Gene flow and spontaneous mutations are responsible for imidazolinone herbicide-resistant weedy rice (Oryza sativa L.).

For more than two decades, weedy rice (Oryza sativa L.) has been controlled in rice fields by using imidazolinone (IMI) herbicide-resistant rice technology (Clearfield®). Outcrossing in weedy rice populations and spontaneous mutations are potential problems with herbicide-resistant crop management technologies, such as the IMI-resistant rice. The aim of this study was to characterize the mechanism of IMI herbicide resistance in weedy rice through dose-response bioassay study and evaluating amino acid substitutions in acetolactate synthase (ALS) protein. A total of 118 suspected IMI-resistant weedy rice samples, which survived in the field after an IMI herbicide application, were collected at harvest time from Türkiye in 2020 and 2021. Single-dose imazamox application experiment revealed that 38 plants survived herbicide treatment. The imazamox resistance of the surviving plants was confirmed by dose-response experiment. ALS gene region underwent a sanger DNA partial sequencing. No substitution was found in 10 samples, however, amino acid substitutions were found in 26 samples with S563N, one sample with S653T, and one sample with E630D. The S653N point is the same substitution point that serves as the origin of resistance for the Clearfield® rice varieties that are commonly cultivated in the region. It has been hypothesized that the gene flow from IMI-resistant rice may be the cause of resistance in the IMI resistant weedy rice samples with S653N. The other substitution, S653T, were considered spontaneous mutation to IMI resistance. Interestingly, the S653T mutation was detected for the first time in weedy rice. The mechanism of resistance of 10 resistant weedy rice was not confirmed in this study, however, it may be a non-target resistance or another mutation point in target site, but evidently, they did not acquire resistance by gene flow from IMI-resistant rice. It has been concluded that the effectiveness of IMI-resistant rice technology in controlling weedy rice has drastically decreased due to possible gene flow, spontaneous mutation and non-target resistance. In addition to cultural controls like clean seed, clean machinery and crop rotation, other herbicide-tolerant rice systems such as Provisia® and Roxy-RPS® rice are needed to create a diverse weedy rice management ensemble available for rice production and move towards sustainable rice farming.

Target and nontarget mechanisms of AHAS inhibitor cross-resistance patterns in Cyperus difformis.

Weed resistance to acetohydroxyacid synthase (AHAS) inhibiting herbicides has been a critical issue for rice growers worldwide since the early 1990's. In California, resistance to bensulfuron-methyl was first detected in Cyperus difformis in 1993. Since then, populations of most major weeds of rice in California have been reported to show resistance to at least one AHAS inhibitor. We sought to describe the magnitude and mechanisms of AHAS inhibitor cross-resistance in California populations of C. difformis. Sixty-two populations were collected and screened for cross-resistance to bensulfuron-methyl (BEN), halosulfuron-methyl (HAL), bispyribac­sodium (BIS), and penoxsulam (PEN), revealing six major patterns of cross-resistance. Representative C. difformis populations from each cross-resistance pattern were then subjected to dose-response, cytochrome P450 inhibition, AHAS gene sequencing, and metabolic studies with the same herbicides as in the screening. Dose-response confirmed the detected resistances in the representative populations, and suggested that the majority of observed resistance was dose-dependent. Cytochrome P450 inhibition via malathion revealed evidence of increased metabolic activity in resistant populations to BEN, BIS, and PEN. AHAS gene sequencing revealed amino acid substitutions in five of six populations: R3 (Pro197-Ser), R4 (Pro97-His), R10 (Asp376), R41 (Ala122-Asn), and R18 (Trp574-Leu). Metabolic studies confirmed evidence of increased activity of cytochrome P450s in all populations. Metabolic BEN and HAL analysis did not yield similar results to malathion inhibition, suggesting different P450's or other pathways. Taken together, the results of the studies confirm the complexity of AHAS inhibitor cross-resistance in C. difformis, and the presence of both target-site and metabolic resistance in most of the representative populations underscores the importance of proper herbicide selection, rotation, and scouting in fields.

Assessment of oxyfluorfen-tolerant rice systems and implications for rice-weed management in California.

BACKGROUND: Weeds are a significant barrier to rice production in California, exacerbated by lack of chemical control options and herbicide-resistance in persistent aquaphilic species. Oxyfluorfen-tolerant rice, created at the Rice Experiment Station (RES) in Biggs, California, was developed to provide an agronomic program for managing problematic grass and sedge rice-weeds including Oryza sativa f. spontanea Roshev. (weedy 'red' rice). Hand-pulling is the most common removal method for O. sativa spontanea because there are no herbicides registered for this pest in California. Oxyfluorfen was used in combination with oxyfluorfen-tolerant rice in 2019 and 2021 field studies to evaluate rice injury and weed control efficacy on prevalent rice-weed species. Additional studies were conducted in 2021 on University of California Davis campus to determine pre-emergent oxyfluorfen efficacy on four California O. sativa spontanea accessions. RESULTS: Fields studies indicated minimal crop injury in the first 28 days after seeding (DAS), but no observable injury at 60 DAS in both years. Weed control with oxyfluorfen alone was 87% or greater for all weeds rated with the exception of Schoenoplectus mucronatus (L.) Palla (ricefield bulrush), and Leptochloa fascicularis (Lam.) A. Gray (bearded sprangletop) in both years. All O. sativa spontanea exposed to soil-applied oxyfluorfen successfully emerged through the soil surface, but became completely necrotic 28 days after flooding. CONCLUSION: Oxyfluorfen-tolerant rice system was demonstrated to be a viable management strategy for California rice growers who struggle with grass- and sedge-weed control as well as provide a novel herbicide option for California O. sativa spontanea management. © 2022 University of California, Davis. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Protocol for ethyl methanesulphonate (EMS) mutagenesis application in rice.

Background: Non-transgenic chemical mutagen application, particularly ethyl methanesulfonate (EMS), is an important tool to create mutations and gain a new genetic makeup for plants. It is useful to obtain a sufficient number of mutant plants instead of working with a severe mutation in a few plants. EMS dose and exposure period have been previously studied in several crops; however, EMS used to create point mutations in presoaked rice seeds has not been sufficiently studied and there is no standard protocol for such treatment. The aim of this study is to establish a standard protocol for EMS mutagenesis application in rice. Methods: Two studies were conducted to evaluate the effect of four durations of rice seed presoaking (0, 6, 12, and 24 hours), four EMS concentration doses (0.0%, 0.5%, 1.0%, and 2.0%), and four EMS exposure periods (6, 12, 24, and 48 hours). Germination rate, plumula and radicle length, seedling survival, LD 50 (Lethal Dose) determination, shoot length, root length and fresh seedling weight were evaluated. Results: Results showed that a 12-hour presoaking duration, 0.5% EMS dose, and six hours of EMS exposure were the best practices for the optimum number of mutant plants. Conclusions: In light of both this study and the literature, a standard application protocol was established. This application protocol, detailed in this article, contains the following guidelines: (1) Presoaking: 12 hours, (2) EMS application: 0.5% dose EMS and six hours, (3) Final washing: six hours, (4) Drying: 72 hours at 38°C. A user-friendly protocol has been presented for utilization by researchers.

Mechanism of clomazone resistance in Leptochloa fusca spp. fasicularis to clomazone.

Bearded sprangletop (Leptochloa fusca spp. fasicularis) is a problematic weed in California rice (Oryza sativa) production. Historically, bearded sprangletop is controlled with clomazone in California rice fields. The continuous use of clomazone and lack of crop rotation in rice fields resulted in resistance to clomazone in several bearded sprangletop. The objective of this research was to determine the clomazone mechanism of resistance of two bearded sprangletop populations in California rice by investigating clomazone absorption, translocation, and metabolism under controlled environmental conditions in two resistant (CRBS1 and CRBS2) and one susceptible (S) populations. Absorption and translocation of 14C-clomazone were similar in R and S. Clomazone metabolism, as determined by inhibition of cytochrome P450 enzymes with malathion and determining clomazone metabolites profile, was different between S and R 3 days after treatment. Bearded sprangletop pretreated with malathion was 2-fold more sensitive to clomazone than when treated with clomazone alone, suggesting that cytochrome P450-mediated clomazone metabolism might be involved in the mechanism of resistance. An HPLC-MS/MS analysis revealed differences in clomazone metabolism between R and S biotypes. Hydroxymethylclomazone was the most abundant metabolite found in R plants with three and five-fold more abundant in CRBS1 and CRBS2, respectively, when compared to S plants. 5-ketoclomazone, the known toxic metabolite of clomazone, accumulated 2-fold more in S plants than in R plants at 72 h after treatment. This research shows that clomazone is metabolized differently between R and S populations of bearded sprangletop and that P450 monooxidation is likely involved in the mechanism of resistance.

Genetic variation and possible origins of weedy rice found in California.

Control of weeds in cultivated crops is a pivotal component in successful crop production allowing higher yield and higher quality. In rice-growing regions worldwide, weedy rice (Oryza sativa f. spontanea Rosh.) is a weed related to cultivated rice which infests rice fields. With populations across the globe evolving a suite of phenotypic traits characteristic of weeds and of cultivated rice, varying hypotheses exist on the origin of weedy rice. Here, we investigated the genetic diversity and possible origin of weedy rice in California using 98 simple sequence repeat (SSR) markers and an Rc gene-specific marker. By employing phylogenetic clustering analysis, we show that four to five genetically distinct biotypes of weedy rice exist in California. Analysis of population structure and genetic distance among individuals reveals diverse evolutionary origins of California weedy rice biotypes, with ancestry derived from indica, aus, and japonica cultivated rice as well as possible contributions from weedy rice from the southern United States and wild rice. Because this diverse parentage primarily consists of weedy, wild, and cultivated rice not found in California, most existing weedy rice biotypes likely originated outside California.

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.

Metabolism of quizalofop and rimsulfuron in herbicide resistant grain sorghum.

Studies were conducted to determine if herbicide metabolism is an additional mechanism that could explain the resistance of ACCase- and ALS-resistant grain sorghum to quizalofop and rimsulfuron, respectively. ACCase- and ALS-resistant and -susceptible genetic lines were grown under controlled conditions and treated at the 4-leaf stage with (14)C-labeled quizalofop and rimsulfuron. Plants were harvested at 3, 5, and 7d after treatments. In the ACCase metabolism experiment, resistant grain sorghum transformed 88% of quizalofop-ethyl to quizalofop while 91% of the inactive was converted to active form by the susceptible plants 3DAT. By 7DAT, all inactive quizalofop-ethyl was converted to active quizalofop. In the ALS metabolism study, two distinct metabolites were produced from rimsulfuron. Metabolism rate was similar between resistant lines (TX430R and N223R) in all harvest dates except at 7 DAT; however, more rapid metabolism were observed when resistant were compared with the susceptible genotypes (TX430S and N223S). The percentage of recovered rimsulfuron 3DAT corresponded to 80% and 83% in the resistant compared to 87% in the susceptible grain sorghum. At 5DAT, metabolism was near steady in all sorghum plants but by 7DAT, resistant genotypes metabolized 4-12% more than the susceptible sorghum. Metabolism of rimsulfuron in ALS-resistant grain sorghum is an added mechanism that could help elevate the level of rimsulfuron resistance.