Metabolic Resistance To Fenoxaprop-p-ethyl In American Sloughgrass(Beckmannia Syzigachne)from Wheat Fields

American sloughgrass?Beckmannia syzigachne?is a common Gramineae weed which widely distributed in wheat?Triticum aestivum L.?rotated with rice?Oryza sativa L.?or rice rotated with oilseed rape?Brassica napus L.?fields in the Yangtze River delta and southwest region of China.Fenoxaprop-P-ethyl is the most common herbicide used to control grass weeds in wheat field of China which target of action is the acetyl-CoA carboxylase?ACCase?due to its low toxicity,high efficiency and long endurance.However,resistance has evolved in American sloughgrass under continuously use of fenoxaprop-P-ethyl and the heibicide can not control American sloughgrass successfully at the recommended rate.Since then,mesosulfuron-methyl,which target of action is the acetolactate synthase?ALS?,become the important alternative herbicide for control of fenoxaprop-P-ethyl-resistant weeds.Unfortunately,American sloughgrass also evolved resistance to mesosulfuron-methyl gradually,and these multiple resistance populations threat the safe production of wheat seriously.We did not find any reported amino acid substitutions related with the target-site resistance at the the study of target site mechanism which showed that there may exist non-target-site resistance in this population.Based on this research,we mainly studied the non-target-site resistance mechanism and hope to provide theoretical guidance for the control of this multiple resistance American sloughgrass populations.The main results were as follows:The AH-02 population was treated with fenoxaprop-P-ethyl(62.1 g a.i.ha^-1)and mesosulfuron-methyl(15.75 g a.i.ha^-1)at the recommended rate respectively at the whole plant level.The results showed that the AH-02 population has produced resistance both to fenoxaprop-P-ethyl and mesosulfuron-methyl.The gene fragments of plastid ACCase CT domain and the ALS gene fragments of AH-02 population were amplified and sequenced,and the blasted results indicated that there were not any reported amino acid substitutions related with the target-site resistance.The resistant and sensitive plants with a clear genetic background were bred in greenhouse to obtain the F1(R_F1 and S_F1)by minimizing genetic differences.To ensure the background of R_F1 and S_F1,the gene fragments of plastid ACCase CT domain and the ALS gene fragments of R_F1 and S_F1 were also amplified and sequenced,and the blasted results indicated that there were not any reported amino acid substitutions related with the target-site resistance which consistent with the field population.The activity of P450s in F1 was investigated by comparing the effective fenoxaprop-P-ethyl and mesosulfuron-methyl rate causing a 50%growth reduction?GR50?in the absence and presence of P450s inhibitors?PBO and malathion?at plant level.The results showed that the use of PBO in combination with fenoxaprop-P-ethyl has a significant effect on R_F1 and decrease the GR₅₀ by 45.7%-60.0%compared with the fenoxaprop-P-ethyl treatment alone;the use of malathion in combination with mesosulfuron-methyl has a significant effect on RF1 and decrease the GR50 by 48.9%-65.6%compared with the mesosulfuron-methyl treatment alone which indicated that the non-target-site resistance exist in this population.The sensitivity to other herbicides in RF1 was tested at plant level and the results showed that the R_F1 produced high resistance to fenoxaprop-P-ethyl?Resistance index;RI=31.2?,low resistance to quizalofop-P-ethyl?RI=2.8?and clodinafop-propargyl?RI=4.2?;produced low resistance to sethoxydim?RI=2.7?and sensitive to clethodim?RI=1.9?and pinoxaden?RI=1.7?;produced low resistance to mesosulfuron-methyl?RI=2.6?,moderate resistance to flucarbazone-sodium?RI=7.7?,high resistance to pyroxsulam?RI=23.6?and no resistance to isoproturon.The RNA-Seq transcriptome technology was used to screen metabolic genes that conferred non-target-site resistance to the herbicide fenoxaprop-P-ethyl in purified RF2American sloughgrass plants.Thirteen candidate genes were selected which were annotated to P450s,GSTs,GTs,ABC transporters,growth factors and plant hormones including P450s?4?,GSTs?2?,GTs?1?,ABC transporters?2?,carotenoids?1?,inositol?1?,abscisic acid?1?and salicylic acid?1?.The average gene expression levels were validated by quantitative real-time PCR analysis which were consistent with the RNA-Seq.We selected two genes?c32066_g2 and c21365_g2?which were annotated to GSTU17and GT73C1 from thirteen candidate genes for further functional verification.The plant transformation vector was constructed by pPZP211,and obtain twenty-two positive transgenic Brachypodium distachyon plants of GSTU17 gene and seventeen positive transgenic Brachypodium distachyon plants of GT73C1 gene?Bd-F0?based on efficient Agrobacterium-mediated transformation system.The test of positive F1 transgenic plants includes two sides:at plant level,the growth power of positive transgenic plants were better than control plants after the treatment of fenoxaprop-P-ethyl and mesosulfuron-methyl at recommended doses;at molecular level,the expression levels of GSTU17 and GT73C1 genes were significantly up-regulated after the treatment of fenoxaprop-P-ethyl and mesosulfuron-methyl compared with the untreated.In conclusion,this study determined the resistance levels to ten common herbicides used in wheat fields including fenoxaprop-P-ethyl and mesosulfuron-methyl.The blast of target genes showed that there were not any reported amino acid substitutions related with the target-site resistance and the P450-based metabolic resistance is most likely involved in the purified R_F1 American sloughgrass plants.The RNA-Seq transcriptome technology was selected to screen metabolic genes that conferred non-target-site resistance to fenoxaprop-P-ethyl which enriched the genomic database of weeds.The screened thirteen metabolic genes were validate by the quantitative real-time PCR which was consistent with the RNA-Seq.The Agrobacterium-mediated transformation technology was used to validate the GSTU17 and GT73C1 genes'function which results were consistent with the expected.As a result this sutdy provide a theoretical basis for the metabolic genes'functional verification and hope to provide scientific guidlines for the control of this specific resistant American sloughgrass.