Preliminary Study On The Resistance Of Echinochloa Oryzoides To Penoxsulam In Northeast China

Northeast China is one of the most important rice producing areas in China,and rice production in the area is crucial to China’s grain industry.However,in recent years,weeds in rice fields in the region are more serious,especially the late watergrass(Echinochloa oryzoides),which is difficult to control.Penoxsulam,an acetalactate synthase(ALS)inhibitor herbicide,has excellent control effects on Echinochloa weeds.However,due to its very single site of action,continuous and excessive use over the years,the resistance of weeds has become more serious.The decline in the control effect of penoxsulam on late watergrass has appeared in the northeast rice region.In order to determine whether late watergrass in Northeast China has resistance to penoxsulam and its resistance level,the laboratory continuously collected late watergrass seeds in the northeast rice region from 2015 to 2017,and clarified the sensitivity level of 40 late watergrass populations to penoxsulam in the area.Taking two resistant populations as the research object,the cross-resistance and multi-resistance level of penoxsulam-resistant late watergrass were clarified,which provided a theoretical basis for chemical control of resistant late watergrass.A sensitive late watergrass population and two resistant late watergrass populations were used as the research object,and carry out researches of in vitro ALS activity,ALS gene sequence,ALS gene expression level and cytochrome P450 oxidase inhibitors,preliminarily clarified the mechanism of resistance of late watergrass to penoxsulam.The specific results of this study are as follows:The whole plant bioassay was used to determine the effective dose of 50%inhibition in plant growth(GR50)of 40 late watergrass populations to penoxsulam,which were collected from the northeast China.According to the laboratory standards:among the 40 tested populations,there are 4 sensitive populations(RI≤2)and 6 sensitive decreased populations(2<RI≤4),19 low resistance population(4<RI≤10),10 medium resistant populations(10<RI≤30),1 high resistant population(30<RI≤150).This indicates that the late watergrass in the northeast rice region of China has indeed produced resistance to penoxsulam,and its resistance level is not low,which is consistent with the fact that the effect of field control to late watergrass is reduced.Then,this study selected two resistant late watergrass populations for cross-resistance and multi-resistance research.In terms of population:the medium-resistant population HSRH-520 showed a certain degree of cross-resistance to the 5 herbicides include flucarbazone,bispyribac-sodium,pyroxsulam,flupirsulfuron and propyrisulfuron,while high-resistance population HJHL-715 had cross-resistance to 8 herbicides include flucarbazone,pyribenzoxim,bispyribac-sodium,pyroxsulam,triafamone,rimsulfuron,flupirsulfuron and propyrisulfuron.These two resistant populations also had multi-resistance to other herbicides,which were not belong to ALS inhibitors.In general,at least one resistant late watergrass population had multi-resistance to all of these tested herbicides,except for propanil,mefenacet,oxadiazon and pendimethalin.It can be seen that the above four effective herbicides can be used to control resistant late watergrass.However,it should be noted that pendimethalin has not registered on the rice transplanting field,and the improper use of propanil can cause phytotoxicity,so it must be cautious when using these herbicides.The preliminary study on the resistance mechanism of late watergrass resistance to penoxsulam was carried out from four aspects:ALS activity,ALS gene sequence,ALS gene expression and the effect of cytochrome P450 oxidase inhibitors to resistance,and the first three are all studies on the mechanism of target enzyme resistance.In the study of in vitro ALS activity,it was found that there was almost no difference in ALS between resistant and sensitive populations,which were not treated with penoxsulam.However,under the treatment of penoxsulam,the degree of inhibition of in vitro ALS activity in the three tested populations was significantly different.Reference to sensitive populations,the RI of HSRH-520 and HJHL-715 were 13.73 and 1.60 times,respectively.This indicates that the decrease of ALS sensitivity in resistant late watergrass is one of the mechanisms of HSRH-520 resistance to penoxsulam,but not for HJHL-715.Using the genomic DNA of late watergrass as a template,the full-length sequence of the rice ALS gene was amplified by Touch Down Polymerase Chain Reaction(TD-PCR).The length of the ALS gene was about 2000 bp,and the populations were slightly different.They all contain no introns,and the coding region is about 1935 bp,encoding about 645 amino acids.By comparison,two copies of the ALS gene of the three test populations were found.After alignment with the sensitive population HSRH-538,an amino acid mutationit(proline to serine)at 197 site was found in the second ALS gene copy of the HSRH-520.This indicates that ALS gene mutation is one of the reasons for the resistance of HSRH-520 to penoxsulam,while not for HJHL-715,because no site mutations that have been reported to cause ALS resistance have been found in this population,and it means the mechanism of resistance in HJHL-715 needs to be further research.The change of ALS gene expression was determined by real-time PCR.The results showed that after treatment with penoxsulam,the expression levels of ALS genes in all three populations began to increase and peaked at 3 days,and the up-regulation of HSRH-538 was higher than that of HSRH-520 and HJHL-715.This may be due to differences between populations and negative feedback regulation mechanisms.This suggests that the difference in the amount of ALS gene expression is not responsible for the resistance of late watergrass to penoxsulam.Through the previous three studies,it has been clarified that the resistance of HSRH-520 to penoxsulam is caused by changes in its target enzymes.However,the resistance of HJHL-715 is difficult to explain.Therefore,this study conducted the fourth step to investigate the effect of cytochrome P450 oxidase inhibitors to resistance.The results showed that under the action of three cytochrome P450 oxidase inhibitors,the resistance of HJHL-715 to penoxsulam decreased significantly,and the its RI decreased by 90.76%,91.97%and 94.35%,respectively.This suggests that the resistance mechanism of HJHL-715 to penoxsulam is likely to be the metabolic resistance,which was mediated by cytochrome P450 oxidase.In summary,this study first identified the sensitivity level of 40 late watergrass populations to penoxsulam;selected two resistant populations for cross-resistance and multi-resistance research,and identified their sensitivity to common herbicides.The preliminary study on the resistance mechanism of two resistant populations was carried out,and it was clarified that a reported target enzyme ALS amino acid mutation and elevated ALS activity are important reasons for the resistance to penoxsulam of HSRH-520,and the difference in the amount of ALS gene expression is not the cause of resistance,while the metabolism of herbicides involved in cytochrome P450 oxidase is likely to be responsible for the resistance of HJHL-715 to penoxsulam.