Supplementarymaterial

Climate change increases the risk of herbicide-resistance weeds due to enhanced detoxification

Maor Matzrafi1, Bettina Seiwert2, Thorsten Reemtsma2, Baruch Rubin1 and Zvi Peleg1

1.The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, P.O. Box 12, 7610001 Rehovot, Israel.

2.Department of Analytical Chemistry, Helmholtz-Centre for Environmental Research - UFZPermoserstrasse 15,04318 Leipzig, Germany.

Fig. S1 Response of Lolium populations to diclofop-methyl under different temperature regimes

Table S1List of populations and accessions used in this study.

Table S2Response of different grass weeds to diclofop-methyl under different temperature regimes.

Table S3Response of Lolium populations to diclofop-methyl under different temperature regimes.

Table S4Chemical name and structure of the pinoxaden parent molecule and its major metabolites

Table S5Effect of temperature onpinoxaden detoxification in B. hybridum.


Table S1 List of populations and accessions used in this study.

Species / Population / Accession / Location / Resistance / Resistance mechanism / Herbicides tested / Reference
Alopecurus myosuroides / Roth99 / Rothamsted (U.K.) / - / S / - / James et al. (1995)
Avenasterilis / OA / Alumim (Organic) / - / S / - / Matzrafi et al. (unpublished data)
Brachypodium hybridum / BrI-782 / Schiller / + / NTS / Diclofop-methyl methyl, pinoxaden / Matzrafi et al. (2014)
Brachypodium hybridum / BrI-638 / Mevo Hama / - / S / - / Matzrafi et al. (2014)
Lolium multiflorum / EMR / Ein ha-Mifratz / + / NTS / Diclofop-methyl, pinoxaden / Matzrafi et al. (2013)
Lolium multyflorum / KY / KfarYehoshua / - / S / - / Matzrafi et al. (unpublished data)
Lolium rigidum / NO / Nahal Oz / + / TS (Cys2088 to Arg) / Clodinafop-propargyl, diclofop-methyl, clethodim, pinoxaden / Matzrafi et al. (2014)
Lolium rigidum / AL / Alumim (Organic) / - / S / - / Matzrafi et al. (unpublished data)
Setariaviridis / RF / Rehovot / - / S / - / Matzrafi et al. (unpublished data)

Table S2 Response of different grass weeds to diclofop-methyl under different temperature regimes.

Temperature regime / Low (10/16oC)a / High (28/34oC)
Species / Shoot fresh weight (% of control)±SD / Survival (%) / Shoot fresh weight (% of control)±SD / Survival (%) / TSIb
Target site resistance
Lolium rigidum (NO) / 77.31±22.81 / 100 / 81.51±49.71 / 100 / 1.05
Non-herbicide resistance
Alopecurus myosuroides (Roth99) / 4.06±6.79 / 0 / 52.07±8.76*** / 60 / 12.83
Avenasterilis(OA) / 6.97±2.02 / 0 / 15.75±2.02** / 60 / 2.26
Lolium rigidum (AL) / 3.64±1.53 / 0 / 17.551±1.53* / 16 / 4.82
Setariaviridis (RF) / 1.08±2.24 / 0 / 10.37±2.51** / 80 / 9.6

Data is means (n=5), *, ** and *** indicate significant difference between

low and high temperature regimes, for every treatment and every population

separately at P ≤ 0.05, P < 0.01, and P < 0.001, respectively.

a.Night and day temperatures.

bTemperature affected sensitivityindex (TSI) was calculated as the ratio between the responses of the population/accession in two different temperature regimes. HigherTSI indicates decreased herbicide sensitivityat high temperature regime.

Table S3 Response of Lolium populations to diclofop-methyl under different temperature regimes.

Temperature regime / Low (10/16oC)a / High (28/34oC)
Lolium population / Herbicide dose (g h-1) / Shoot fresh weight (% of control) ± SD / Survival (%) / Shoot fresh weight (% of control) ± SD / Survival (%) / RI b
KY / 0 / 100±15.76 / 100 / 100±29.92 / 100 / 22
180 / 21.23±10.29 / 0 / 81.19±28.51*** / 100
360 / 13.28±6.35 / 0 / 72.80±24.50*** / 100
720 / 9.66±4.84 / 0 / 62.17±18.32*** / 100
1440 / 9.546±2.04 / 0 / 6.25±5.76 / 33
2880 / 7.73±3.15* / 0 / 3.35±2.02 / 0
ED50 / 30 / 670
EMR / 0 / 100±20.85 / 100 / 100±54.92 / 100 / 4
180 / 53.30±12.97 / 100 / 126.49±33.28** / 100
360 / 50.11±15.06 / 83 / 77.51±21.06* / 100
720 / 43.21±25.33 / 66 / 84.52±39.08* / 83
1440 / 21.63±6.85 / 0 / 35.15±52.08* / 50
2880 / 6.45±2.30 / 0 / 19.92±3.57*** / 16
ED50 / 299 / 1223
NO / 0 / 100±44.36 / 100 / 100±40.89 / 100 / -
180 / 50.97±7.31 / 100 / 136.13±63.11* / 100
360 / 42.88±15.04 / 100 / 140.26±61.81** / 100
720 / 49.15±10.74 / 83 / 133.36±54.73* / 66
1440 / 57.76±29.71 / 66 / 202.31±32.20** / 83
2880 / 41.26±1.73 / 66 / 127.94±72.32 / 66
ED50 / >2880 / >2880

Data is means (n=5), *, ** and *** indicate significant difference betweenlow and high temperature regimes, for every treatment and every population separately at P ≤ 0.05, P < 0.01, and P < 0.001, respectively.

aNight and day temperatures.

bResistance index (RI) was calculated as the R/S ratio of the ED50determine the level of resistance of the resistant plants compared to that of the sensitive plants.

Table S4Chemical name and structure of the pinoxaden parent molecule and its major metabolites.

Common namea / Chemical name (IUPAC) / Retention time [min] / Sum formula [M+H]+ / Exact mass (m/z) / Fragments (m/z)
M1 (Pinoxaden) / 2,2-dimethyl-propionic acid 8-(2,6-diethyl-4-methyl-phenyl)-9-oxo- 1,2,4,5-tetrahydro-9H-pyrazolo[1,2-d][1,4,5] oxadiazepin-7-yl ester / 9.83 / C23H32N2O4 / 401.244 / 317.1865
M2 / 8-(2,6-diethyl-4-methyl-phenyl)- tetra-hydro-pyrazolo[1,2- d][1,4,5]oxadiazepine-7,9-dione / 7.19 / C18H24N2O3 / 317.1865 / 101.071
M3 / 8-(2,6-diethyl-4-methyl-phenyl)- 8-hydroxy -tetrahydro- pyrazolo[1,2-d][1,4,5] oxadiazepine-7,9-dione / 4.8 / C18H24N2O4 / 333.1814 / 315.1705; 303.1708; 101.0708
M4 / 8-(2,6-diethyl-4-hydroxymethyl- phenyl)-9-hydroxy-1,2,4,5- tetrahydro-pyrazolo[1,2- d][1,4,5]oxadiazepin-7-one / 7.48 / C18H24N2O4Na / 355.1627 / 333.1814; 315.1702
TP-463* / 8-(4-(3,4-diethylphenoxy)benzene-1,2-diol)- 9-hydroxy-1,2,4,5- tetrahydro-pyrazolo[1,2- d][1,4,5]oxadiazepin-7-one / 6.01 / C24H29N2O6, C24H28N2O6Na / 463.1845; 441.2026; 315.1709 / 333.1814; 315.1705
M5 / 8-[2,6-diethyl-4-(3,4,5- trihydroxy-6-hydroxymethyl- tetrahydro-pyran-2-yloxy- methyl)-phenyl]-9-hydroxy- 1,2,4,5-tetra-hydro-pyrazolo[1,2- d][1,4,5]oxadiazepin-7-one / 4.35 / C24H34N2O9 / 495.2343 / 333.1814; 315.1705; 303.1708; 101.0708

a.EFSA.

*proposed structure.

Table S5Effect of temperature onpinoxaden detoxification in B. hybridum.

Common name / Accession/ temperature regimea / Molecular peak area (normalized to mass)
Time after application / 2 / 48 / 96
M1 (Pinoxaden) / Resistant/Low / 2442.83±694.34 / 786.05±58.33*** / 210.62±36.82
Resistant/High / 6013.36±621.04** / 293.814±44.09 / 105.76±42.52
Sensitive/Low / 11397.30±836.09** / 564.44±40.13* / 260.73±18.24*
Sensitive/High / 6991.50±836.09 / 431.92±40.13 / 205.44±16.89
M2 / Resistant/Low / 40332.60±3788.90 / 8326.67±711.29*** / 2057.05±411.26*
Resistant/High / 46353.60±3507.90 / 1127.82±601.15 / 278.14±530.93
Sensitive/Low / 28106.60±1908.00 / 4972.69±346.22*** / 988.48±59.04***
Sensitive/High / 25687.9±1741.7 / 1058.97±346.22 / 481.03±49.87
M3 / Resistant/Low / 621.50±90.18 / 453.33±98.66 / 96.47±14.58
Resistant/High / 958.52±83.49** / 295.32±91.34 / 120.53±20.61
Sensitive/Low / 93.16±36.76 / 1644.53±238.47** / 1641.97±255.91***
Sensitive/High / 255.90±31.06** / 326.03±238.47 / 130.80±255.91
M4 / Resistant/Low / 383.95±167.02 / 7699.70±1453.70** / 1638.33±370.73**
Resistant/High / 1137.53±154.63** / 2091.88±1345.90 / 280.14±454.05
Sensitive/Low / ND / 115.71±429.47 / 561.04±102.23
Sensitive/High / ND / 2284.04±429.47** / 597.43±102.23
TP-463 / Resistant/Low / 122.16±116.74 / 337.5±162.87*** / 129.06±40.37***
Resistant/High / 84.52±57.82 / 55.74±24.93 / 39.5±28.34
Sensitive/Low / 38.91±27.66 / 157.97±60.94*** / 139.67±39.45***
Sensitive/High / 56.44±24.73 / 34.84±7.8 / 35.92±19.63
M5 / Resistant/Low / ND / 686.74±229.82 / 812.64±269.91
Resistant/High / ND / 1760.65±209.80** / 2028.57±348.46*
Sensitive/Low / ND / 115.71±489.38 / 561.04±292.57
Sensitive/High / ND / 2481.33±489.38** / 3161.93±292.57***

Data is means (n=5).

*, ** and *** indicate significant difference between low and high temperature regimes, for every treatment and every populationseparately at P ≤ 0.05, P < 0.01, and P < 0.001, respectively.

a.Night and day temperatures: high=28/34oC, low=10/16oC.

ND = non-detected.

Supplementary References

James EH, Kemp MS, Ashton LBB, Moss SR(1995)Phytotoxicity of trifluoromethyl- and methyl-substituted dinitroaniline herbicides on resistant and susceptible populations of black-grass (Alopecurus myosuroides).Pest Sci43:273–277.

Matzrafi M, Peleg Z, Rubin B(2013) Evolution of molecular and biochemical mechanisms of herbicides resistant in Lolium spp. The 16th European Weed Research Society Symposium. Samsun, Turkey, 274.

Matzrafi M, Gadri Y, Frenkel E, Rubin B, Peleg Z(2014) Evolution of herbicide resistance mechanisms in grass weeds. Plant Sci 229:43–52.

EFSA(2013) Conclusion on the peer review of the pesticide risk assessment of the active substance pinoxaden.EFSA Journal11:3269, 112 pp.