Enhanced trifluralin metabolism can confer resistance in Lolium rigidum

Subscriber access provided by - Access paid by the | UCSB Libraries

Agricultural and Environmental Chemistry

Enhanced trifluralin metabolism can confer resistance in Lolium rigidum Jinyi Chen, Danica Erin Goggin, Heping Han, Roberto Busi, Qin Yu, and Stephen Powles J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02283 • Publication Date (Web): 02 Jul 2018 Downloaded from http://pubs.acs.org on July 7, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32

Journal of Agricultural and Food Chemistry

Enhanced trifluralin metabolism can confer resistance in Lolium rigidum Jinyi Chen, Danica Goggin, Heping Han, Roberto Busi, Qin Yu,* and Stephen Powles Australian Herbicide Resistance Initiative, School of Agriculture & Environment, University of Western Australia, Crawley, WA, Australia, 6009 *Corresponding author, e-mail address: [email protected]

ACS Paragon Plus Environment


Abstract Resistance to the pre-emergence herbicide trifluralin is increasing in Australian annual ryegrass (Lolium rigidum) populations. Three L. rigidum populations (R1, R2 and R3) collected from Australian grain fields were identified with trifluralin resistance. Both targetsite and non-target-site resistance mechanisms were investigated. No target-site α-tubulin mutations were detected in populations R1 and R3, while an Arg-243-Lys mutation was found in R2. Compared with the three trifluralin-susceptible populations, enhanced [14C]trifluralin metabolism, quantified by measuring the amount of [14C] label partitioning into the polar phase of a hexane:methanol system, was identified in all the three resistant populations. This is the first report of metabolic resistance to trifluralin. Coevolution of target-site and non-target-site resistance to trifluralin is occurring, and metabolic resistance is not rare in L. rigidum populations in Australia. A method was established for trifluralin metabolic resistance detection, overcoming the difficulties of quantifying this highly volatile herbicide by chromatographic methods. Keywords: Lolium rigidum; enhanced metabolism; trifluralin; resistance; coevolution


Page 2 of 32

Page 3 of 32


1. Introduction Trifluralin (α,α,α-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine) is a pre-emergence soilincorporated dinitroaniline herbicide used to control annual grass and certain dicot weed species. Trifluralin has been used extensively in fields crop, especially wheat, soybean and cotton 1. In Australia, trifluralin was first introduced into the cereal market in the 1960’s, and was adopted rapidly during the 1970’s. With the development of no-till farming systems and innovations in low-disturbance seeding machinery (e.g. knife points) in Australia, trifluralin is effectively used in various crops, especially wheat 2. Trifluralin targets plant tubulin, a structural protein that is mainly comprised of α- and βsubunits. The heterodimers of α-/β-tubulin assemble to form microtubules, which dynamically switch between stages of polymerisation and depolymerisation in the mitotic process 3. The balance of this dynamic property is essential for the survival of all eukaryotes 4

. By preventing polymerisation of tubulin, trifluralin affects mitosis in plants, inhibiting

growth and resulting in plant death. Ultrastructural studies have shown that after trifluralin treatment, the mitotic disruptions in plant cells are due to the absence of microtubules 5. Lolium rigidum (annual ryegrass) is the most widespread crop weed in the Australian grain belt. With high reliance on trifluralin for L. rigidum management in no-till systems, trifluralin-resistant L. rigidum populations from Australia were first reported in the 1990’s 6. Resistance has been slow to appear but now, trifluralin resistance in L. rigidum has reached a concerning level, with increasing numbers of resistant populations detected in resistance surveys across Australia 7-9. Annual ryegrass populations from Western and South Australia have been confirmed with high levels of trifluralin resistance 10, 11.



Page 4 of 32

Trifluralin resistance mechanisms can be target-site and/or non-target-site based. For target-site resistance (TSR), a single mutation in a tubulin gene results in trifluralin resistance in the self-pollinated grasses Eleusine indica 12 and Setaria viridis 13. In L. rigidum, the known α-tubulin mutations Val-202-Phe and Thr-239-Ile, together with two novel mutations Arg-243-Lys and Arg-243-Met have been recently identified

10, 14, 15

. In contrast,

potential non-target-site resistance (NTSR) mechanisms, such as reduced herbicide uptake, restricted translocation, or enhanced metabolism, have not yet been identified in trifluralinresistant weeds. Indeed, early studies have indicated that trifluralin is not readily metabolised by plants. In carrot

16

, peanut and sweet potato

17

, and L. rigidum 6, the

majority of applied trifluralin was unaltered in extracts of treated plants. Trifluralin is highly volatile (vapour pressure 9.5 mPa at 25 °C) and this is an important characteristic which affects the extraction procedure and the recovery rate in laboratory experiments on the fate of trifluralin. Few studies have specified the actual recovery of applied [14C]-trifluralin while reporting on the percentage metabolised. Due to its high volatility, conventional methods for herbicide metabolite extraction and concentration prior to chromatography are not entirely suitable for trifluralin. The establishment of an accurate diagnostic method for detection of weeds with enhanced trifluralin metabolism is thus essential. As NTSR is suspected, this study aimed to 1) Examine for NTSR mechanisms in trifluralinresistant L. rigidum populations, by investigating trifluralin uptake, translocation and metabolism. 2) Establish a reliable method for trifluralin metabolic resistance diagnosis in L. rigidum. 2. Materials and Methods


Page 5 of 32


2.1 Plant material Three herbicide-susceptible populations (SVLR1, VLR1 and DARGO (VicSeeds®), hereafter named as S1, S2 and S3) and three trifluralin-resistant populations (SLR31, M2/6 and M4/8, hereafter referred to as R1, R2 and R3) were used in this research. S1 and S2 are wellcharacterized susceptible populations originally collected from the field and used as controls in herbicide resistance studies 18, 19, while S3 is a commercial seed population 20. R1 has an extensive field history of herbicide selection and exhibits multiple resistance to herbicides with different modes of action

21

, including trifluralin. Trifluralin-resistant populations R2

and R3 were collected in a random herbicide resistance survey in Western Australia conducted in 2010 9. 2.2 Trifluralin resistance screening Seeds from the six populations were imbibed, seeded and sprayed pre-emergent with trifluralin (TriflurX; 480 g active ingredient L-1, Nufarm®) as described in Chen et al 10. Four pots with just-germinating seeds from each population (radical length approximately 5 mm) were treated with trifluralin. There were 20 seeds per pot, and four pots as replicates. Trifluralin was applied to the soil surface at the recommended field rate (960 g ha-1). Immediately after spraying, seeds were covered with a thin layer (1 cm) of soil, watered lightly and kept in the glasshouse (25/10°C day/night). The survivorship (classed as plants that emerged and grew actively) was recorded 21 days after treatment. 2.3 Identification of target-site mutations Leaf tissue samples (about 100 mg per sample) were taken for DNA and RNA isolation from untreated individuals in the susceptible populations and from trifluralin survivors in the



Page 6 of 32

resistant populations. Five individuals from S1 and at least seven from each resistant population were tested. The published primer pair ATG/UAG named

Intron

F/R

(Intron

F:

14

and a new pair of primers

5′-GGTTAATCTACTGTGCAGGTCATCT-3′,

Intron

R:

5′-TCGGACAACTCTCCCTCCTCCATAC-3′) were used to check for α-tubulin mutations with cDNA, DNA as the template, respectively. The Intron F/R primers were designed according to our unpublished L. rigidum tubulin DNA sequences (data not shown), and covered putative mutation sites Thr-239 and Arg-243 in α-tubulin. The nucleic acid isolation, reverse transcription, PCR and sequencing were the same as described in Chen et al.

10

. For the

primer pair Intron F/R, the PCR cycling was as follows: 94°C 5 min, 35 cycles of 94°C 30 s, 58°C 30 s, and 72°C 60 s, followed by a final extension step of 7 min at 72°C. 2.4 Uptake, translocation and metabolism of [14C]-trifluralin 2.4.1 Root application of [14C]-trifluralin In accordance with previous studies on trifluralin metabolism in plants

6, 17, 22

, some

experiments were performed with seedlings grown in solution culture and treated via the roots. Seeds of L. rigidum were germinated on moist filter paper at 22°C for five days, and when the coleoptile had reached 2-3 cm, ten seedlings were randomly selected and transferred to a 10 ml glass beaker containing 700 µl water supplemented with [14C]trifluralin (ring-14C [U]; 16 mCi/mmol stock dissolved in ethanol; American Radiolabeled Chemicals, St Louis, Missouri) at a final concentration ranging from 1.4-4.3 Bq/µl (corresponding to 2.3-7.3 µM trifluralin). After ensuring that all plant roots were immersed, the beakers were sealed with Parafilm to minimise trifluralin volatilisation, and stored in an incubator at 20/15°C day/night temperature in the dark. 2.4.2 Coleoptile [14C]-trifluralin application


Page 7 of 32


In order to more closely simulate trifluralin application conditions in the field, where trifluralin works effectively on the coleoptile

23, 24

, some seedlings were treated with one

droplet of [14C]-trifluralin placed on the coleoptile. Seeds were germinated as described in section 2.4.1. Ten randomly-selected seedlings from each population were transferred into a round plastic container (8.5 cm×11.5 cm×5 cm) on a 9 cm-diameter filter paper moistened with 3 ml distilled water. The [14C]-trifluralin stock was diluted to a final concentration of around 125-976 Bq/µl (corresponding to 0.2-1.6 mM trifluralin) in 0.25% (v/v) of the nonionic surfactant BS1000. Pilot studies showed that due to volatilisation of the droplet on the coleoptile, less than 10% of applied trifluralin actually penetrated the tissue. A 1 µl droplet of diluted [14C]-trifluralin was smeared onto the coleoptile of each seedling. All seedlings were kept vertical during the application. After treatment, all containers were sealed with the lids on and kept in an incubator at 20/15°C in the dark. 2.4.3 Measurement of uptake and translocation of [14C]-trifluralin in L. rigidum Pilot experiments showed that 48 h was a suitable time period for treatment and assessment, as the plants were confirmed to absorb and metabolise trifluralin during this time period. After treatment with [14C]-trifluralin for 48 h via either the roots or coleoptile, the seedlings were washed thoroughly with 20% methanol containing 0.2% Triton X-100 (5 ml per seedling), and blotted dry. The dry seedlings were pressed and oven dried at 70°C for 48 h, then exposed to a storage phosphor screen (BAS-IP MS2040; GE Healthcare, Little Chalfont, UK) for 24 h. Visualisation of trifluralin translocation was achieved using a phosphor imager (Typhoon 5, GE Healthcare Life Sciences). Ten seedlings from each population were assessed.



To compare the total coleoptile uptake of trifluralin in susceptible versus resistant populations, five oven-dried whole plants from populations S1 and R1 were combusted in a Biological Sample Oxidizer (RJ Harvey Instrument Corporation, Hillsadale, NJ, USA). The 14

CO2 released was trapped in the cocktail solution (1:1 mixture of IRGA-Safe scintillation

cocktail and Carbo-Sorb E®) and radioactivity measured by liquid scintillation spectrometry 25

.

2.5 Extraction of [14C]-trifluralin metabolites After washing as described in 2.4.3, [14C]-trifluralin-treated seedlings were thoroughly homogenised using a mortar and pestle, with an initial 1 ml of cold 80% methanol. To check for in vitro degradation of [14C]-trifluralin, untreated seedlings (ten per sample) were extracted in 80% methanol containing 9.76 kBq [14C]-trifluralin. The homogenate was transferred to a 2 ml chilled microcentrifuge tube. The mortar and pestle were washed with another 600 µl 80% cold methanol, and the washing solution added to the homogenate. The 5 min per sample extraction procedure was conducted in a fume hood. The mixture was well vortexed for 1 min prior to centrifugation at 4,500 g for 90 s. After the volume was recorded, the supernatant was transferred into a fresh 2 ml tube. There were ten seedlings per replicate and four replicates for each population. The amount of [14C] in the seedling washing solution and clarified extract was determined by liquid scintillation counting. Clarified seedling extracts were concentrated by evaporation prior to thin-layer chromatography (TLC) and high-performance chromatography (HPLC) analysis, in order to allow loading of >167 Bq for each sample. 2.6 Analysis of trifluralin metabolites 2.6.1 Thin-layer chromatography


Page 8 of 32

Page 9 of 32


For qualitative TLC analysis of metabolites, concentrated samples from S1 and R1, dissolved in methanol, were spotted onto 10 × 10 cm silica gel TLC plates (Sigma-Aldrich, Sydney, Australia)

alongside

authentic

chloroform:ethanol (9:1)

26

[14C]-trifluralin,

and

plates

were

developed

in

. Radiolabelled compounds were detected by exposing the TLC

plates to a storage phosphor screen for 24 h. 2.6.2 High performance liquid chromatography HPLC was used to obtain a better resolution of trifluralin metabolites than was possible with TLC

27

. A pilot HPLC separation was performed with parent [14C]-trifluralin to determine

retention time and compare its loss rate (volatility) during sample concentration with that of the non-volatile herbicide 2,4-D. [14C]-trifluralin was mixed with [14C]-2,4-D (ring

14

C [U];

specific activity 2.035 GBq mmol−1, American Radiolabeled Chemicals) at a final concentration of approximately 3.8 Bq/µl each in 1200 µl of 100% methanol. The mixture was divided into six 200 µl subsamples. Three were directly injected into the HPLC, and the other three were evaporated to dryness and redissolved in 200 µl 100% methanol. Fifty µl of each sample was injected and separated by gradient reverse-phase HPLC equipped with a 600E dual-head pump with a 717 Plus autosampler (Waters, Milford, MA, USA). Separation was conducted on a Waters Spherisorb 5 µM ODS2 column (250mm long × 4.6mm i.d.). Radioactivity was detected with an inline Beta-RAM model 2B detector (IN/US Systems Inc., Pine Brook, NJ). The solvents used were MQ water with 0.1% formic acid (v/v) (solvent A), and acetonitrile with 0.1% formic acid (v/v) (solvent B). The chromatographic conditions involved a 30 min linear gradient from 10-85% solvent B, then held for 5 min before immediately changed to 100% solvent B, and held again for 5 min before changed back to the initial condition of 10% B for 15 min column equilibration prior to the next injection. The



flow rate of both the HPLC and the scintillant pump on the Beta-RAM detector was 1 ml min1

. Trifluralin retention time was recorded, and trifluralin and 2,4-D loss rate was calculated

by comparing the areas under the peaks in the directly-injected and dried-down samples. For HPLC analysis of trifluralin metabolites in plant extracts, the concentrated samples were separated and detected by the same HPLC system as described above. Injection volumes were adjusted so that the same amount of radioactivity (167 Bq) was injected for each sample. The retention time of different peaks corresponding to parent trifluralin and metabolites from each population was used to compare the nature of trifluralin metabolism in each sample. 2.6.3 Partitioning analysis of metabolites In order to minimise the inconsistencies in [14C] recovery encountered when concentrating extracts for chromatography, unconcentrated extracts were partitioned against hexane to quantify the [14C] in the organic fraction (mainly parent trifluralin) and aqueous fraction (mainly polar metabolites). Extracts were prepared from root- and coleoptile-treated seedlings as described above. Four replicates each with ten seedlings were used for metabolite extraction. After extraction, 300 µl 100% hexane was added to the clarified seedling extracts and mixed by vortexing for 1 min before centrifugation at 4,500 g for 90 s. Two phases formed in the solution, with hexane above (organic phase), and 80% methanol below (aqueous phase), in a volume ratio of approximately 3:10. Ten µl from the hexane phase and 30 µl from the aqueous phase were taken for scintillation counting. The total radioactivity in each phase was then calculated. Control samples containing [14C]-trifluralin alone were subjected to hexane partitioning to determine the distribution of parent trifluralin in the two phases. Three


Page 10 of 32

Page 11 of 32


independent experiments with different resistant populations were conducted with S1 as the control population to ensure reproducibility. 2.7 Statistical analysis Data from the partitioning experiments were expressed as percentages (radioactivity from each phase divided by total radioactivity in the sample). An unpaired t-test was performed to identify differences between S1 and resistant populations in terms of 14C recovered from the combustion experiments and the partitioning experiments, as well as the radioactivity distribution after partitioning. 3. Results 3.1 Trifluralin resistance characterization and α-tubulin gene sequencing The pot experiments with commercially formulated trifluralin showed that, as expected, the three susceptible populations were fully controlled at 960 g ha-1, while the resistant populations demonstrated up to 40% survivorship at the same rate (Table 1). No α-tubulin gene mutations known to confer trifluralin resistance in L. rigidum were identified in survivors from R1 and R3 compared with the untreated S1. However, the known Arg-243-Lys mutation in the α-tubulin gene

15

was identified in survivors from R2, with four plants

containing this mutation out of nine plants analysed. 3.2 Trifluralin uptake and translocation Uptake of [14C]-trifluralin was measured by combusting the whole seedling of coleoptiletreated S1 and R1 seedlings. R1 seedlings absorbed significantly more trifluralin than S1 (9% of applied trifluralin in R1 vs 5% in S1; p=0.02) (Table 2). Further confirmation that reduced trifluralin uptake does not occur in the resistant populations comes from the total recovery



of trifluralin from the seedling extracts used for quantitative metabolite analysis (see section 3.5). Around 20% of applied trifluralin was recovered from all extracts, with no significant differences between S1 and each resistant population (Table 2). The phosphor images showed no obvious visual differences in the translocation pattern of [14C]-trifluralin between susceptible and resistant populations, whether trifluralin was applied via the roots (Fig. 1) or the coleoptile (Supporting Information Fig. S1). Forty-eight hours after treatment, seedlings from all populations displayed visually uniform distribution of radioactivity throughout all plant tissues. This establishes that trifluralin was absorbed and readily translocated in all susceptible and resistant populations tested. 3.3 Comparison of evaporation-loss of [14C]-trifluralin and [14C]-2, 4-D The retention time of parent [14C]-trifluralin was 36 min under our HPLC conditions. In a comparison of the recovery of 2,4-D and trifluralin following concentration by evaporation, 24% of the original 2, 4-D signal was lost after evaporation and resuspension, whilst 97% of trifluralin was lost in the same process (Fig. 2). This result demonstrated that as trifluralin is highly volatile (and the relative volatility of trifluralin metabolites is unknown), concentration of trifluralin-containing plant extracts for chromatography renders quantification largely meaningless, and these methods can only be used for metabolite quantification (e.g. retention time or Rf value). 3.4 TLC and HPLC analysis of trifluralin metabolites Extracts from [14C]-trifluralin treated seedlings run on TLC plates contained bands comigrating with the parent trifluralin (Rf 0.91), and bands remaining at the origin of the plates (Fig. 3, lane 2), representing polar metabolites possessing the [14C]-labelled trifluralin ring.


Page 12 of 32

Page 13 of 32


Extracts from untreated seedlings spiked with [14C]-trifluralin during the extraction process did not show any signals except those co-migrating with parent trifluralin (lane 1), indicating that degradation of trifluralin during the extraction process was negligible. Although the loss of trifluralin during sample concentration made quantification invalid, there was a clear visual difference in the proportion of [14C] signal present in the parent and metabolite bands in the susceptible (S1) vs. resistant (R1) populations. Under our HPLC conditions, trifluralin and its major polar metabolite were eluted at about 36 min and 21 min, respectively (Fig. 4). In the spiked samples of untreated S1 and R1, few metabolites were present (Fig. 4a, b), consistent with the TLC results (Fig. 3). The HPLC chromatograms from treated susceptible (S1) and resistant (R1) plants show the same pattern of non-polar metabolites clustered around the trifluralin peak plus the major polar metabolite peak at 21 min, indicating that the resistant population does not produce extra metabolites. To confirm that the trifluralin-resistant populations are capable of enhanced metabolism of the herbicide compared to the susceptible populations, accurate quantification of the amount of polar metabolites produced by each population was performed using hexane partitioning of unconcentrated extracts. 3.5 Partitioning analysis of trifluralin metabolites Partitioning of parent [14C]-trifluralin (dissolved in 80% methanol) against 100% hexane resulted in 97% of the total radioactivity being detected in the nonpolar (hexane) phase. Partitioning analysis was then conducted on extracts from seedlings treated via the roots or coleoptile. To keep sample numbers manageable, three independent experiments on roottreated seedlings (S1, S2 and S3; S1 and R1; S1, R2 and R3) were conducted separately, with S1 as the control population since no significant differences were found between the three



susceptible populations. As the distribution of radioactivity between the nonpolar and polar phases in the three experiments on S1 was the same, the data were combined. In contrast to the 25% of recovered radioactivity that was present in the polar phase of all three susceptible biotypes, 45-55% of the total radioactivity partitioned into the polar phase in extracts from the three resistant populations (Fig. 5). Similarly, at 48 h after coleoptile treatment of S1 and R1, 65% of recovered radioactivity was in the polar phase of extracts from R1, compared to

Enhanced trifluralin metabolism can confer resistance in Lolium rigidum

Recommend Documents