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Determining the mechanisms of herbicide resistance in weeds allows for the development and implementation of applied management practices aimed to control and to prevent further spread of herbicide-resistant populations in crop fields. This research was conducted to determine propanil resistance and cross-resistance to other photosystem II (PSII) inhibitors in ricefield bulrush biotypes and to elucidate the mechanism of propanil resistance. To this end, propanil-resistant (R) and propanil-susceptible (S) biotypes were selected from field-collected populations after propanil spraying at the field rate, and whole-plant, dose–response experiments were conducted to evaluate cross-resistance to PSII inhibitors and interactions between propanil and the insecticides malathion and carbaryl. In addition, thepsbAgene from R and S biotypes was sequenced for amino acid alterations following polymerase chain reaction (PCR) amplification. Plant survival data indicated the R biotype displayed a 14-fold increase in propanil resistance relative to the susceptible (S) biotype. In addition, the propanil-R biotype also had increased resistance to the PSII-inhibitors bromoxynil, diuron, and metribuzin but was more susceptible to bentazon than were propanil-S plants. Synergism between propanil and the insecticides carbaryl and malathion was greater in the S biotype than it was in the R biotype, indicating that, unlike propanil resistance in weedy grasses, enhanced degradation of the herbicide molecule is not a mechanism of resistance for propanil in ricefield bulrush. A Val219to Ile substitution in the propanil-R chloroplast D1 protein was identified following sequencing of thepsbAgene. This research suggests a single-point mutation at the target site causes resistance to propanil, diuron, metribuzin, and bromoxynil but increasing susceptibility to bentazon in propanil-R ricefield bulrush, a novel Val219–Ile feature. To our knowledge, this is the first instance of propanil resistance in weeds because of a mechanism other than enhanced herbicide metabolism. Tank-mixing bentazon and propanil, where permitted, can control both propanil-R and propanil-S biotypes.

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.

Herbicide resistance is a challenge for modern agriculture further complicated by cases of resistance to multiple herbicides. Conyza bonariensis and Conyza canadensis are invasive weeds of field crops, orchards, and non-cropped areas in many parts of the world. In California, USA, Conyza populations resistant to the herbicides glyphosate and paraquat have recently been described. Although the mechanism conferring resistance to glyphosate and paraquat in these species was not elucidated, reduced translocation of these herbicides was observed under experimental conditions in both species. Glyphosate and paraquat resistance associated with reduced translocation are hypothesized to be a result of sequestration of herbicides into the vacuole, with the possible involvement of over-expression of genes encoding tonoplast transporters of ABC-transporter families in cases of glyphosate resistance or cationic amino acid transporters (CAT) in cases of paraquat resistance. However, gene expression in response to herbicide treatment has not been studied in glyphosate and paraquat resistant populations. In the current study, we evaluated the transcript levels of genes possibly involved in resistance using real-time PCR. First, we evaluated eight candidate reference genes following herbicide treatment and selected three genes that exhibited stable expression profiles; ACTIN, HEAT-SHOCK-PROTEIN-70, and CYCLOPHILIN. The reference genes identified here can be used for further studies related to plant-herbicide interactions. We used these reference genes to assay the transcript levels of EPSPS, ABC transporters, and CAT in response to herbicide treatment in susceptible and resistant Conyza spp. lines. No transcription changes were observed in EPSPS or CAT genes after glyphosate or paraquat treatment, suggesting that these genes are not involved in the resistance mechanism. Transcription of the two ABC transporter genes increased following glyphosate treatment in all Conyza spp. lines. Transcription of ABC transporters also increased after paraquat treatment in all three lines of C. bonariensis. However, in C. canadensis, paraquat treatment increased transcription of only one ABC transporter gene in the susceptible line. The increase in transcription of ABC transporters after herbicide treatment is likely a stress response based on similar response observed across all Conyza lines regardless of resistance or sensitivity to glyphosate or paraquat, thus these genes do not appear to be directly involved in the mechanism of resistance in Conyza spp.

BACKGROUND: Echinochloa colona is an annual weed affecting field crops and orchards in California. An E. colona population carrying a mutation in the EPSPS gene endowing resistance to glyphosate, the most widely used non-selective herbicide, was recently identified in the Northern Sacramento Valley of California. Plants from this population, from a suspected glyphosate-resistant (GR) population, and from one susceptible (S) population collected in the Northern Sacramento Valley of California, were used to generate three GR and one S selfed lines to study possible mechanisms involved in glyphosate resistance. RESULTS: Based on the amount of glyphosate required to kill 50% of the plants (LD50), GR lines were 4–9-fold more resistant than S plants and accumulated less shikimate after glyphosate treatment. GR and S lines did not differ in glyphosate absorption, translocation or metabolism. A different target-site mutation was found in each of two of the GR lines corresponding to Pro106Thr and Pro106Ser substitutions; the mutations were found in different homoeologous EPSPS genes. No mutation was found in the third GR line, which exhibited 1.4-fold higher basal EPSPS activity and a fivefold greater LD 50 than S plants. Quantitative RT-PCR revealed that GR lines had similar or lower EPSPS expression than S plants. CONCLUSION: It is demonstrated that individuals with different glyphosate resistance mechanisms can coexist in the same population, individuals from different populations may carry different resistance mechanisms and different mechanisms can act in concert within single E. colona plants. However, other plant factors or resistance mechanisms appear to modulate plant expression of EPSPS sensitivity to glyphosate. © 2014 Society of Chemical Industry Supporting information may be found in the online version of this article.

A suspected glyphosate-resistant (R) junglerice population was collected from a glyphosate-R corn field near Durham in northern California where glyphosate had been applied at least twice a year for over 6 yr. Based on the amount of glyphosate required to reduce growth by 50% (ED50), the R population was 6.6 times more R than the susceptible (S) standard population. Based on the glyphosate concentration that inhibits EPSPS by 50% based on shikimate accumulation (I50) in leaf discs, R plants were four times more R than S plants. By 3 d after treatment with 0.42 kg ae ha−1glyphosate, the S population had accumulated approximately five times more shikimate than the R population. No differences in [14C]-glyphosate uptake and translocation were detected between R and S plants. However, partial sequencing of theEPSPSgene revealed a mutation in R plants causing a proline to serine change at EPSPS position 106 (P106S). Our results reveal the first case of a P106S target site mutation associated with glyphosate resistance in junglerice.

Summary Evolution of resistance to herbicides in weeds is becoming an increasing problem worldwide. To develop effective strategies for weed control, a thorough knowledge of the basis of resistance is required. Although non-target-site-based resistance is widespread, target site resistance, often caused by a single nucleotide change in the gene encoding the target enzyme, is also a common factor affecting the efficacies of key herbicides. Therefore, fast and relatively simple high-throughput screening methods to detect target site resistance mutations will represent important tools for monitoring the distribution and evolution of resistant alleles within weed populations. Here, we present a simple and quick method that can be used to simultaneously screen for up to 10 mutations from several target site resistance-associated codons in a single reaction. As a proof of concept, this SNaPshot multiplex method was successfully applied to the genotyping of nine variable nucleotide positions in the CT domain of the chloroplastic ACCase gene from Lolium multiflorum plants from 54 populations. A total of 10 nucleotide substitutions at seven of these nine positions (namely codons 1781, 1999, 2027, 2041 2078, 2088 and 2096) are known to confer resistance to ACCase-inhibiting herbicides. This assay has several advantages when compared with other methods currently in use in weed science. It can discriminate between different nucleotide changes at a single locus, as well as screening for SNPs from different target sites by pooling multiple PCR products within a single reaction. The method is scalable, allowing reactions to be carried out in either 96- or 384-well plate formats, thus reducing work time and cost.