Comparison of an Unlabeled Probe High-Resolution Melting Analysis Assay (HRMA) for Factor V Leiden 1691 G/A Mutation to a Fluorogenic 5' Nuclease PCR Hydrolysis Assay.

BACKGROUND: The clinically significant Factor V Leiden (FVL) point mutation (1691 G/A) causes replacement of Arg with Gln (glutamine), preventing activated protein C from inactivating Factor V leading to a lengthened clotting process. Individuals with the Factor V Leiden mutations have an increased risk for venous thrombosis. The aim of this study is to compare an unlabeled probe high-resolution melting analysis (HRMA) assay for Factor V Leiden mutation to a TaqMan hydrolysis assay (fluorogenic 5' nuclease PCR hydrolysis assay). HRMA is a post-PCR, homogenous, closed-tube system for the detection of sequence variants. Post-PCR, the amplicons are heated gradually until the melting temperature is reached and the fluorescent dye unbinds from the amplicon and exhibits low fluorescence. A melt-curve analysis is generated that is characteristic of a particular sequence variant. Therefore, HRMA allows for comparison of one base changes in genetic sequences based on their differences in melting rate. METHODS: Blood samples were collected in EDTA tubes and DNA extracted using the Roche MagNaPure. Reactions of both HRMA and TaqMan were carried out on 3 controls (1691 G/G, 1691 G/A, and 1691 G/G and G/A) and 20 samples. RESULTS: The genotypes for 3 reference controls purchased from Coriell (F5 1691 G/G, FVL 1691 G/A, and Heterozygote 1691 G/G and G/A) were confirmed by both the HRMA and TaqMan FVL assays. All 20 samples were confirmed to be F5 1691 G/G by both HRMA and TaqMan assays. CONCLUSIONS: Comparing the results of the unlabeled probe HRMA FVL assay with a real-time TaqMan probe end point genotyping assay resulted in 100% sensitivity and 100% specificity for both assays.

Multiple Metabolic Enzymes Can Be Involved in Cross-Resistance to 4-Hydroxyphenylpyruvate-Dioxygenase-Inhibiting Herbicides in Wild Radish.

A wild radish population (R) has been recently confirmed to be cross-resistant to 4-hydroxyphenylpyruvate dioxygenase (HPPD)-inhibiting herbicides without previous exposure to these herbicides. This cross-resistance is endowed by enhanced metabolism. Our study identified one 2-oxoglutarate/Fe(II)-dependent dioxygenase gene (Rr2ODD1) and two P450 genes (RrCYP704C1 and RrCYP709B1), which were significantly more highly expressed in R versus susceptible (S) plants. Gene functional characterization using Arabidopsis transformation showed that overexpression of RrCYP709B1 conferred a modest level of resistance to mesotrione. Ultra-performance liquid chromatography-tandem mass spectrometry analysis showed that tissue mesotrione levels in RrCYP709B1 transgenic Arabidopsis plants were significantly lower than that in the wild type. In addition, overexpression of Rr2ODD1 or RrCYP704C1 in Arabidopsis endowed resistance to tembotrione and isoxaflutole. Structural modeling indicated that mesotrione can bind to CYP709B1 and be easily hydroxylated to form 4-OH-mesotrione. Although each gene confers a modest level of resistance, overexpression of the multiple herbicide-metabolizing genes could contribute to HPPD-inhibiting herbicide resistance in this wild radish population.

Aldo-keto reductase may contribute to glyphosate resistance in Lolium rigidum.

BACKGROUND: We have previously demonstrated that an aldo-keto reductase (AKR) from Echinochloa colona (EcAKR4-1) can metabolize glyphosate and confers glyphosate resistance. This study aims to investigate if the EcAKR4-1 orthologs from Lolium rigidum also play a role in glyphosate resistance in non-target-site based, glyphosate-resistant (R) L. rigidum populations from Western Australia. RESULTS: The full-length L. rigidum AKR gene (LrAKR4C10) orthologous to EcAKR4-1, together with a distinct LrAKR1, were cloned from plants of a glyphosate-susceptible (S) (VLR1) and three glyphosate R L. rigidum populations (WALR50, WALR60 and WALR70). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) results showed that basal expression levels of the two LrAKR genes did not differ between the R and S populations, but their expression was significantly induced by glyphosate (up to 4.3-fold) or 2,4-D treatment (up to 3.4-fold) in R populations. Escherichia coli cells transformed respectively with LrAKR4C10 and LrAKR1 were more tolerant to glyphosate. Rice (Oryza sativa) seedlings overexpressing each of the two LrAKR gene survived glyphosate rates that were lethal to the green fluorescence protein (GFP) control plants. Structural modeling predicts a similar way of glyphosate binding and detoxification by LrAKR4C10 and EcAKR4-1, but an alternative way of glyphosate binding by LrAKR1. Relatively lower capacity of the two LrAKRs in conferring glyphosate resistance than the known EcAKR4-1 was discussed in relation to structural interaction. CONCLUSION: Glyphosate-induced higher expression of the two LrAKR genes in L. rigidum populations contributes to a moderate level of glyphosate resistance likely through enhanced glyphosate metabolism. The herbicide 2,4-D can also induce the LrAKR expression, indicating the potential antagonistic effect of 2,4-D to glyphosate. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Challenging glyphosate resistance EPSPS P106S and TIPS mutations with soybean competition and glyphosate: implications for management.

BACKGROUND: Eleusine indica (L.) Gaertn. (goosegrass) is a major weed in global cropping systems. It has evolved resistance to glyphosate due to single Pro-106-Ser (P106S) or double Thr-102-Ile + Pro-106-Ser (TIPS) EPSPS target site mutations. Here, experiments were conducted to evaluate the single effect of soybean competition and its combined effect with a glyphosate field dose (1080 g ae ha-1 ) on the growth and fitness of plants carrying these glyphosate resistance endowing target site mutations. RESULTS: TIPS E. indica plants are highly glyphosate-resistant but the double mutation endows a substantial fitness cost. The TIPS fitness penalty increased under the effect of soybean competition resulting in a cost of 95%, 95% and 96% in terms of, respectively, vegetative growth, seed mass and seed number investment. Glyphosate treatment of these glyphosate-resistant TIPS plants showed an increase in growth relative to those without glyphosate. Conversely, for the P106S moderate glyphosate resistance mutation, glyphosate treatment alone reduced survival rate, vegetative growth, aboveground biomass (34%), seed mass (48%) and number (52%) of P106S plants relative to the glyphosate nontreated plants. However, under the combined effects of both soybean competition and the field-recommended glyphosate dose, vegetative growth, aboveground biomass, seed mass and number of P106S and TIPS plants were substantially limited (by ≤99%). CONCLUSION: The ecological environment imposed by intense competition from a soybean crop sets a significant constraint for the landscape-level increase of both the E. indica single and double glyphosate resistance mutations in the agroecosystem and highlights the key role of crop competition in limiting the population growth of weeds, whether they are herbicide-resistant or susceptible. © 2022 Society of Chemical Industry.

A dinitroaniline herbicide resistance mutation can be nearly lethal to plants.

BACKGROUND: Lolium rigidum is the most important weed in Australian agriculture and pre-emergence dinitroaniline herbicides (e.g., trifluralin) are widely and persistently used for Lolium control. Consequently, evolution of resistance to dinitroaniline herbicides has been increasingly reported. Resistance-endowing target-site α-tubulin gene mutations are identified with varying frequency. This study investigated the putative fitness cost associated with the common resistance mutation Val-202-Phe and the rare resistance mutation Arg-243-Met causing helical plant growth. RESULTS: Results showed a deleterious effect of Arg-243-Met on fitness when plants are homozygous for this mutation. This was evidenced as high plant mortality, severely diminished root and aboveground vegetative growth (lower relative growth rate), and very poor fecundity compared with the wild-type, which led to a nearly lethal fitness cost of >99.9% in competition with a wheat crop. A fitness penalty in vegetative growth was evident, but to a much lesser extent, in plants heterozygous for the Arg-243-Met mutation. By contrast, plants possessing the Val-202-Phe mutation exhibited a fitness advantage in vegetative and reproductive growth. CONCLUSION: The α-tubulin mutations Arg-243-Met and Val-202-Phe have contrasting effects on fitness. These results help understand the absence of plants homozygous for the Arg-243-Met mutation and the high frequency of plants carrying the Val-202-Phe mutation in dinitroaniline-resistant L. rigidum populations. The α-tubulin Arg-243-Met mutation can have an exceptional fitness cost with nearly lethal effects on resistant L. rigidum plants. © 2022 Society of Chemical Industry.

A naturally evolved mutation (Ser59Gly) in glutamine synthetase confers glufosinate resistance in plants.

Glufosinate is an important and widely used non-selective herbicide active on a wide range of plant species. Evolution of resistance to glufosinate in weedy plant species (including the global weed Eleusine indica) is underway. Here, we established the molecular basis of target site glufosinate resistance in Eleusine indica. Full-length E. indica glutamine synthetase (GS) iso-genes (EiGS1-1, 1-2, 1-3, and EiGS2) were cloned, and expression of EiGS1-1 and EiGS1-2 was higher than that of EiGS2. A novel point mutation resulting in a Ser59Gly substitution in EiGS1-1 was identified in glufosinate-resistant plants. Rice calli and seedlings transformed with the mutant EiGS1-1 gene were resistant to glufosinate. Purified mutant EiGS1-1 expressed in yeast was more tolerant to glufosinate than the wild-type variant. These transgenic results correlate with a more glufosinate-resistant GS in the crude tissue extract of resistant versus susceptible E. indica plants. Structural modelling of the mutant EiGS1-1 revealed that Ser59 is not directly involved in glufosinate binding but is in contact with some important binding residues (e.g. Glu297) and especially with Asp56 that forms an intratoroidal contact interface. Importantly, the same Ser59Gly mutation was also found in geographically isolated glufosinate-resistant populations from Malaysia and China, suggesting parallel evolution of this resistance mutation.

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.

Contrasting plant ecological benefits endowed by naturally occurring EPSPS resistance mutations under glyphosate selection.

Concurrent natural evolution of glyphosate resistance single- and double-point EPSPS mutations in weed species provides an opportunity for the estimation of resistance fitness benefits and prediction of equilibrium resistance frequencies in environments under glyphosate selection. Assessment of glyphosate resistance benefit was conducted for the most commonly identified single Pro-106-Ser and less-frequent double TIPS mutations in the EPSPS gene evolved in the global damaging weed Eleusine indica. Under glyphosate selection at the field dose, plants with the single Pro-106-Ser mutation at homozygous state (P106S-rr) showed reduced survival and compromised vegetative growth and fecundity compared with TIPS plants. Whereas both homozygous (TIPS-RR) and compound heterozygous (TIPS-Rr) plants with the double TIPS resistance mutation displayed similar survival rates when exposed to glyphosate, a significantly higher fecundity in the currency of seed number was observed in TIPS-Rr than TIPS-RR plants. The highest plant fitness benefit was associated with the heterozygous TIPS-Rr mutation, whereas plants with the homozygous Pro-106-Ser and TIPS mutations exhibited, respectively, 31% and 39% of the fitness benefit revealed by the TIPS-Rr plants. Populations are predicted to reach stable allelic and genotypic frequencies after 20 years of glyphosate selection at which the WT allele is lost and the stable genotypic polymorphism is comprised by 2% of heterozygous TIPS-Rr, 52% of homozygous TIPS-RR and 46% of homozygous P106S-rr. The high inbreeding nature of E. indica is responsible for the expected frequency decrease in the fittest TIPS-Rr in favour of the homozygous TIPS-RR and P106S-rr. Mutated alleles associated with the glyphosate resistance EPSPS single EPSPS Pro-106-Ser and double TIPS mutations confer contrasting fitness benefits to E. indica under glyphosate treatment and therefore are expected to exhibit contrasting evolution rates in cropping systems under recurrent glyphosate selection.

An ABCC-type transporter endowing glyphosate resistance in plants.

Glyphosate is the most widely used herbicide in world agriculture and for general vegetation control in a wide range of situations. Global and often intensive glyphosate selection of very large weedy plant populations has resulted in widespread glyphosate resistance evolution in populations of many weed species. Here, working with a glyphosate-resistant (GR) Echinochloa colona population that evolved in a Western Australia agricultural field, we identified an ATP-binding cassette (ABC) transporter (EcABCC8) that is consistently up-regulated in GR plants. When expressed in transgenic rice, this EcABCC8 transporter endowed glyphosate resistance. Equally, rice, maize, and soybean overexpressing the EcABCC8 ortholog genes were made resistant to glyphosate. Conversely, CRISPR/Cas9-mediated knockout of the EcABCC8 ortholog gene OsABCC8 increased rice susceptibility to glyphosate. Subcellular localization analysis and quantification of glyphosate cellular levels in treated ABCC8 transgenic rice plants and isolated leaf protoplasts as well as structural modeling support that EcABCC8 is likely a plasma membrane-localized transporter extruding cytoplasmic glyphosate to the apoplast, lowering the cellular glyphosate level. This is a report of a membrane transporter effluxing glyphosate in a GR plant species, and its function is likely conserved in crop plant species.

Identification of the first glyphosate-resistant capeweed (Arctotheca calendula) population.

BACKGROUND: Glyphosate is routinely used in Australia to control the Arctotheca species Arctotheca calendula (L.) Levyns (referred hereinafter as capeweed). This study identifies the first global case of field-evolved glyphosate-resistant capeweed, collected from the grainbelt of Western Australia. RESULTS: In 2020, a capeweed biotype that was collected from Borden in the southern Western Australian grainbelt was confirmed to be glyphosate-resistant (referred hereinafter as Spence population). When compared to the pooled mortality of six field-collected, glyphosate susceptible capeweed populations (S1, S2, S3, S4, S5 and S6), the Spence population was found > 11-fold more resistant to glyphosate than the pooled results of the susceptible populations (S1-S6) at the lethal dose of 50% (LD50 ) level. The growth of the Spence population was also less affected, requiring > 13-fold more glyphosate to reduce growth than the pooled susceptible populations at the growth reduction of 50% (GR50 ) level. Sequencing of the plastidic 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene indicated no known single gene mutation imparting glyphosate resistance. This study, however, did not investigate any other known mechanisms that impart glyphosate resistance. When screened at the field-applied rate, this Spence population was also found to survive an inhibitor of acetolactate synthase (ALS) (metosulam) and an inhibitor of phytoene desaturase (diflufenican). CONCLUSIONS: This is the first confirmation of glyphosate resistance evolution in a capeweed population globally. With capeweed resistance already confirmed to photosystem-I inhibiting herbicides (paraquat and diquat), this study emphasizes the importance of using integrated measures that do not depend only on the use of non-selective herbicides for controlling herbicide resistance-prone capeweed populations. © 2021 Society of Chemical Industry.

Diversity of α-tubulin transcripts in Lolium rigidum.

BACKGROUND: Tubulin, the target site of dinitroaniline herbicides, is encoded by small gene families in plants. To better characterize the mechanisms of target-site resistance to dinitroaniline herbicides in the globally important weedy species Lolium rigidum, attempts were made to amplify and sequence α-tubulin transcripts. RESULTS: Four α-tubulin isoforms (TUA1, TUA2, TUA3 and TUA4) were identified in L. rigidum. Variations in the number and sequence of transcripts encoding these α-tubulin proteins were found in individuals from the two L. rigidum populations examined. Within and among populations, differences in the 5'- and 3'-untranslated regions of cDNA in TUA3 and TUA4 were identified. Furthermore, a novel double mutation, Arg-390-Cys+Asp-442-Glu, in the TUA3 transcript was identified and has the potential to confer dinitroaniline resistance. CONCLUSION: This research reveals the complexity of the α-tubulin gene family in individuals/populations of the cross-pollinated weedy species L. rigidum, and highlights the need for better understanding of the molecular architecture of tubulin gene families for detecting resistance point mutations. Although TUA4 is a commonly expressed α-tubulin isoform containing most frequently reported resistance mutations, other mutant tubulin isoforms may also have a role in conferring dinitroaniline resistance.

Exploring quinclorac resistance mechanisms in Echinochloa crus-pavonis from China.

BACKGROUND: Barnyardgrass (Echinochloa spp.) is a global weed in rice fields. Quinclorac is commonly used to control barnyardgrass. However, due to persistent use, quinclorac resistance has evolved. We obtained quinclorac-susceptible (QS) and -resistant (QR1, QR2) lines from the progeny of a single resistant E. crus-pavonis for a resistance mechanism study. RESULTS: Line QR1 exhibited resistance to high quinclorac rates (up to 6400 g ha-1 ), whereas line QR2 exhibited a resistance/susceptibility segregation ratio of 3:1 at the field or lower rates (400, 100 g ha-1 ). Intriguingly, a lower level of 14 C-quinclorac metabolism and hence a higher level of 14 C-quinclorac translocation was observed in QR1 than QS plants. The basal expression levels of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) and ACC oxidase 2 (ACO2) genes did not differ significantly between the QR1 and QS lines. However, more expression of ACS and ACO genes was induced by quinclorac treatment in QS than in QR1. Basal levels of ß-cyanoalanine synthase (ß-CAS) gene expression were similar in QS and QR1 plants, but a greater level of down-regulation was detected in QS than in QR1 plants after quinclorac treatment. CONCLUSION: These results indicate QR plants are less responsive to quinclorac than QS plants in terms of up-regulating quinclorac metabolism and ethylene synthesis. Resistance in this E. crus-pavonis line is likely controlled by a single major gene, involving possibly an alteration in auxin signal perception/transduction to the ethylene biosynthesis pathway. The ß-CAS is unlikely to play a major role in quinclorac resistance in this particular population.

Cytochrome P450 CYP81A10v7 in Lolium rigidum confers metabolic resistance to herbicides across at least five modes of action.

Rapid and widespread evolution of multiple herbicide resistance in global weed species endowed by increased capacity to metabolize (degrade) herbicides (metabolic resistance) is a great threat to herbicide sustainability and global food production. Metabolic resistance in the economically damaging crop weed species Lolium rigidum is well known but a molecular understanding has been lacking. We purified a metabolic resistant (R) subset from a field evolved R L. rigidum population. The R, the herbicide susceptible (S) and derived F2 populations were used for candidate herbicide resistance gene discovery by RNA sequencing. A P450 gene CYP81A10v7 was identified with higher expression in R vs. S plants. Transgenic rice overexpressing this Lolium CYP81A10v7 gene became highly resistant to acetyl-coenzyme A carboxylase- and acetolactate synthase-inhibiting herbicides (diclofop-methyl, tralkoxydim, chlorsulfuron) and moderately resistant to hydroxyphenylpyruvate dioxygenase-inhibiting herbicide (mesotrione), photosystem II-inhibiting herbicides (atrazine and chlorotoluron) and the tubulin-inhibiting herbicide trifluralin. This wide cross-resistance profile to many dissimilar herbicides in CYP81A10v7 transgenic rice generally reflects what is evident in the R L. rigidum. This report clearly showed that a single P450 gene in a cross-pollinated weed species L. rigidum confers resistance to herbicides of at least five modes of action across seven herbicide chemistries.

Mechanistic basis for synergism of 2,4-D amine and metribuzin in Avena sterilis.

The combination of herbicides with different modes of action has been adopted not only to improve weed control but also to increase the environmental sustainability of plant-protection products. In this study, we showed a synergistic effect of the auxin herbicide 2,4-D amine with the PSII-inhibiting herbicide metribuzin to control the global grass weed wild oat (Avena sterilis) population and investigated the underlying mechanisms. Pretreatment with 2,4-D amine did not change the foliar absorption of metribuzin but did increase metribuzin translocation to the roots and new leaves, although enhancement of the metribuzin metabolism was also observed. Considering that the expression level of the target site psbA gene is significantly higher in leaves than in roots, increased metribuzin translocation to new leaves is likely the major cause of the observed synergism, even though enhanced metribuzin metabolism may offset the metribuzin efficacy. This is the first report on the synergistic mechanism between 2,4-D amine and metribuzin in weed control.

2,4-D antagonizes glyphosate in glyphosate-resistant barnyard grass Echinochloa colona.

Glyphosate is often tank-mixed with auxinic herbicide 2,4-D for grass and broadleaf weed control. Here we examined the possible interaction of 2,4-D and glyphosate in barnyard grass, Echinochloa colona (L.) Link. The results showed that 2,4-D antagonizes glyphosate remarkably in glyphosate-resistant populations but only marginally in susceptible populations. This antagonism is related to reduced glyphosate uptake and (to a lesser extent) translocation. As 2,4-D has multiple, unpredictable effects on other herbicides, care must be taken when tank-mixing herbicides with 2,4-D.

Metribuzin resistance via enhanced metabolism in a multiple herbicide resistant Lolium rigidum population.

BACKGROUND: The photosystem II (PSII)-inhibiting herbicides are important for Australian farmers to control Lolium rigidum Gaud. and other weed species in trazine tolerant (TT)-canola fields. A L. rigidum population (R) collected from a TT-canola field from Western Australia showed multiple resistance to PSII, acetyl-coenzyme A carboxylase (ACCase) and acetolactate synthase (ALS) inhibitors. The mechanisms of multiple resistance in this R population were determined. RESULTS: The R population showed a low-level (about 3.0-fold) resistance to the PSII-inhibiting herbicides metribuzin and atrazine. Sequencing of the psbA gene revealed no differences between the R and susceptible (S) sequences. Furthermore, [14 C]-metribuzin experiments found no significant difference in metribuzin foliar uptake and translocation between the R and S plants. However, [14 C]-metribuzin metabolism in R plants was 2.3-fold greater than in S plants. The cytochrome P450 monooxygenase inhibitor piperonyl butoxide (PBO) enhanced plant mortality response to metribuzin and atrazine in both R and S populations. In addition, multiple resistance to ALS and ACCase inhibitors are due to known resistance mutations in ALS and ACCase genes. CONCLUSION: The results demonstrate that enhanced metribuzin metabolism likely involving cytochrome P450 monooxygenase contributes to metribuzin resistance in Lolium rigidum. This is the first report of metabolic resistance to the PSII-inhibiting herbicide metribuzin in Australian Lolium rigidum. © 2020 Society of Chemical Industry.

A Val-202-Phe α-tubulin mutation and enhanced metabolism confer dinitroaniline resistance in a single Lolium rigidum population.

BACKGROUND: A Lolium rigidum population collected from Western Australia was previously reported as highly resistant to dinitroaniline herbicides mainly due to a Val-202-Phe substitution in the target site α-tubulin protein. To further determine the contribution of the 202 mutation to resistance, two sub-populations, respectively comprising the 202 mutant and wild-type (WT) individuals, were isolated from within the same resistant population and subject to dinitroaniline herbicide doses. A rice transgenic study was conducted to demonstrate whether the amino acid substitution at the 202 residue confers resistance. In addition, as indicated in the phenotyping and genotyping study, non-target enhanced trifluralin metabolism was further examined in the same population. RESULTS: The 202 mutants were more resistant than the wild-type plants. Rice calli transformed with the L. rigidum mutant α-tubulin gene (Val-202-Phe) were more resistant to dinitroaniline herbicides relative to calli transformed with the wild-type gene. Also, enhanced trifluralin metabolism was detected in the 202 mutants in comparison to the susceptible seedlings. CONLCUSION: Both target-site Val-202-Phe α-tubulin mutation and non-target-site enhanced trifluralin metabolism co-exist in this dinitroaniline-resistant L. rigidum population. © 2019 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.

Aldo-keto Reductase Metabolizes Glyphosate and Confers Glyphosate Resistance in Echinochloa colona.

Glyphosate, the most commonly used herbicide in the world, controls a wide range of plant species, mainly because plants have little capacity to metabolize (detoxify) glyphosate. Massive glyphosate use has led to world-wide evolution of glyphosate-resistant (GR) weed species, including the economically damaging grass weed Echinochloa colona An Australian population of E colona has evolved resistance to glyphosate with unknown mechanisms that do not involve the glyphosate target enzyme 5-enolpyruvylshikimate-3-P synthase. GR and glyphosate-susceptible (S) lines were isolated from this population and used for resistance gene discovery. RNA sequencing analysis and phenotype/genotype validation experiments revealed that one aldo-keto reductase (AKR) contig had higher expression and higher resultant AKR activity in GR than S plants. Two full-length AKR (EcAKR4-1 and EcAKR4-2) complementary DNA transcripts were cloned with identical sequences between the GR and S plants but were upregulated in the GR plants. Rice (Oryza sativa) calli and seedlings overexpressing EcAKR4-1 and displaying increased AKR activity were resistant to glyphosate. EcAKR4-1 expressed in Escherichia coli can metabolize glyphosate to produce aminomethylphosphonic acid and glyoxylate. Consistent with these results, GR E colona plants exhibited enhanced capacity for detoxifying glyphosate into aminomethylphosphonic acid and glyoxylate. Structural modeling predicted that glyphosate binds to EcAKR4-1 for oxidation, and metabolomics analysis of EcAKR4-1 transgenic rice seedlings revealed possible redox pathways involved in glyphosate metabolism. Our study provides direct experimental evidence of the evolution of a plant AKR that metabolizes glyphosate and thereby confers glyphosate resistance.