Cyhalofop-butyl-resistance Mechanism And Herbicide Screening Of Leptochloa Chinensis In Rice Fields

Chinese sprangletop（Leptochloa chinensis）is a harmful gramineous weed second only to barnyardgrass（Echinochloa crus-galli）in Chinese direct-seeded rice fields.Cyhalofop-butyl,an acetyl-Co A carboxylase（ACCase）inhibitor,is the dominant herbicide for controlling L.chinensis in rice fields.Resistance of L.chinensis to cyhalofop-butyl has emerged as a serious problem due to overuse of cyhalofop-butyl to control the frequent occurrence of L.chinensis.The present study would contribute to the precise and regional prevention and control of L.chinensis,by evaluating the resistance level and exploring the resistance mechanism of L.chinensis in different regions,as well as screening effective herbicides to control L.chinensis in rice fields.In this study,we characterized the resistance level of different L.chinensis populations to cyhalofop-butyl and other ACCase inhitors,and determined the differences of base and amino acid residues in ACCase carboxyl transferase（CT）region of cyhalofop-butyl-resistant L.chinensis.The binding ability of sensitive（S）and mutated amino acid residues at specific sites of cyhalofop-butyl-resistant L.chinensis（R）ACCase with cyhalofo-butyl was studied by molecular dynamics simulation.We also further developed the specific rapid molecular detection method（d CAPS）for amino acid resistance mutations at specific sites of ACCase.In addition,transcriptomics sequencing（RNA-seq）and quantitative real-time PCR（q RT-PCR）were performed to analyze the non-target-site resistant（NTSR）genes and metabolic pathways in R population.The efficacy of the new compound CHD18119 on the R populations was confirmed by whole plant bioassay.Finally,the control effects of different herbicides on L.chinensis were studied through field experiments.The main results are as follows:1.Sensitivity of L.chinensis to cyhalofop-butyl and other ACCase inhibitorsUsing whole-plant bioassay method,181 L.chinensis populations collected from2014 to 2018 for cyhalofop-butyl sensitivity were tested,and we screened out 12suspected R populations.Through the dose-response test,the relatively resistance index（RI）of R populations to cyhalofop-butyl was determined by comparing with the ED₅₀ of the S population.The results showed that there were two populations with low-level resistance（2≤RI≤5）,one with moderate-level resistance（5<RI≤10）and nine with high-level resistance（RI>10）.The cross-resistance to metamifop and clethodim of R population was studied.The results showed that among the high-level resistant L.chinensis populations,five were sensitive to metamifop,one was low-level resistant to metamifop,and two were high-level resistant to metamifop;six were sensitive to clethodim and one was low-level resistant to clethodim.According to different resistance levels of L.chinensis to herbicides,accurate prevention and control strategies can be drawn up.2.Target-site resistance mechanism in cyhalofop-butyl-resistant L.chinensisThe CT region of ACCase in L.chinensis was sequenced.The result showed that there were four mutation types related to herbicide resistance in three amino acid positions of the ACCase CT region in five R populations,including isoleucine（Ile）to leucine（Leu）at position 1781,tryptophan（Trp）to cysteine（Cys）or serine（Ser）at position 2027,and the Ile to asparagine（Asn）at position 2041.This study enriches the TSR mechanism L.chinensis to cyhalofop-butyl.Molecular dynamics simulation was used to study the binding capacity of S and R（Ile-1781-Leu,Trp-2027-Ser,and Ile-2041-Asn）ACCase with cyhalofop-butyl.The average binding free energies of the above four ACCase with cyhalofop-butyl were-39.1 kcal/mol,-22.8 kcal/mol,-31.2 kcal/mol,and-29.9 kcal/mol,respectively.These results indicated that cyhalofop-butyl could strongly interact with S-type Accase,but its binding capacity with the three R-type ACCase was weakened.On the basis of molecular dynamics model,this study demonstrated that the above target mutations resulted in the decreased sensitivity of L.chinensis to cyhalofop-butyl.Three d CAPS methods were designed to detect the mutations of Ile（ATA）-1781-Leu（CTA）,Trp（TGG）-2027-Ser（TCG）and Ile（ATT）-2041-Asn（AAT）in L.chinensis,which provided molecular rapid detection methods to precisely detect the TSR of L.chinensis.3.Non-target-site resistance mechanism in cyhalofop-butyl-resistant L.chinensisBased on RNA-seq,relevant genes and metabolic pathways related to NTSR were analyzed,and the expression levels of some P450 genes and ABC transporter genes related to herbicide resistance were analyzed by q RT-PCR.Metabolic pathways and secondary metabolite biosynthesis were the most important pathways for upregulating differentially expressed genes（DEGs）in resistant populations treated with cyhalofop-butyl,which might affect cyhalofop-butyl metabolism in resistant populations.Two cytochrome P450 monooxygenase（P450）subfamily genes（CYP71A and CYP706C）and two ABC transporter family genes（ABCA7 and ABCA2）were only appeared in the up-regulated DEGs of resistant population after treated with cyhalofop-butyl.The relative expression ratio of CYP71A gene reached to 97.85 in q RT-PCR.The results of RNA-seq provided a theoretical basis for the further study on the NTSR mechanism of L.chinensis.We also constructed phylogenetic trees containing the L.chinensis P450 family genes and some P450 family genes that have been identified in other species for herbicide metabolism,and found that some L.chinensis P450 family members were similar to known herbicide resistance genes.These results provided a reference for screening candidate P450 genes related to herbicide resistance.4.Screening of control agents for L.chinensis in rice fields.Through the dose-response test,ED₉₀ values of the new compound CHD18119 to R populations were 133.25-201.20 g a.i./ha,and the maximum ratio of ED₉₀ value was only 1.51,indicating that all R populations were sensitive to CHD18119.Herbicide screening field trial for L.chinensis control in directed-seeded rice paddy was conducted,and the results showed that under experimental doses,the control effects of butachlor on L.chinensis was around 90%.For tillering stage of L.chinensis,the control effects of cyhalofop-butyl,cyhalofop-butyl+clomazone and benzobicyclon were more than 95%,metamifop was around 90%,and benzobicyclon（throw）was around 95%.CHD18119 has the potential to control L.chinensis,especially cyhalofop-butyl-resistant L.chinensis.Cyhalofop-butyl,benzobicyclon can effectively control sensitive L.chinensis at tillering stage in rice paddy fields.The results of laboratory and field experiments provide the guidelines for the chemical control of L.chinensis.