Metribuzin Resistance in a Wild Radish (Raphanus raphanistrum

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Agricultural and Environmental Chemistry

Metribuzin resistance in a wild radish (Raphanus raphanistrum) population via both psbA gene mutation and enhanced metabolism Huan Lu, Qin Yu, Heping Han, Mechelle J. Owen, and Stephen Powles J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05974 • Publication Date (Web): 14 Jan 2019 Downloaded from http://pubs.acs.org on January 16, 2019

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Journal of Agricultural and Food Chemistry

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Metribuzin resistance in a wild radish (Raphanus raphanistrum) population

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via both psbA gene mutation and enhanced metabolism

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Huan Lu, Qin Yu*, Heping Han, Mechelle J. Owen, Stephen B. Powles

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Address: Australian Herbicide Resistance Initiative, School of Agriculture and Environment,

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University of Western Australia, Perth, WA 6009, Australia

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*Correspondence to: Qin Yu, Australian Herbicide Resistance Initiative, School of Agriculture

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and Environment, University of Western Australia, Perth, WA 6009, Australia, Tel: 61-8-

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64887041, E-mail: [email protected]

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ACS Paragon Plus Environment


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Abstract

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There have been many studies on target-site resistance (TSR) to PSII-inhibiting herbicides, but

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only a few on the non-target-site resistance (NTSR). Here, we reported both TSR and NTSR to

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metribuzin in a wild radish population. Dose-response studies revealed a higher level of

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resistance to metribuzin in the resistant (R) compared to the susceptible (S) population.

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Sequencing of the target psbA gene revealed the known Ser-264-Gly mutation in R plants. In

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addition, a higher level of [14C]-metribuzin metabolism, and consequently, a lower level of

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[14C] translocation were also detected in the R plants. These results demonstrated that both

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psbA gene mutation and enhanced metabolism contribute to metribuzin resistance in this

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wild radish population. Furthermore, this resistant population showed resistance to ALS-

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inhibiting herbicides due to multiple ALS gene mutations. This is the first report in wild radish

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of metabolic herbicide resistance, in addition to the target-site psbA gene mutation.

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Keywords: Wild radish, metribuzin resistance, psbA gene mutation, non-target-site resistance

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mechanism, enhanced metabolism

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1 Introduction

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Metribuzin [4-amino-6-tert-butyl-3-methylsulfanyl-1,2,4-triazin-5-one] is a triazinone pre-

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and post-emergent herbicide that controls a range of dicot and monocot weed species in

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several crops, including wheat (Triticium aestivum) and lupin (Lupinus angustifolius).

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Metribuzin is important for Australian agriculture due to its broad weed control spectrum and

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relatively low cost. As a PSII-inhibiting herbicide, metribuzin inhibits photosynthesis by

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blocking the electron transport from QA to QB, leading to photooxidation of lipid and

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chlorophyll, and consequently plant death.

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Persistent herbicide selection has led to herbicide resistance evolution in many weed species.

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The first case of metribuzin resistance was reported in Amaranthus powelli in Idaho, USA.1

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Globally, there are biotypes of 74 weed species reported resistant to PSII-inhibiting

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herbicides.2 Weeds can resist herbicides by many mechanisms broadly separated into target-

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site resistance (TSR) or non-target-site resistance (NTSR) mechanisms. TSR refers to target

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gene mutations, gene amplification or overexpression.3 So far, TSR to PSII-inhibiting

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herbicides is due to mutations within the psbA gene that result in amino acid substitutions in

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the chloroplastic D1 protein. NTSR refers to any mechanisms reducing herbicide dose to a

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sub-lethal level before reaching a herbicide target site.3 These include, but are not limited to,

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reduced herbicide uptake, translocation and/or enhanced herbicide metabolism. NTSR to

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PSII-inhibiting herbicides due to enhanced metabolism has been identified in several weed

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species, including Abutilon theophrasti, Lolium rigidum and Alopecurus myosuroides.4 It is

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known that herbicide metabolism can be mediated by cytochrome P450 monooxygenases

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(P450s) and/or glutathione S-transferases (GSTs).3 P450 catalyzed increased simazine

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metabolism was reported in simazine resistant L. rigidum.5 Rapid atrazine metabolism via GST



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conjugation was reported in atrazine resistant A. theophrasti 6 and recently in a Amaranthus

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palmeri population.7 NTSR studies are often overlooked due to technical difficulties and often

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lower level of resistance compared to TSR. Therefore, our knowledge of NTSR is limited,

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relative to TSR.8

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Wild radish (Raphanus raphanistrum) is a damaging dicot weed infesting crops in Australia.9-

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densities.12 Many herbicides are effective on wild radish. However, over-reliance on

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herbicides has led to multiple resistance evolution in wild radish. Resistance exists to

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acetolactate synthase (ALS), PSII, phytoene desaturase (PDS) inhibitor herbicides and auxinic

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herbicides.11,

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herbicides in wild radish was identification of the well-known target psbA gene mutation (Ser-

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264-Gly) conferring triazine resistance.15 Here, a triazine resistant wild radish population

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(H1/16) from the Western Australia (WA) grain belt, was investigated for TSR and NTSR

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mechanisms to metribuzin. The aims of this study were to: (1) characterise the metribuzin

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resistance level, and (2) determine metribuzin resistance mechanisms in this population.

Wild radish is cross pollinated, genetically diverse, and causes crop yield losses even at low

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The first molecular evidence of target-site resistance to PSII-inhibiting

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2 Materials and methods

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2.1 Plant Materials

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The metribuzin resistant (R) wild radish population (H1/16) was collected from the high

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rainfall zone in the northern WA grain belt, a region where lupin/wheat rotations are common.

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Plants surviving 900 g atrazine ha-1 were hand-crossed to produce seeds for further

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experiments. A well-known susceptible (S) population (WARR7)16 was used as control in all


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experiments. Narrow-leafed lupin, naturally tolerant to metribuzin,17 was used in the

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metribuzin metabolism study as a positive control.

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2.2 Metribuzin dose response assay

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Seeds were germinated on moist filter paper in the dark at room temperature for 2 days.

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Germinated seeds were transplanted into 18 cm diameter pots containing potting mix (50%

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peatmoss, 25% sand and 25% pine bark). Seedlings at the two- to three-leaf stage were

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treated with metribuzin (Mentor WG Herbicide, Adama Australia Pty Ltd, St Leonards, NSW,

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Australia) plus 0.25% (v/v) BS1000 (1000 g L-1 of alcohol alkoylate) at doses of 0, 105, 210,

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420, 840, 1260 and 1680 g a.i. ha-1 for R and 0, 13, 26, 52, 105 and 210 g a.i. ha-1 for S. Plants

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were herbicide treated using a custom-built sprayer (TeeJet XR11001 flat fan, Spraying

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Systems Co, Wheaton, IL, USA) delivering a volume of 118 L ha-1 at 200 kPa with a speed of

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3.6 km h-1. There were 12 plants per pot and three pots per treatment. The plants were

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returned to the glasshouse after herbicide treatment. Survivorship was assessed 21 days after

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treatment (DAT), and plants were harvested and oven-dried at 60℃ for three days before dry

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weight was measured.

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2.3 Full-length psbA gene sequencing

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DNA was extracted from untreated R and S plants using the CTAB method.18 The full-length

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psbA gene was sequenced according to Lu, et al.19 Ten plants from each R and S populations

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were sequenced, and results were analysed using DNAMAN (version 7.0, Lynnon Corp,

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Quebec, Canada).

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2.4 [14C]-metribuzin uptake and translocation



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Seedling preparation was the same as described in section 2.2, except that seedlings were

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transplanted into plastic cups (60 × 60 × 60 mm) and grown in a controlled environment room

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(CER) at 20/15℃ day/night temperatures, 12/12 h photoperiod (300 µmol m-2 s-1) and 75%

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relative humidity. When plants reached the two- to three-leaf stage, the first true leaf was

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marked and the metribuzin treatment solution ([14C]-metribuzin (Institute of Isotopes Co Ltd,

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Budapest, Hungary) mixed with metribuzin commercial formulation plus 0.25% (v/v) BS1000)

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was applied as a 1 µL droplet. Total concentration applied per plant was equal to 30 g

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metribuzin ha-1 with radioactivity of 0.65 kBq. Both above- and under-ground plant material

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was harvested at 24, 48 and 72 h after treatment (HAT) with seven replicates for each time

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point. Plant roots were washed out of potting mix with 100 mL water and the treated leaf was

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rinsed with 20 mL 20% (v/v) methanol plus 0.2% (v/v) Triton X-100. Radioactivity in root and

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leaf rinse solution was measured using liquid scintillation counter (LSC). Plants were pressed

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against tissue paper and oven-dried at 60℃ for 3 days, then exposed to a phosphor screen

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film (GE Healthcare, Little Chalfont, UK) for 24 h. The [14C] translocation was visualised using

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a Typhoon Phosphor Imager (GE Healthcare, Little Chalfont, UK). After imaging, the oven-

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dried plants (5 plants per population per time point) were separated into treated leaf (TL),

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untreated aboveground part (UAP) and root and combusted in a Biological oxidizer (RJ Harvey

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Instrument Corporation, Hillsadale, NJ, USA) for radioactivity measurement using LSC

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according to Yu, et al.20 Herbicide uptake was expressed as percentage of applied, and

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herbicide translocation was expressed as percentage of total absorbed (the sum of the

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radioactivity from each section).

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2.5 [14C]-metribuzin metabolism


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At the two- to three-leaf stage, the first and second true leaves of seedlings were marked and

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metribuzin treatment solution ([14C]-metribuzin mixed with 0.25% (v/v) BS1000) was applied

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as a 1 µL droplet to each leaf. Total concentration applied per plant was equal to 30 g

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metribuzin ha-1 with radioactivity of 4.17 kBq. The aboveground material was collected at 24,

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48, 72, 96 and 120 HAT. There were three replicates per population per time point and two

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seedlings per replicate. The treated leaves in each harvest plant were washed, blotted-dry,

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and the radioactivity in leaf wash was measured. Then the aboveground material was snap-

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frozen in liquid nitrogen and stored at -80 ℃ until use.

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Aboveground plant material was ground with liquid nitrogen in a pre-chilled mortar and

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pestle, then homogenized in 2 mL 80% (v/v) cold methanol. Additional 2 mL 80% (v/v) cold

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methanol was used to wash the mortar and pestle. The homogenate was pooled and

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centrifuged at 7, 500 g for 15 min at 4 ℃. The supernatant was transferred into a new 15 mL

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centrifuge tube and the residue was suspended with 1.5 mL 80% (v/v) cold methanol,

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followed by a final suspension in 1.5 mL 50% (v/v) cold methanol. The supernatant was pooled,

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and dried under vacuum using SpeedVac (Savant, Farmingdale, NY, USA) and resuspended in

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300 µL 50% (v/v) methanol prior to centrifugation at 19, 000 g for 3 min.

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HPLC analysis was performed using gradient reverse-phase HPLC equipped with a 600E dual-

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head pump with 717 plus autosampler (Waters, Milford, MA, USA). A 4.6 × 250 mm Apollo

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C18, 5 μm particle column (Grace Davison Discovery Sciences, Deerfield IL) was used with a

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mobile phase flow rate of 1.0 mL min-1 at 24℃. The mobile phase was a linear gradient over

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20 min of 15-30% acetonitrile in water (with 1% acetic acid) then held at 30% acetonitrile for

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10 min before an immediate change to 100% acetonitrile, then held at 100% acetonitrile for

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10 min followed by re-equilibration with 15% acetonitrile for 15 min. A β-RAM model 2B



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detector (IN/US Systems Inc, Pine Brook, NJ, USA) was used to monitor [14C] eluting from the

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column. Scintillant at 1.0 mL min-1 was mixed post column. Under these conditions, standard

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[14C] metribuzin eluted at 34.7 min. Injection volumes were adjusted to provide the same

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sample loading with respect to total radioactivity for all samples. The proportion of the

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herbicide and metabolites were expressed as a percentage peak area of total radioactivity in

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the sample injection.

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2.6 Multiple herbicide resistance screening

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Germinated seeds were transplanted into trays (approximate 30 seeds per tray) containing

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potting mix. At the two- to three-leaf stage, herbicides were applied at the recommended

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field rates or the rates fully controlling the S plants, including chlorsulfuron (Chlorsulfuron

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750WDG Herbicide, Landmark Operations Ltd, Macquarie Park, NSW, Australia) at 10 g a.i. ha-

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1,

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3.5/10.5 g a.i. ha-1, 2,4-D (Amicide advance 700, Nufarm Australia Ltd, Laverton North, VIC,

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Australia) at 625 g a.e. ha-1 and mesotrione (Callosto 480SC Herbicide, Syngenta Canada Inc,

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Guelph, Ontario, Canada) at 12 g a.e. ha-1,

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2.7 ALS gene sequencing

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DNA was extracted as described in section 2.3. The ALS gene fragment covering all five

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conserved domains was amplified using the primer pair WR122F/WR653R according to Han,

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et al.21 The PCR products were purified and sequenced using three internal sequencing

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primers WR205R, WR376R and WR574F.21 DNA and deduced amino acid sequences from R

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and S plants were aligned and analysed using DNAMAN.

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2.8 Data analysis

imazapic/imazapyr (Onduty Herbicde, BASF Australia Ltd, Southbank, VIC, Australia) at


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The LD50 (herbicide rate causing 50% plant mortality), GR50 (herbicide rate causing 50% plant

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growth reduction) and M50 (time required for metabolising 50% herbicide) were estimated

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using a three-parameter Sigmoidal-logistic model (SigmaPlot 13.0, Systat Software, San Jose,

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CA, USA):

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𝑦 = a / [1+(x/x0)b]. Where a is the upper asymptote, x0 is LD50, GR50 or M50 and b is the slope

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at x0. Difference between treatments in all experiments was analysed by one-way analysis of

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variance (ANOVA) or t-test using the software Prism 5.0 (GraphPad Software, La Jolla, CA,

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USA).

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3 Results

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3.1 Dose-response to metribuzin

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Metribuzin resistance in the R population (H1/16) was confirmed based on plant survival rate

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and dry weight in response to metribuzin treatment. R plants showed no mortality at a rate

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(210 g metribuzin ha-1) that killed all S plants. The LD50 value of the R population was 1105 g

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ha-1 compared to 15 g ha-1 for the S population, thus confirming high level metribuzin

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resistance (74-fold, P < 0.001, Fig 1A, Table 1). The R/S GR50 ratio also indicated a similar level

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of resistance (65-fold, P < 0.001, Fig 1B, Table 1).

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3.2 Full-length psbA gene sequencing

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The full-length psbA gene from both R and S plants was amplified and sequenced. Alignment

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of the psbA gene between R and S samples revealed one single nucleotide change (AGT to

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GGT) in all R plants tested, resulting in the widely known PSII-inhibiting herbicide resistance

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endowing Ser-264-Gly mutation in the D1 protein.



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3.3 [14C]-Metribuzin uptake and translocation

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No significant differences were found in [14C]-metribuzin uptake between R and S plants in all

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time points tested (except for lower absorption in S plants at 48 HAT), with more than 90% of

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applied [14C]-metribuzin being foliar absorbed by 24 HAT (Table 2).

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Very little metribuzin translocation occurred in R and S plants, with more than 95% [14C]

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remaining in the treated leaf of R plants at all time points tested (Table 2). However, S plants

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tended to translocate more [14C], especially to the untreated aboveground parts (Table 2, Fig

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2). At each time point, significantly more [14C] was detected in the untreated aboveground

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part of S plants than R plants. However, variation in the translocation pattern (variable

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percentages of [14C] translocation to parts of the plant, i.e. treated leaf, untreated

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aboveground part and root) in individual R and S populations was observed (images not

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shown).

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3.4 [14C]-metribuzin metabolism

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Metribuzin metabolism was detected in both R and S plants. The parent [14C]-metribuzin

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resolved at a peak retention time (RT) of approximate 34.7 min (Fig 3A). As expected,

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metribuzin-tolerant lupin, as a positive control, metabolised more than 50% metribuzin at 48

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HAT with 5 major metabolites eluted at RT = 10.8 min, RT = 11.3 min, RT = 13.3 min, RT = 20.7

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min and RT = 38.0 min, respectively (Fig 3D). Both R and S wild radish plants metabolised

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metribuzin, but the R plants metabolised metribuzin at a higher rate than the S plants (Fig 3B

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& C, Fig 4A) and four major metribuzin metabolites were detected in wild radish plants

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(metabolite 1, RT = 7.4 min; metabolite 2, RT = 9.8 min; metabolite 3, RT=13.3min; metabolite

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4, RT = 15.4 min). R plants metabolised metribuzin faster than S plants, as non-linear

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regression analysis (Fig 4A) estimated that the R plants had a M50 (time required for


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metabolising 50% metribuzin) value of 60 h, at least 2-fold faster than the S plants (M50 value

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in the S plants could not be estimated, but >120 h). At each time point, the percentage of

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parent [14C]-metribuzin remaining in plant tissue was significantly less in R than in S plants

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(Fig 4A). Correspondingly, significantly greater amounts of major metribuzin metabolites

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were accumulated in R than in S plants (Fig 4B).

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3.5 Multiple resistance test

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Multiple herbicide resistance of the R population was tested with several herbicides of

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different modes of action. Resistance was evident to the ALS-inhibiting herbicides

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chlrosulfuron and imazapic + imazapyr, but not to the auxinic herbicide 2,4-D or the 4-

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hydroxyphenylpyruvate dioxygenase (HPPD)-inhibiting herbicide mesotrione (Table 3).

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3.6 ALS gene sequencing

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The ALS gene was sequenced in 8 plants surviving chlorsulfuron and 4 plants surviving

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imazapic/imazapyr from the R population and 5 untreated plants from the S population. In 12

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resistant samples analysed, two of them contained the two known ALS resistance mutations

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of Pro-197-Thr and Asp-376-Glu (each in a heterozygous status), six plants contained the

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single mutation of Pro-197-Thr (heterozygote) and two plants possessed the mutation of Pro-

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197-Thr/Ser (compound heterozygote). Only two R plants were homozygous for the Pro-197-

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Thr mutation.

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4 Discussion

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In this study, we demonstrate that metribuzin resistance in a wild radish biotype is due to

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both target-site and non-target-site based resistance. Target-site resistance to metribuzin is

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conferred by a point mutation in the psbA gene, resulting in the Ser-264-Gly substitution in



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the D1 protein. Non-target-site resistance is conferred by enhanced rates of metribuzin

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metabolism. In addition, there is target-site resistance to ALS-inhibiting herbicides.

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4.1 Target-site based resistance to metribuzin

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Target-site based resistance to PSII-inhibiting herbicides has been reported in many weed

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species3,

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resistance to PSII-inhibiting herbicides have been identified in resistant plants,3, 19, 22 with two

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mutations (Ser-264-Gly and Phe-274-Val) detected in wild radish.15,

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resistance to PSII-inhibiting herbicides has evolved relatively slowly in wild radish in Australian

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agricultural fields and the reason why is unclear, although limited usage of PSII-inhibiting

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herbicides in Australian major crops is a contributing factor.

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4.2 Non-target-site based resistance to metribuzin

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As the target-site Ser-264-Gly mutation confers high level of resistance to triazine and

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triazinone herbicides, additional resistance mechanisms are often not studied. Here, we

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examined for possible NTSR in the population containing the target-site Ser-264-Gly mutation.

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Metribuzin resistance in the R biotype is clearly not due to reduced herbicide uptake.

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Although the S plants translocated significantly more [14C] from treated leaf to other plant

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parts, especially to the untreated aboveground part (Table 2), metribuzin can be metabolised

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by R and S wild radish plants (Fig 3 & 4), and hence it is difficult to determine whether the

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differential translocation pattern observed in R and S plants is related to [14C]-metribuzin or

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to its metabolites. Therefore, it is hard to conclude if metribuzin translocation is a primary

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mechanism. Similarly, reduced translocation was also observed in a tembotrione resistant A.

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palmeri population with enhanced tembotrione metabolism.23 The resistant A. palmeri plants

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due to psbA gene mutations. Currently, eight psbA gene mutations conferring


19

Target-site based

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metabolised tembotrione faster but translocated [14C] less than the susceptible plants.

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Herbicide metabolites may be less easily translocated than the parent herbicide, and reduced

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herbicide translocation could be related to enhanced herbicide metabolism.23 It is possible

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that reduced [14C] translocation is caused by enhanced metribuzin metabolism in R wild radish

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plants, rather than a primary resistance mechanism (see below).

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Metabolic herbicide resistance is being increasingly reported in resistant weed species.3, 24, 25

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Metabolic herbicide resistance is little reported in dicot weeds, limited to only a few species,

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such as Sinapis arvensis,26 Abutilon theophrasti,6, 27 and Amaranthus spp.23, 28, 29 Here, we

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establish that metribuzin is metabolised by both R and S wild radish plants. However, the R

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plants metabolised metribuzin faster (more than 2-fold) than the S plants, demonstrating that

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enhanced herbicide metabolism is a resistance mechanism in addition to the target-site Ser-

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264-Gly mutation. To our knowledge, this is the first time that metabolic herbicide resistance

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is revealed in the economically important dicot weed species wild radish. We observed that

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the R plants metabolised metribuzin significantly more than the S plants across each time

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point, and correspondingly the R plants translocated [14C] less than the S plants (Table 2, Fig

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2). Therefore, differential metribuzin translocation in R and S populations is likely the

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consequence of differential metribuzin metabolism.

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Although herbicide susceptible, most plant species have a basal level of metabolism of some

284

herbicides.25 Metribuzin is a metabolizable herbicide, and the crop lupin is naturally

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metribuzin tolerant due to its capacity to metabolise metribuzin.30 Even the S wild radish

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plants exhibited a level of metribuzin metabolism (Fig 3B), and individuals with a higher level

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of metribuzin metabolism can be selected by repeated metribuzin application, especially

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under low herbicide rates. It was also noticed that metribuzin metabolism in wild radish is



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similar but not identical to lupin (Fig 3). For instance, a major polar metribuzin metabolite

290

resolved at 20.7 min in lupin was not found in wild radish, in addition to a significantly higher

291

level of a non-polar metabolite eluted at 38.0 min (Fig 3).

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In studies of herbicide resistance, metabolic herbicide resistance can be overlooked,

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especially when a strong target-site mutation (like the Ser-264-Gly mutation) is identified.

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Nevertheless, the reality is that metabolic herbicide resistance along with target-site

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resistance is commonly occurring in many weed species, such as L. rigidum,31 A.

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myosuroides,32 Amaranthus spp.29 Unlike chloroplastic psbA gene mutations, which are

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maternally inherited,4 metabolic resistance is usually conferred by nuclear genes.33-35

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Therefore, dispersal of metabolic resistance alleles via pollen is more rapid relative to the

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psbA gene mutation via seeds.

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In summary, this study reported for the first time that non-target-site enhanced herbicide

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metabolism additional to target-site psbA gene mutation endow metribuzin resistance in a

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wild radish population. Coevolution of multiple resistance mechanisms is an evolutionary

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reality in the field, tactics other than chemical control should be considered to manage

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resistance evolution proactively.

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Funding sources

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Huan Lu has received a Scholarship for International Research Fee China (IRFSC) sponsored

308

by China Scholarship Council (CSC, No. 201606750002) and the University of Western

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Australia. This research was partially funded by the Australian Grains Research and

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Development Corporation (GRDC).


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of triazine-resistant Powell amaranth (Amaranthus powellii) in Idaho. Weed Sci. 1992, 40, 507-

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Biol. 2010, 61, 317-347.

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Press, Boca Raton, FL, USA, 1994; pp. 27-60.

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is a mechanism of simazine resistance in Lolium rigidum. Pestic. Biochem. Physiol. 1993, 46,

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theopgrasti) biotype due to enhanced glutathione S-transferase activity. Plant Physiol. 1991,

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detoxification via glutathione S-transferase (GST) conjugation confers a high level of atrazine

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resistance in Palmer amaranth (Amaranthus palmeri). Pest Manage. Sci. 2017, 73, 2236-2243.

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herbicides: a major challenge for weed science in the forthcoming decade. Pest Manage. Sci.

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413 414 415 416 417 418 419 420



421

Figure Captions

422

Fig 1. Metribuzin dose response based on survival rate (A) or dry weight (B) of the R (○) and S

423

(●) wild radish populations at 21 DAT. Data were means ± standard error (n = 3).

424 425

Fig 2. Visualisation of [14C]-metribuzin translocation in representative R and S plants at 24 (A

426

& B), 48 (C & D) and 72 (E & F) HAT with camera (left) and phosphor images (right).

427 428

Fig 3. HPLC chromatograms of [14C]-metribuzin standard (A), metribuzin and its major

429

detectable metabolites in S (B) and R (C) wild radish plant at 72 HAT, and lupin (D) at 48 HAT.

430 431

Fig 4. Non-linear regression of HPLC profiling of [14C]-metribuzin metabolism in both R (○) and

432

S (●) wild radish plants, parent [14C]-metribuzin remaining (A), major metribuzin metabolites

433

accumulating (B) in the R and S plants at 24, 48, 72, 96 and 120 HAT. (* in each time point

434

indicates significant difference in percentage of metabolised metribuzin or major metribuzin

435

metabolites between the R and S plants by the t-test. *, **, *** indicate significant differences

436

at the level of P < 0.05, P < 0.01 and P < 0.001, respectively).

437


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Page 21 of 26


Tables Table 1. Parameter estimates of metribuzin dose response curve of the R and S wild radish populations. Standard errors are in parentheses. Population

a

b

LD50 or GR50a

R2

R/S ratio

Plant survival rate R

99.6 (1.86)

4.0 (0.45)

1105 (33.65) a

0.99

74

S

100.0 (0.01)

6.7 (0.01)

15 (0.00) b

1

-

Plant dry weight

aDifferent

R

1.0 (0.07)

0.94 (0.19)

389 (96.96)a

0.96

65

S

1.0 (0.03)

1.6 (0.39)

6 (1.49) b

1

-

letters in each R and S pair mean significantly difference in LD50 or GR50 value between the

R and S populations using t-test, P < 0.001.

Table 2. [14C]-metribuzin uptake and translocation in the R and S wild radish plants at 24, 48, 72 HAT. The plants from R and S populations were separated to root, untreated aboveground part (UAP) and treated leaf (TL). Standard errors are in parentheses.

Population

R

Time (HAT)

24

S R S

48

Uptake (% of [14C]-

Translocation (% of [14C]-metribuzin absorbed)a

metribuzin applied)a

Root

UAP

TL

93.96 (0.79) a

2.30 (0.61) a

1.57 (0.37) b

96.13 (0.91) a

91.55 (1.11) a

4.45 (2.13) a

5.12 (1.03) a

90.43 (2.91) a

95.47 (0.62) a

2.30 (0.46) a

1.68 (0.20) b

96.02 (0.35) a

93.66 (0.17) b

3.72 (0.75) a

3.09 (0.52) a

93.19 (1.19) a



R

72

S

Page 22 of 26

95.85 (0.87) a

1.19 (0.09) b

1.85 (0.59) b

96.96 (0.56) a

94.07 (0.55) a

4.43 (1.44) a

7.18 (0.43) a

88.38 (2.52) b

aDifferent letters in each R and S pair at a time point mean significant differences using t-test, P < 0.05.

Table 3. Percentage survival of plants from resistant (R) and susceptible (S) wild radish populations in response to herbicides of different modes of action. Survival (% survival) Herbicide

Mode of action

Herbicide dose (g

ha-1)

R population

S population

Chlorsulfuron

ALS

10

100

0

Imazapic/imazapyr

ALS

3.5/10.5

58

0

2,4-D

Auxin

625

4

0

Mesotrione

HPPD

12

0

0


Page 23 of 26


Figure graphics

A

80

60

40

20

B

100

Dry Weight (% of control)

Survival Rate (% of control)

100

80

60

40

20

R S 0

0

Control 0.1

1

10

100

1000

Control 0.01

Metribuzin (g ha-1)

1

Metribuzin (g ha-1)

Fig 1.

Fig 2.


10

100

1000


Fig 3.


Page 24 of 26

Page 25 of 26


A

100

Metribuzin (%)

80

* 60

40

R S

20

0.1 Control

* *

*** **

Major Metabolites (%)

**

*

60

**

40

*** *

20

0

1

10

100

1000

0.1 Control

1

10

Hours after treatment

Hours after treatment

Fig 4.


100

B


Table of contents


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Metribuzin Resistance in a Wild Radish (Raphanus raphanistrum

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