Resistance Of Barnyardgrass (Echinochloa Crusgalli (L.) Beauv.) To Quizalofop-p-ethyl In Soybean Fields Of Heilongjiang Province

Heilongjiang Province is the important soybean production in China and one of the earliestherbicide application regions. Echinochloa crusgalli (L.) Beauv. (barnyardgrass) is a maingrass weed in soybean fields. For controlling this grass weed, APP and CHD herbicides werewidely used in the region. Especially, quizalofop-p-ethyl was used more than 10 years onsome farms. Because of the continuous use of this herbicide for years, barnyardgrass evolvedresistance to this herbicide. In order to determine the popularization of the resistance,barnyardgrass seeds were collected from 11 farms where quizalofop-p-ethyl was used formany years and the resistance level was determined using whole-plant bioassay and seedbioassay. In order to clarify the mechanism of resistance, the resistance mechanism wasstudied using molecular biologymethods and enzymologymethods. Main results as follows:(1) Seeds bioassay results: GR50 of Geqiushan R was 2847.0 mg L-1, GR50 of S was 22.7mg L-1, RI was 125.5; GR50 of 853 R was 63.6 mg L-1, GR50 of S was 22.8 mg L-1, RI was 2.8;RI of Hongxing R was 2.6; GR50 of other Rs were 26.5-50.1 mg L-1, GR50 of Ss were21.3-32.1 mg L-1, RI <2.8.(2) Whole plant bioassay results: The resistance level of Geqiushan R was the highest.GR50 of Geqiushan R was 1066.4 g ai ha-1, GR50 of S was 12.2 g ai ha-1; RI was 87.3; GR50 of853 R was 92.5 g ai ha-1, GR50 of S was 15.3 g ai ha-1, RI was 6.0; GR50 of other Rs were29.5-61.2 g ai ha-1, GR50 of Ss were 13.5-20.8 g ai ha-1, RI were 1.5-3.6. Combined the dataof seeds bioassay, Geqiushan R was defined as the high resistance population, the resistancelevel of other Rs was low.(3) Geqiushan R, 853 R and Wudalianchi R were chosen as the study objects and theactivities tendency of SOD, POD, CAT, GSTs after spraying quizalofop-p-ethyl 60 g ai ha-1from 0d to 8d were determined. Results showed that: the activities of GSTs in 853 R andWudalianchi R increased and reached the peak at the eighth day. The activity of GSTs inGeqiushan R changed little compared the control. Among the three protective enzymes thevariation tendency of POD has significant difference between the high resistant biotype andthe low resistant biotypes. The results showed that the resistance mechanism of 853 R andWudalianchi R related to the enhanced detoxication of GSTs. POD activities inhigh-resistance biotype were significantly activated.(4) Using HPLC, the activities of plastid ACCase in Geqiushan R (RR) and Geqiushan S(SS) at series quizalofop-p-ethyl concentrations were determined. The quizalofop-p-ethylconcentration that inhibited the activity of plastid ACCase in RR and SS by 50% (IC50) wasdetermined. The results showed that IC50 of RR was 145.38μM, IC50 of SS was 0.69μM, RIwas 210.7. Results showed that sensitiveness of high-resistance biotype to quizalofop-p-ethylreduced greatly compared to the sensitive biotype. (5) Full-length cDNAs of barnyardgrass plastid ACCase in RR and SS were clonedsuccessfully. The plastid ACCase cDNA of RR is about 7 kb, includes an ORF of 6951bp,193 nucleotides of 5' untranslated sequence (UTR) and 383 nucleotides of 3' UTR. This geneis deduced to encode a protein of 2316 amino acids with a pI of 5.97 and molecular weight ca.256 KD. According to the results of BLAST, phylogenetic tree analysis and transit peptideanalysis, the cloned cDNA sequences were barnyardgrass plastid ACCase. The accessionnumbers in GenBank were HQ395758 and HQ395759 respectively. After compared thefull-length cDNA of barnyardgrass ACCase in RR and SS, one non-synonymous mutationoccurred at nucleotide position 5341, with codon 1781 (standardized to blackgrass), causingan ATA codon (isoleucine) to be changed into a TTA codon (leucine). After compared thesetwo sequences and other ACCase sequences which were resistant or sensitive toACCase-inhibiting herbicides, this point mutation was considered as an important reason ofthe resistance of plastid ACCase in RR to quizalofop-p-ethyl.(6) Using SYBR Green I Real-time PCR test, the expression of plastid ACCase gene in RRand SS was determined after spraying the quizalofop-p-ethyl at the dosage of 60 g ai ha-1.Theexpression of plastid ACCase gene in RR was inhibited at the beginning and then recovered;the tendency of SS was similar. So the expression of target gene was not the reason of thehigh resistance.To sum up, high resistant biotype only appeared in Geqiushan Farm and the resistancelevels in other ten farms were low. The resistance of RR to quizalofop-p-ethyl was induced bythe reduced sensitiveness of plastid ACCase. From the results of sequence analyse and geneexpression analyse, one point mutation was found compared the sensitive biotype. Theexpression of target gene was similar in RR and SS. So the reduced sensitiveness of plastidACCase in RR was induced mainly by this point mutation. In this study an enhanceddetoxication enzyme (GSTs) was considered as the main reason of the resistance in 853 R andWudalianchi R.