Preliminary Investigation On Thifensulfuron-methyl Resistance Mechanisms In Amaranthus Retroflexus

Amaranthus retroflexus, an annual broadleaf weed in the Amaranthaceae family, is widely distributed in our country. A. retroflexus can secrete allelochemicals which inhibit the growth of other plants. Moreover, it is the hosts for many pathogens and insects in the field. As one of the most troublesome weeds, A. retroflexus causes serious damage to the yield of soybean, corn, cotton and other crops. Thifensulfuron-methyl, an ALS inhibitor, is widely used to control broadleaf weeds in the fields of soybean and corn. Due to long-term, repetitive use of thifensulfuron-methyl,farmers in parts of Heilongjiang province reflected that A. retroflexus was not sensitive to thifensulfuron-methyl any more. The harm caused by A. retroflexus has become increasingly acute.However, relatively few domestic and international studies have been done to investigate the level and mechanism of the resistance to thifensulfuron-methyl in A. retroflexus. In this study, seven A.retroflexus populations from Heilongjiang province were collected to characterize the level of thifensulfuron-methyl resistance. Petri-dish and whole-plant test showed that four A. retroflexus populations were resistant to thifensulfuron-methyl. These four A. retroflexus populations were used for identifying the resistance mechanisms. The main results were as follows:(1) The resistant level detection to thifensulfuron-methyl in different A. retroflexus populationsThe results of petri-dish test are consistent with those of the whole-plant test on the whole.The results showed that the A. retroflexus populations in parts of Heilongjiang province had already developed different resistant level to thifensulfuron-methyl. HLJ13-1 is susceptible to thifensulfuron-methyl. HLJ13-2 and HLJ14-1 are as susceptible as HLJ13-1, which received no herbicide application before. HLJ13-3, HLJ13-4, HLJ13-5, HLJ14-2 evolved resistance to thifensulfuron-methyl, With the method of petri-dish test, the resistance indexes(RI) were 5.11,63.92, 26.25, 8.12, respectively.With the method of whole-plant test, the resistance indexes(RI)were 8.91, 69.86, 27.20, 9.46 for HLJ13-3, HLJ13-4, HLJ13-5 and HLJ14-2, respectively.HLJ13-3 and HLJ14-2 belonged to moderate resistance, HLJ13-4 and HLJ13-5 belonged to high resistance.(2) In vitro ALS activity assayIn vitro ALS activity assay, the sensitivity of ALS between the R and S populations was significantly different. ALS extracted from four resistant A. retroflexus populations showed lower sensitivity to thifensulfuron-methyl. The thifensulfuron-methyl I50 values for HLJ13-1, HLJ13-3,HLJ13-4, HLJ13-5 and HLJ14-2 were 0.06, 0.40, 4.99, 1.44 and 0.51 μM, respectively. Comparedwith HLJ13-1, the corresponding I50 ratio was 6.99, 83.74, 24.96, 8.94 for HLJ13-3, HLJ13-4,HLJ13-5 and HLJ14-2, respectively. The overall result showed that the resistance to thifensulfuron-methyl was due to the reduced ALS sensitivity in resistant A. retroflexus populations..(3) ALS gene detectionCompared with HLJ13-1 population, sequencing results showed that four different mutations caused amino acid substitution among HLJ13-3, HLJ13-4, HLJ13-5 and HLJ14-2 populations.GCT to GTT mutation was found in HLJ13-3 individuals at 205, leading to the substitution of Ala by Val. In HLJ13-4, TGG was replaced by TTG, and the corresponding amino acid substitution was Trp-574-Leu. In HLJ13-5, GAT was displaced by GAA, resulting in a Asp-376-Glu substitution. HLJ14-2 possessed a AGC to ATC mutation, resulting in a Ser-653-Ile substitution.In conclusion, the resistance to thifensulfuron-methyl was due to the reduced ALS sensitivity in resistant A. retroflexus populations. ALS target-site mutations and diverse mutation sites within the ALS gene led to the occurrence of resistance and different resistance level.