A high-throughput, modified ALS activity assay for Cyperus difformis and Schoenoplectus mucronatus seedlings.

Cyperus difformis L. (CYPDI) and Schoenoplectus mucronatus (L.) Palla (SCHMU) are major weeds of California (CA) rice, where resistance to acetolactate synthase (ALS)-inhibitors was identified in several CYPDI and SCHMU populations that have also evolved resistance to photosystem II (PSII)-inhibiting herbicides. The mechanism of ALS resistance in these populations remains to be clarified but this information is crucial in a weed management program, especially in a scenario where resistance to multiple herbicides has been identified. ALS activity assays are commonly used to diagnose resistance to ALS-inhibitors, but protocols currently available are burdensome for the study of CYPDI and SCHMU, as they require large amounts of plant material from young seedlings and have low yields. Our objective was to investigate the ALS resistance mechanism in suspected ALS-resistant (R) CYPDI and SCHMU biotypes using a modified ALS activity assay that requires less plant material. ALS enzymes from suspected R biotypes were at least 10,000-fold less sensitive to bensulfuron-methyl than susceptible (S) cohorts, indicating ALS resistance that is likely due to an altered target-site. Protein concentration (mgg-1 tissue) did not differ between R and S biotypes within each species, suggesting that R biotypes do not over produce ALS enzymes. CYPDI biotypes had up to 4-fold more protein per mg of tissue than SCHMU biotypes, but up to 7-fold more acetoin per mg-1 protein was quantified in SCHMU, suggesting greater ALS catalytic ability in SCHMU biotypes, regardless of their herbicide resistance status. Our optimized protocol to measure ALS activity allowed for up to a 3-fold increase in the number of assays performed per g of leaf tissue. The modified assay may be useful for measuring ALS activity in other weed species that also produce small amount of foliage in early growth stages when protein in tissue is most abundant.

A psbA mutation (Val219 to Ile) causes resistance to propanil and increased susceptibility to bentazon in Cyperus difformis.

BACKGROUND: Propanil-resistant (R) Cyperus difformis populations were recently confirmed in California rice fields. To date, propanil resistance in other weed species has been associated with enhanced aryl acylamidase (AAA)-mediated propanil conversion into 3,4-dichloroaniline. Our objectives were to determine the level of propanil resistance and cross-resistance to other PSII inhibitors in C. difformis lines, and to elucidate the mechanism of propanil resistance. RESULTS: The propanil-R line had a 14-fold propanil resistance and increased resistance to bromoxynil, diuron and metribuzin, but not to atrazine. The R line, however, displayed a fourfold increased susceptibility to bentazon. Interestingly, susceptible (S) plants accumulated more 3,4-dichloroaniline and were more injured by propanil and carbaryl (AAA-inhibitor) applications than R plants, suggesting that propanil metabolism is not the resistance mechanism. psbA gene sequence analysis indicated a valine-219-isoleucine (Val219 Ile) amino acid exchange in the propanil-R chloroplast D1 protein. CONCLUSION: The D1 Val219 Ile modification in C. difformis causes resistance to propanil, diuron, metribuzin and bromoxynil but increased susceptibility to bentazon, suggesting that the Val219 residue participates in binding of these herbicides. This is the first report of a higher plant exhibiting target-site propanil resistance. Tank mixing of bentazon and propanil, where permitted, can control both propanil-R and propanil-S C. difformis and prevent the spread of the resistant phenotype. © 2016 Society of Chemical Industry.