Molecular Mechanism Of Resistance To Tribenuron-Methyl In Shepherd’s Purse(Capsella Bursa-Pastoris)from Wheat Fields

Shepherd’s purse（Capsella bursa-pastoris（L.）Medik.）is a troublesome broadleaf weed in the major wheat-producing areas,and widely distributed in southwest,east China,north China and Yangtse River basin.Tribenuron-methyl,a ALS-inhibiting herbicide,was the most widely used herbicide to control shepherd’s purse in wheat fields in China.In recent years,shepherd’s purse was more difficult to be controlled in many main wheat producing areas with tribenuron and even control failed after this herbicide was continuously applied for many years.The aims of the present study were to characterize the resistance level and identify the resistance mechanisms to tribenuron–methyl in shepherd’s purse populations collected from Henan Province in China.This study mainly researches about resistance distribution,resistance levels,target and non-target resistance mechanism,cross resistance and fitness in order to use herbicide rationally and delay the resistant shepherd’s purse evolution.The main results are showed below:Whole-plant dose-response experiments were conducted to determine the resistance levels of 28 shepherd’s purse field populations.Populations showed high-level resistance to tribenuron-methyl,with resistance index（RI）11.3⁵95.8.Population HN-13 collected from Zhumadian city showed the highest RI among all tested populations.The shepherd’s purse ALS gene fragment was amplified and sequenced,and resistant shepherd’s purse populations possessed 7 kinds of ALS target-site mutation:Pro-197-His（CCT-CAT）,Pro-197-Leu（CCT-CTT）,Pro-197-Arg（CCT-CGT）,Pro-197-Thr（CCT-ACT）,Pro-197-Ser（CCT-TCT）,Pro-197-Ala（CCT-GCT）and Trp-574-Leu（TGG-TTG）.Two different ALS partial gene sequences were obtained by connection and transformation,and Southern blotting was carried out to confirm the existence of two copies of genes encoding plastidic ALS in shepherd’s purse.There are mutations in both of the ALS-1 and ALS-2 genes.When comparing the sequences derived from both the R and S populations,the gene ALS-1 contained a CCT to TCT（Pro-197-Ser）mutation,and the gene ALS-2 contained a CCT to CAT（Pro-197-His）mutation.The expression levels of the ALS-1 and ALS-2 genes were validated by RT-qPCR by employing theβ-action gene as the keep-house gene.This study attempted to identity the expression of the ALS genes during herbicide treatment and under non-treatment conditions,and relative expression levels of ALS genes in root,stem and leaf tissues from shepherd’s purse.The results showed that there was no significant difference in the expression of ALS-1and ALS-2,and the expression of ALS in the resistant population was significantly higher than that in the sensitive population.After tribenuron-methyl treatment,the expression of ALS in the resistant population increased significantly.The expression of ALS gene in leaf was significantly higher than that in root and stem.Two dCAPS assays were designed to detect mutations in ALS 197 and 574 codon using BsiY I and Nco I as the restriction enzyme respectively.The dCAPS were used to detect the wild genetype for identifying the resistant shepherd’s purse populations with mutation.The results of these dCAPS assays were reliable and in accordance with the sequencing results.Results from in vitro ALS assays indicated that the total ALS activity was similar between the resistant and sensitive populations.ALS extracted from the resistant populations with mutation（HN-01,HN-02,HN-03,HN-04,HN-05,HN-06,HN-07,HN-08,HN-09,HN-11,HN-12,HN-13,HN-15,HN-18,HN-19,HN-22 and HN-25）was much less susceptible to tribenuron-methyl in comparison to the sensitive population.The the herbicide concentration resulting in 50%reduction of ALS activity(I₅₀)was 23.45-fold to 55.97-fold greater than the S population.ALS extracted from the resistant populations without mutation（HN-10,HN-14,HN-16,HN-17,HN-20,HN-21,HN-23,HN-24,HN-26,HN-27 and HN-28）was similar to the sensitive populations.As the cytochrome P450s inhibitor,malathion was used in combination with tribenuron-methyl to evaluate the activity of P450s in resistant population HN-06.The results of malathion(1000 g a.i.ha^-1)in combination with tribenuron-methyl indicated that malathion significantly decrease the GR₅₀ for tribenuron-methyl in resistant population.Compared with the sensitive population,the GR₅₀ value of resistant population decreased 25%.Whole-plant dose-response experimrnts were conducted to determine the cross-resistance to other ALS-inbihiting herbicides:pyrithiobac sodium,florasulam,pyroxsulam,flucarbazone-Na and imazethapyr using the resistant populations（HN-06 and HN-13）.Compared with the sensitive population,the resistant populations showed high-level resistance to pyrithiobac sodium,florasulam,pyroxsulam and flucarbazone-Na.HN-06showed no resistance to imazethapyr with a resistance index of 1.1,and HN-13 showed low-level resistance to imazethapyr with a resistance index of 5.6.There were no fitness cost on seed germination and seedling emergence of resistant shepherd’s purse population HN-05 with the environmental factors of light,temperature,pH,salt stress,osmotic stress,and depth of sowing.Light was not required for germination;The optimum germination occurred at fluctuating temperature 15-20℃;pH from 5 to 10 had insignificant effect on germination;Seed germination was tolerant to salinity;Seed germination was sensitive to osmotic potential;When seeds were placed on the soil surface,seedling emergence percent was the highest,but it declined with burial depth increasing,and no more emergence when the depth was above 1.2 cm.This study characterized the resistance to tribenuron-methyl in shepherd’s purse populations collected from Henan Province.Two partial intronless ALS genes（ALS-1 and ALS-2）were identified,and the expression of ALS genes increased in the resistant populations.The resistance was established as being due to an insensitive form of ALS,resulting from the mutations in the ALS gene.Moreover,the enhanced metabolism mediated by cytochrome P450S was account for the resistance in resistant populations,and different resistance mechanisms may coexist in a single shepherd’s purse plant.Cross-resistance experiments indicated that resistant populations exhibited various resistance patterns to other ALS-inhibiting herbicides.Molecular markers were designed to rapidly identify the mutations in shepherd’s purse populations.In addition,this study preliminary evaluate the influence of various environmental factors on seed germination and seedling emergence of shepherd’s purse.