2,4-D and dicamba resistance mechanisms in wild radish: subtle, complex and population specific?

Background and Aims: Resistance to synthetic auxin herbicides such as 2,4-dichlorophenoxyacetic acid (2,4-D) is increasing in weed populations worldwide, which is of concern given the recent introduction of synthetic auxin-resistant transgenic crops. Due to the complex mode of action of the auxinic herbicides, the mechanisms of evolved resistance remain largely uncharacterized. The aims of this study were to assess the level of diversity in resistance mechanisms in 11 populations of the problem weed Raphanus raphanistrum, and to use a high-throughput, whole-genome transcriptomic analysis on one resistant and one susceptible population to identify important changes in gene expression in response to 2,4-D. Methods: Levels of 2,4-D and dicamba (3,6-dichloro-2-methoxybenzoic acid) resistance were quantified in a dose-response study and the populations were further screened for auxin selectivity, 2,4-D translocation and metabolism, expression of key 2,4-D-responsive genes and activation of the mitogen-activated proein kinase (MAPK) pathway. Potential links between resistance levels and mechanisms were assessed using correlation analysis. Key Results: The transcriptomic study revealed early deployment of the plant defence response in the 2,4-D-treated resistant population, and there was a corresponding positive relationship between auxinic herbicide resistance and constitutive MAPK phosphorylation across all populations. Populations with shoot-wide translocation of 2,4-D had similar resistance levels to those with restricted translocation, suggesting that reduced translocation may not be as strong a resistance mechanism as originally thought. Differences in auxin selectivity between populations point to the likelihood of different resistance-conferring alterations in auxin signalling and/or perception in the different populations. Conclusions: 2,4-D resistance in wild radish appears to result from subtly different auxin signalling alterations in different populations, supplemented by an enhanced defence response and, in some cases, reduced 2,4-D translocation. This study highlights the dangers of applying knowledge generated from a few populations of a weed species to the species as a whole.

Target-site resistance to trifluralin is more prevalent in annual ryegrass populations from Western Australia.

BACKGROUND: Trifluralin is widely used in Australia as one of the important pre-emergence herbicides to control annual ryegrass (Lolium rigidum Gaud.) populations. Trifluralin resistance evolution and mechanisms have been identified in some ryegrass populations. RESULTS: In this study, 21 putative resistant field survey populations from Western Australian were screened with trifluralin, and 90% (19 of 21) contained individuals surviving 480 g ha-1 trifluralin treatment. Twelve populations contained individuals possessing the known α-tubulin resistance mutations at Val-202, Thr-239 and Arg-243 in TUA4 (alpha-tubulin 4 n), plus multiple potential resistance mutations in TUA4 pending genetic confirmation. Three populations had only individuals carrying newly identified (but uncharacterized) mutations in TUA3/TUA4. Radioactive work found that six populations evolved metabolic resistance to trifluralin, and at least four of them also possessed the known and/or putative target-site mutations. CONCLUSION: These results confirm that a high incidence of resistance to the dinitroaniline herbicide (trifluralin) is present, and target-site tubulin mutations make a major contribution to resistance in these annual ryegrass populations. Co-evolution of both target-site and non-target-site resistance to per-emergence herbicides warrants diverse management tactics.

Increasing the value and efficiency of herbicide resistance surveys.

The scale of herbicide resistance within a cropping region can be estimated and monitored using surveys of weed populations. The current approach to herbicide resistance surveys is time-consuming, logistically challenging and costly. Here we review past and current approaches used in herbicide resistance surveys with the aims of (i) defining effective survey methodologies, (ii) highlighting opportunities for improving efficiencies through the use of new technologies and (iii) identifying the value of repeated region-wide herbicide resistance surveys. One of the most extensively surveyed areas of the world's cropping regions is the Australian grain production region, with >2900 fields randomly surveyed in each of three surveys conducted over the past 15 years. Consequently, recommended methodologies are based on what has been learned from the Australian experience. Traditional seedling-based herbicide screening assays remain the most reliable and widely applicable method for characterizing resistance in weed populations. The use of satellite or aerial imagery to plan collections and image analysis to rapidly quantify screening results could complement traditional resistance assays by increasing survey efficiency and sampling accuracy. Global management of herbicide-resistant weeds would benefit from repeated and standardized surveys that track herbicide resistance evolution within and across cropping regions. © 2021 Society of Chemical Industry.

Evolution of resistance to HPPD-inhibiting herbicides in a wild radish population via enhanced herbicide metabolism.

BACKGROUND: Relatively new herbicides that target 4-hydroxyphenylpyruvate dioxygenase (HPPD) are now available for use on the world's great grain crops (rice, wheat, corn and soybean) and for other uses. With widespread and persistent use of HPPD-inhibiting herbicides, the evolution of HPPD-inhibiting herbicide resistant weeds is inevitable. Currently, resistance to HPPD-inhibiting herbicides is known in two weed species, waterhemp and Palmer amaranth. Here, we report a HPPD-inhibiting herbicide resistant wild radish population from the Western Australia grain belt. This population was not selected with HPPD-inhibiting herbicides, rather it evolved resistance to earlier used herbicides with different modes of action and exhibits cross-resistance to HPPD-inhibiting herbicides. RESULTS: Dose-response experiments showed the resistant (R) population exhibits 4 to 6.5-fold resistance to the HPPD-inhibiting herbicides mesotrione, tembotrione and isoxaflutole, compared to the susceptible (S) population. This resistance is not target-site based as cloning of full coding sequences of the HPPD genes from S and R plants did not reveal resistance-endowing single nucleotide polymorphisms. The HPPD gene expression levels are similar in S and R plants. In addition, no differences in [14 C]-mesotrione uptake and translocation were observed in the S and R plants. However, the time required for R plants to metabolise 50% [14 C]-mesotrione is 7.7-fold faster than for the S plants. CONCLUSION: We confirm resistance to HPPD-inhibiting herbicides exists in a population of the economically damaging global weed wild radish. The resistance in this population is due to a non-target-site based enhanced rate of herbicide metabolism. © 2019 Society of Chemical Industry.

Non-target-site resistance to PDS-inhibiting herbicides in a wild radish (Raphanus raphanistrum) population.

BACKGROUND: Diflufenican resistance has been reported in wild radish populations since 1998, but the resistance mechanisms have not been investigated. Recently, we identified a wild radish population (H2/10) from the Western Australian grain belt that is resistant (R) to the phytoene desaturase (PDS)-inhibiting herbicide diflufenican. RESULTS: Dose-response results showed this R population is 4.9-fold more resistant than the susceptible (S) population based on the LD50 R/S ratio. In addition, the R population also exhibits cross-resistance to the PDS-inhibiting herbicide fluridone. The cytochrome P450 inhibitor malathion reversed diflufenican resistance and partially reversed fluridone resistance in the R population. The full coding sequences of the PDS gene were cloned from the S and R plants and there are natural variations in the PDS gene transcripts/alleles with no correlation to resistance. In addition, the R plants had a level of PDS gene expression that is not significantly different from the S plants. CONCLUSION: These results demonstrated that diflufenican resistance in this R wild radish population is likely due to non-target-site based enhanced herbicide metabolism involving cytochrome P450s. © 2019 Society of Chemical Industry.

Agricultural Weed Assessment Calculator: An Australian Evaluation.

Weed risk assessment systems are used to estimate the potential weediness or invasiveness of introduced species in non-agricultural habitats. However, an equivalent system has not been developed for weed species that occur in agronomic cropland. Therefore, the Agricultural Weed Assessment Calculator (AWAC) was developed to quantify the present and potential future adverse impact of a weed species on crop production and profitability (threat analysis), thereby informing or directing research, development, and extension (RDE) investments or activities. AWAC comprises 10 questions related primarily to a weed's abundance and economic impact. Twenty weed species from across Australia were evaluated by AWAC using existing information and expert opinion, and rated as high, medium, or low for RDE prioritization based on total scores of 70 to 100, 40 to <70, or <40, respectively. Five species were rated as high (e.g., Lolium rigidum Gaud.), eight were rated as medium (e.g., Conyza spp.), and seven were rated as low (e.g., Rapistrum rugosum L.). Scores were consistent with the current state of knowledge of the species' impact on grain crop production in Australia. AWAC estimated the economic or agronomic threat of 20 major or minor agricultural weeds from across Australia. The next phase of development is the testing of AWAC by weed practitioners (e.g., agronomists, consultants, farmers) to verify its utility and robustness in accurately assessing these and additional weed species.

Distinct non-target site mechanisms endow resistance to glyphosate, ACCase and ALS-inhibiting herbicides in multiple herbicide-resistant Lolium rigidum.

This study investigates mechanisms of multiple resistance to glyphosate, acetyl-coenzyme A carboxylase (ACCase) and acetolactate synthase (ALS)-inhibiting herbicides in two Lolium rigidum populations from Australia. When treated with glyphosate, susceptible (S) plants accumulated 4- to 6-fold more shikimic acid than resistant (R) plants. The resistant plants did not have the known glyphosate resistance endowing mutation of 5-enolpyruvylshikimate-3 phosphate synthase (EPSPS) at Pro-106, nor was there over-expression of EPSPS in either of the R populations. However, [(14)C]-glyphosate translocation experiments showed that the R plants in both populations have altered glyphosate translocation patterns compared to the S plants. The R plants showed much less glyphosate translocation to untreated young leaves, but more to the treated leaf tip, than did the S plants. Sequencing of the carboxyl transferase domain of the plastidic ACCase gene revealed no resistance endowing amino acid substitutions in the two R populations, and the ALS in vitro inhibition assay demonstrated herbicide-sensitive ALS in the ALS R population (WALR70). By using the cytochrome P450 inhibitor malathion and amitrole with ALS and ACCase herbicides, respectively, we showed that malathion reverses chlorsulfuron resistance and amitrole reverses diclofop resistance in the R population examined. Therefore, we conclude that multiple glyphosate, ACCase and ALS herbicide resistance in the two R populations is due to the presence of distinct non-target site based resistance mechanisms for each herbicide. Glyphosate resistance is due to reduced rates of glyphosate translocation, and resistance to ACCase and ALS herbicides is likely due to enhanced herbicide metabolism involving different cytochrome P450 enzymes.

Metribuzin Resistance in a Wild Radish ( Raphanus raphanistrum) Population via Both psbA Gene Mutation and Enhanced Metabolism.

There have been many studies on target-site resistance (TSR) to PSII-inhibiting herbicides, but only a few on the non-target-site resistance (NTSR). Here, we reported both TSR and NTSR to metribuzin in a wild radish population. Dose-response studies revealed a higher level of resistance to metribuzin in the resistant (R) compared to the susceptible (S) population. Sequencing of the target psbA gene revealed the known Ser-264-Gly mutation in R plants. In addition, a higher level of [14C]-metribuzin metabolism and, consequently, a lower level of [14C] translocation were also detected in the R plants. These results demonstrated that both psbA gene mutation and enhanced metabolism contribute to metribuzin resistance in this wild radish population. Furthermore, this resistant population showed resistance to ALS-inhibiting herbicides due to multiple ALS gene mutations. This is the first report in wild radish of metabolic herbicide resistance, in addition to the target-site psbA gene mutation.

A novel psbA mutation (Phe274-Val) confers resistance to PSII herbicides in wild radish (Raphanus raphanistrum).

BACKGROUND: Wild radish (Raphanus raphanistrum) is a globally important weed of crops. Two atrazine-resistant wild radish populations (R1 and R2), collected from the Western Australia grain belt, were investigated for resistance to photosystem II (PSII) herbicides. RESULTS: Sequencing of the full-length psbA gene revealed the well-known Ser264-Gly substitution in population R1, whereas population R2 displayed a novel Phe274-Val substitution. Herbicide dose-response studies confirmed that the population with the Ser264-Gly mutation exhibited high-level resistance to atrazine, but super-sensitivity to bromoxynil. Plants possessing the novel Phe274-Val mutation exhibited a modest level of resistance to atrazine, metribuzin and diuron, and were bromoxynil susceptible. Structural modelling of the mutant D1 proteins predicts that the Ser264-Gly mutation endows atrazine resistance by abolishing H-bonds, but confers bromoxynil super-sensitivity by enhancing hydrogen bonding. The Phe274-Val substitution provides resistance to atrazine and diuron by indirectly affecting H-bond formation between the Ser264 residue and the herbicides. CONCLUSION: The results demonstrate that the Phe274-Val mutation is likely responsible for resistance to PSII-inhibiting triazine and urea herbicides. To our knowledge, this is the first evidence of the psbA Phe274-Val mutation in wild radish conferring resistance to PSII herbicides. © 2018 Society of Chemical Industry.

Dinitroaniline herbicide resistance in a multiple-resistant Lolium rigidum population.

BACKGROUND: The pre-emergence dinitroaniline herbicides (such as trifluralin and pendimethalin) are vital to Australian no-till farming systems. A Lolium rigidum population collected from the Western Australian grain belt with a 12-year trifluralin use history was characterised for resistance to dinitroaniline, acetyl CoA carboxylase (ACCase)- and acetolactate synthase (ALS)-inhibiting herbicides. Target-site resistance mechanisms were investigated. RESULTS: This L. rigidum population exhibited 32-fold resistance to trifluralin, as compared with the susceptible population. It also displayed 12- to 30-fold cross-resistance to other dinitroaniline herbicides (pendimethalin, ethalfluralin and oryzalin). In addition, this population showed multiple resistance to commonly used post-emergence ACCase- and ALS-inhibiting herbicides. Two target-site α-tubulin gene mutations (Val-202-Phe and Thr-239-Ile) previously documented in other dinitroaniline-resistant weed species were identified, and some known target-site mutations in ACCase (Ile-1781-Leu, Asp-2078-Gly and Cys-2088-Arg) and ALS (Pro-197-Gln/Ser) were found in the same population. An agar-based Petri dish screening method was established for the rapid diagnosis of resistance to dinitroaniline herbicides. CONCLUSION: Evolution of target-site resistance to both pre- and post-emergence herbicides was confirmed in a single L. rigidum population. The α-tubulin mutations Val-202-Phe and Thr-239-Ile, documented here for the first time in L. rigidum, are likely to be responsible for dinitroaniline resistance in this population. Early detection of dinitroaniline herbicide resistance and integrated weed management strategies are needed to maintain the effectiveness of dinitroaniline herbicides. © 2017 Society of Chemical Industry.