Studies On Resistance To Glyphosate And Metamifop Of Eleusine Indica (L.) Gaertn.

Goosegrass(Eleusine indica(L.)Gaertn.)is a gramineous annual weed,which distributes in farmland,orchards and roads.It has strong adaptability and rapid reproduction,which poses a threat to the growth and development of crops.Glyphosate is an organic phosphorus herbicide,which has been widely used in the United States since it was developed by the Monsanto Company in 1970s.It is mainly used to control weeds in fruit trees,vegetables,cotton and other crops.Metamifop,one of the ACCase inhibitors,which has been widely used for weed control in rice fields in recent years.Due to the long-term use of glyphosate and metamifop,goosegrass has developed resistance to both herbicides.In this study,the resistance levels of different populations to glyphosate and metamifop were determined.The mechanism of resistance was investigated by EPSPS and ACCase gene sequence analysis,EPSPS gene expression and transcriptome sequencing.In addition,the control effects of 9 different herbicides on sensitive and resistant goosegrass were determined.The results are as follows:The results of glyphosate resistance to goosegrass showed that:The GRso value of sensitive population(S)was 248.7 g a.i./hm2;the YZ4 and YZ5 populations belonged to low resistant populations,the GR50 values of them were 654.9 and 602.9 g a.i./hm2,and the resistance index(RI)was 2.6 and 2.4 times,respectively;YZ3,YZ6 and YZ7 populations were medium resistant populations,the GR50 values of them were 884.2 g a.i./hm2,766.4 g a.i./hm2 and 1009.9 g a.i./hm2,respectively,and the resistance ratio was 3.1～4.1 times;YZ1,YZ2,YZ8 and YZ9 populations were classified as highly resistant populations,the GR50 values of them were 2138.6 g a.i./hm2,4763.9 g a.i./hm2,2263.5 g a.i./hm2,3531.9 g a.i./hm2,and the resistance ratios were 8.6～19.2 times,respectively.The results of metamifop resistance to goosegrass were determined that the GR50 value of sensitive population(S)was 8.9 g a.i./hm2.The GR50 range of YZ4,YZ5,YZ6,YZ7 and YZ8 populations was 8.02～12.5 g a.i./hm2,indicating that there is no resistance to metamifop.YZ1,YZ2,YZ3,YZ9 populations were considered as highly resistant populations,the GR50 values of them were 213.6 g a.i./hm2,332.9 g a.i./hm2,784.2 g a.i./hm2,513.2 g a.i./hm2,and the resistance ratios were 24-88.1 times,respectively.By comparing and analyzing the EPSPS gene sequences of different populations,the results showed that:the sequence of EPSPS in population YZ3 was changed from proline to alanine in 106th amino acids,and the sequence of EPSPS in population YZ8 and YZ9 was changed from threonine and proline to isoleucine and serine in 102th and 106th amino acids,respectively.The results of other populations were the same as that in sensitive population(S).This suggests that target gene mutation is one of the mechanisms for glyphosate resistance in YZ3,YZ8 and YZ9 populations.The expression levels of EPSPS in different populations were determined by RT-qPCR.The expression levels of EPSPS in YZ3,YZ4,YZ8 and YZ9 populations were 2.5,1.7,7.5 and 2.6 times than that of sensitive populations(S),respectively,which indicated that YZ3,YZ4,YZ8 and YZ9 populations were at a low level;The expression level of EPSPS in resistant populations YZ1,YZ2,YZ5,YZ6,YZ7 was up-regulated 22.4,39,54.1,45.5,11.4 times.These results suggest that the overexpression of EPSPS is one of the mechanisms leading to the development of resistance in various populations.The resistance ratio of YZ5 population was significantly lower than that of YZ1 and YZ2 populations,but the expression of EPSPS was higher than that of YZ1 and YZ2 populations.Besides,the gene sequencing shows that there is no target resistance occured in the three populations.In conclusion,the resistance of YZ1 and YZ2 population to glyphosate may be caused by non-target resistance mechanism.ACCase gene CT region sequencing results showed that:it was found that the gene sequence in population YZ1 was changed from tryptophan to cysteine in 2027th amino acids;the gene sequence of YZ2 was changed from tryptophan to serine in 1999th amino acids;the gene sequence of YZ3 and YZ9 was changed from aspartic acid to glycine in 2078th amino acids.There is no site mutation occurred in other populations.The results of ACCase gene sequencing were consistent with the results of metamifop resistant goosegrass(YZ1,YZ2,YZ3,YZ9),indicating that the resistance of goosegrass population to metamifop was caused by the mutation of the target gene.A total of 10 non-target-site resistance-related genes were identified by transcriptome sequencing,which was performed on S,YZ1 and YZ2 populations.One ABC transporter,one AKRs,and one P450 gene were significantly up-regulated in YZ1 and YZ2 populations,which was further verified by RT-qPCR.ABCC4 and AKR4C10 genes were transferred into Arabidopsis Thaliana by agrobacterium-mediated method,and the function of AKR4C10 gene was verified to determine the sensitivity to glyphosate.The results showed that the transgenic plants with AKR4C10 gene had a certain resistance to glyphosate,and this gene may be related to the resistance of glyphosate.The results of the control test showed that:1500 g a.i./hm2 diquat and 810 g a.i./hm2 glufosinate ammonium can effectively control goosegrass,the control effect of fresh weight and plant were more than 90%;The control effect of 52.5 g a.i./hm2 quizalofop-p-ethyl,105 g a.i./hm2 cyhalofop-butyl and 112.5 g a.i./hm2 metamifop was poor to YZ1,YZ2,YZ3,YZ9 populations,and the sensitive populations YZ4,YZ5,YZ6,YZ7,YZ8 populations were significantly inhibited.The fresh weight and plant control efficiency of YZ4,YZ5,YZ6,YZ7,YZ8 populations were more than 81%.The recommended doses of azilpyrazosulfuron,enoxalone,dichloroquinoline and clofluridine ester had poor control effect and failed to inhibit the growth of goosegrass.