Genetic and ecological factors determining the distribution of ALS-inhibiting herbicide resistance in Cyperus difformis L. in California rice and its implication for management

Resistance to ALS (acetolactate synthase)-inhibiting herbicides has evolved in Cyperus difformis L. populations infesting California rice fields, and basic factors contributing to the rapid evolution and broad distribution of resistant biotypes are unknown. The aim of this study was to evaluate the mating systems, the patterns and mechanism of cross-resistance, and the population genetic structure, of C. difformis from California rice field as a basis for defining rational principles in order to delay and manage the evolution of ALS-inhibiting-herbicide resistance in this species. A multilocus mating analysis based on SRAP fingerprinting, as well as a greenhouse and a field crossing analysis using resistance to ALS-inhibiting herbicides as a phenotypic marker of pollen flow indicate that C. difformis is mostly a self-fertilizing species. Whole-plant and ALS-enzyme activity dose-response assays to herbicides from all five classes of ALS-inhibitors conducted with four C. difformis biotypes suggests that the main mechanism of resistance was target-site insensitivity, which results from specific point mutations on the ALS gene. Therefore, a 1709 bp contig belonging to the ALS gene was isolated to investigate mutations on this gene that could be related with the resistance patterns observed. However, the nucleotide and the amino acid sequences did not vary within analyzed biotypes, suggesting that more than one ALS gene may be present in C. difformis . A phylogenetic analysis indicated that the ALS gene obtained from C. difformis was not homologous to those of certain Poaceae or dicotyledonous species. A cross-resistance study based on an herbicide-agar bioassay with 240,000 seeds from 56 populations found twelve different patterns of herbicide cross-resistance. Resistance to bensulfuron-methyl, halosulfuron-methyl and propoxycarbazone-sodium, and susceptibility to imazethapyr, bispyribac-sodium and penoxsulam was the most frequent resistance pattern. However, of all the populations sampled, the frequencies of those with at least one resistant individual for any of these herbicides were 76, 86, 67 and 50% in the Northern, Central and Southern Sacramento and in the San Joaquin valleys, respectively. A genetic diversity and gene flow analysis was conducted on 29 populations using 73 SRAP molecular markers. Total diversity (HT) was 0.1756, and most of this variation occurred among populations (G ST = 0,96). Genetic relationships among populations were not correlated with geographical distance. The UPGMA dendogram suggests that population clustering is not region specific. These studies demonstrate that independent mutations are the main origin of the C. difformis biotypes with resistance to ALS-inhibiting herbicides found in most California rice fields. Currently available ALS-inhibiting herbicides still can be used in rice paddies to control many of these C. difformis biotypes. However, the large amount of resistant cases and cross-resistance patterns found suggests that their success will be difficult to predict, and that their usefulness will be short lived. In the absence of relevant gene flow contributions to resistance spread among populations, prevention and management practices focused on in-field strategies of weed control should be successful to delay the evolution of ALS-resistance in C. difformis of California rice fields.