Resistance Mechanism Of Cyperus Difformis To Bensulfuron-methyl In Rice Field

Cyperus difformis L.,as one of the main weeds in paddy field,has the characteristics of large seed yield and strong tillering ability,which seriously affects the yield of rice.In rice production,ALS-inhibiting herbicides are mostly used to control C.difformis,and the most commonly used sulfonylurea is bensulfuron-methyl.The herbicide has good control effect on Cyperaceae and broadleaf weeds,but due to long-term overuse,a variety of weeds in paddy fields have developed resistance to it,especially the control effect of bensulfuronmethyl on C.difformis has decreased significantly.In this study,the C.difformis populations from different paddy fields in Hunan and Guangxi Provinces were collected.The level of resistance to bensulfuron-methyl and susceptibility to other herbicides in different C.difformis populations were determined.The target resistance mechanism of C.difformis resistant populations to bensulfuron-methyl and the metabolism-related non-target resistance mechanism were studied,by technical means such as gene cloning,transcriptome sequencing(RNA-seq),Quantitative Real-time PCR(qRT-PCR)and exogenous gene expression.Aming to provide scientific basis for the comprehensive management of resistance in C.difformis.The main findings are as follows:(1)The resistance levels of different C.difformis populations to bensulfuronmethyl were quite different.The differential-dose screening of 74 C.difformis populations collected from Hunan and Guangxi Provinces showed that 33 populations were resistant to bensulfuron-methyl.Dose-response experiments were performed on 22 of the resistant populations(R)and 2 sensitive populations(S),and the results showed that 19 populations with high resistance,2 populations with moderate resistance,and 1 population with low resistance.There are 3 R populations(HN-3,HN-9,HN-10)with GR50 value over 2880 g a.i./ha and resistance index(RI)over 1309;2 S populations with GR50 values below 2.5 g a.i./ha;GR50 values of other R populations ranged from 12.1 to 1071.0 g a.i./ha,and RI ranged from 5 to 487;(2)Part of the C.difformis R populations developed cross-resistance or multiple resistance to other herbicides.Single-dose screening was used to determine the susceptibility of C.difformis populations to the ALS-inhibiting herbicide penoxsulam,the PSII inhibitor herbicide bentazone,and the hormonal herbicide 2-Methyl-4-chlorophenoxyacetic acid(MCPA).The results showed that 18 R populations developed cross-resistance to penoxsulam,7 R populations developed multiple resistance to MCPA,5 R populations had mutiple resistance to bensulfuron-methyl,MCPA and penoxsulam,and all populations were still sensitive to bentazone.(3)The mutation of target gene is one of the reasons for the resistance of C.difformis to ALS-inhibiting herbicides.The sequences of the target genes ALS and psbA of the tested populations were obtained by gene cloning,and the differences in the conserved regions of the R and S populations were compared.The results showed that 80%(8/10)of the populations in Hunan Province and 20%(13/64)of the populations in Guangxi Province had mutation sites in ALS gene.Among them,5 populations in Hunan Province with high level of resistance were mutated from tryptophan(Trp)to leucine(Leu)at site 574,and the highest RI exceeded 1309.17 populations were mutated from proline(Pro)to leucine(Leu),serine(Ser),arginine(Arg)and alanine(Ala)at site 197.The resistance levels of populations in Guangxi Province dominated by this mutation were low,and the RI was between 5-89.Four populations mutated from aspartic acid(Asp)to glutamate(Glu)at site 376.No mutation was found inpsbA gene of R populations.qRT-PCR technology was used to analyze the expression of ALS,the target gene of C.difformis.The results showed that there was no significant difference in ALS gene expression between the R and S population.(4)Metabolic resistance was found in some R populations of C.difformis.Some populations were treated with the P450 inhibitor malathion showed that the resistance of 12 R populations to bensulfuron-methyl could be reversed.One S population and R population with metabolic resistance were selected for RNAseq.According to the data analysis results,some up-regulated genes related to metabolic detoxification(CYPs,ABCs,UGTs,GST)were selected for gene cloning and qRT-PCR verification.The expression differences of the verified candidate genes were analyzed at the transcriptional level,and the results showed that CYP71FL1,CYP74B57,and CYP94C180 genes continued to be highly expressed within 0-2 days after the treatment of bensulfuron-methyl,and the expression levels in the R and S populations were significantly different at P<0.05.GST and UGTs genes fluctuated at different time points after treatment,but the expression was at a low level,and there was no significant difference between R and S populations.(5)The results of heterologous expression of P450 genes of C.difformis indicated that CYP71FL1 and CYP74B57 mediated the resistance of sedge to bensulfuron-methyl.The full-length coding sequences of CYP71FL1,CYP74B57 and CYP94C180 were obtained by RACE and PCR technology.The recombinant plasmids were constructed by seamless cloning and transformed into Arabidopsis thaliana and Saccharomyces cerevisiae respectively.Herbicides were used to evaluate resistance of transgenic A.thaliana.The metabolic rate of bensulfuronmethyl by recombinant yeast was determined by high performance liquid chromatography(HPLC),and the degradation products were identified by high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry(UPLC-QTOF-MS/MS).The results showed that Arabidopsis seedlings overexpressing CYP71FL1 and CYP74B57 became resistant to bensulfuron-methyl and bentazone.The metabolic rate of bensulfuron-methyl by recombinant CYP71FL1 yeast culture was 23%after induction for 16 h,while the recombinant CYP74B57 and CYP94C180 yeast did not degrade bensulfuron.The results of this paper show that the herbicide resistance of C.difformis in paddy fields in China is already serious,and the target resistance mechanism and the non-target resistance mechanism related to metabolism jointly promote the evolution and development of herbicide-resistant weeds.The comprehensive management of weed resistance provides a scientific basis and has important practical significance.