fenoxaprop-P-ethyl dissipation of rice was determined by ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)and clarified the mode of action of the safener isoxadifen-ethyl hydrolysate.The physiological and molecular mechanism of isoxadifen-ethyl hydrolysate were determinestudied by enzymatic assay and transcriptome sequencing technology.The specific research results are as follows:1.The study clarified the protective effect of isoxadifen-ethyl hydrolysate on rice by whole plant bioassay.Isoxadifen-ethyl hydrolysate can significantly alleviate the phytotoxicity of fenoxaprop-P-ethyl on rice,and it was more effective than other safeners such as mefenpyr diethy,fenchlorazole,and cloquintocet mexyl.The fresh weight of rice 9311 had a GR50 value of 84.74 g a.i·ha-1 when fenoxaprop-P-ethyl was applied alone.After treated of fenoxaprop-P-ethyl in combination with isoxadifen-ethyl hydrolysate 180 g a.i·ha-1,the fresh weight GR50 value of rice increased by 3.23 times,reaching 273.83 g a.i·ha-1.The results showed that isoxadifen-ethyl hydrolysate can significantly reduced the phytotoxicity of fenoxaprop-P-ethyl on rice.2.The mode of action of isoxadifen-ethyl hydrolysate was analyzed by measuring the dissipation dynamics of fenoxaprop-P-ethyl in rice.To measuring the dissipation dynamics of fenoxaprop-P-ethyl in rice by UPLC-MS/MS,it was found that the safener had no significant effect on the initial deposition of fenoxaprop-P-ethyl.And the mixed use of the safener isoxadifen-ethyl hydrolysate had a large significant effect on the residues of fenoxaprop-P-ethyl in rice after being treated at 6 h and 24 h.After treated with fenoxaprop-P-ethyl 45 g a.i·ha-1 alone and in combination with isoxadifen-ethyl hydrolysate 180 g a.i·ha-1,the half-live in rice leaves were 16.91 h and 11.75 h,respectively.This results indicated that isoxadifen-ethyl hydrolysate could increase the metabolic rate of fenoxaprop-P-ethyl in rice by nearly 30%.3.The study explored the effect of activity with different cytochrome P450 oxidase inhibitors to fenoxaprop-P-ethyl by whole plant bioassay,and analyzed the effect of isoxadifen-ethyl hydrolysate on the cytochrome P450 monooxygenase(CYPs)enzyme system.When the CYPs inhibitors Malaoxon(MAL),Piperonyl butoxide(PBO),1-aminobenzotriazole(ABT)were in combination with fenoxaprop-P-ethyl,they can improve the phytotoxicity of fenoxaprop-P-ethyl in different degrees.The fresh weight inhibition rate of rice was 38.81% when treated with fenoxaprop-P-ethyl 45 g ai ha-1.When mixed with MAL,PBO,and ABT,the fresh weight inhibition rates of rice were 49.76%、63.32%、57.61%,respectively.Among them,the combined application of PBO and fenoxaprop-P-ethyl had the most significant inhibition on the fresh weight of rice.When the fenoxaprop-P-ethyl in combination with isoxadifen-ethyl hydrolysate,the inhibition rate of rice fresh weight was 14.77%.After isoxadifen-ethyl hydrolysate in combination with fenoxaprop-P-ethyl and three CYPs inhibitors,the fresh weight inhibition rates of rice were 17.91%,19.91% and 18.23%,respectively.The results showed that isoxadifen-ethyl hydrolysate can eliminate the effect of CYPs inhibitors on rice phytotoxicity.That means isoxadifen-ethyl hydrolysate had an effect on rice seedlings through detoxification enzymes CYPs,thereby alleviating the fenoxaprop-P-ethyl phytotoxicity of rice.4.The effects of isoxadifen-ethyl hydrolysate on detoxification enzymes and protective enzymes in rice leaves under the treated with fenoxaprop-P-ethyl by spectrophotometric method.After being treated with fenoxaprop-P-ethyl 45 g a.i·ha-1in combination with isoxadifen-ethyl hydrolysate 180 g a.i·ha-1,isoxadifen-ethyl hydrolysate can significantly increase glutathione-s-transferases(GSTs)activity and glutathione(GSH)content.In this treatment,it had no significant effect on the activities of superoxide dismutase(SOD)and peroxide(POD),it could significantly increase the activity of catalase(CAT)after treatment in 3 d.In addition,the content of malondialdehyde(MDA)in the rice was decreased after the addition of isoxadifen-ethyl hydrolysate.It shows that the damage of rice cell membrane was reduced,and isoxadifen-ethyl hydrolysate can reduce the damage of fenoxaprop-P-ethyl to rice to a certain extent.5.The differentially expressed genes which were regulated by safener and the metabolic pathways involved were studied by using transcriptome sequencing technology,and the mode of action of isoxadifen-ethyl hydrolysate was discussed at the molecular level.By analyzing gene expression levels within rice seedlings under different treatments by transcriptome sequencing,69 genes which encoding metabolic enzymes were screened in response to isoxadifen-ethyl hydrolysate treatment.Most of these genes were up-regulated and their products were involved in different stages of fenoxaprop-P-ethyl metabolism.Hence isoxadifen-ethyl hydrolysate may increase fenoxaprop-P-ethyl tolerance in rice by exacerbating pre-existing metabolic pathways.Key genes involved in the protective effects of isoxadifen-ethyl hydrolysate include Car E15,CYP86A1,GSTU6,GST4,UGT13248,UGT79,and ABCC4.After the treatment of fenoxaprop-P-ethyl,Esterase KAI2,CYP76M5,CYP709B2,GST23,UGT85A2,ABCB5,ABCB9,ABCG48,and Detoxification 49 were also involved in different stages of herbicide metabolism,and the expression level of key genes in the resistant variety 9311 was significantly higher than that of the sensitive rice variety Meixiangzhan No.2.The significant increase in the expression levels of these genes also indicates that isoxadifen-ethyl hydrolysate can significantly accelerate the metabolism of fenoxaprop-P-ethyl in which it is involved,increase the metabolic rate of herbicides,and finally showed a great tolerance in rice to fenoxaprop-P-ethyl.