T+',\T I I tt,j:';l ï I iJT[ -3õ. 3- ?3 L¡iliì,\FTS- I Mechanisms of resistance to herbicides which inhibit acetolactate synthase in annual ryegrass (Lolium rígidum) John T. Christopher (Jack Christopher) Thesis submitted for the degree of Doctor of Philosophy in The University of Adelaide Faculty of Agricultural and Natural Resource Sciences f 1l Table of Contents Title page (i) Table of contents (ii) Abst¡act (ix) Declaration (xi) Acknowledgements (xii) Abbreviations (xiv) Chapter 1 Int¡oductlon l.t Herbicide resistance 1.1.1 The development of resistance 2 1.L.2 Herbicide resistance in Australia 6 L,2 Mechanisms of herbicide resistance 1.2.L Potential mechanisms of resistance I L.2.2 Resistance to inhlbitors of photosystem II. 10 1.2.9 Resistance to herbicides interactlng with photosystem I r3 1.2.4 Resistance to herbicides which inhibit lipid synthesis t4 1.3 Plant interactions with herbicides whlch inhibit acetolactate synthase IALS) r.3.1 Herbicides which inhibit Acetolactate synthase r6 I.3.2 Acetolactate synthase, the target enzyme 19 1.3.3 Phytotoxicity caused by ALS inhibition 42 f .3.4 Selectivity of Al5-inhibiting herbicides 47 r.3.5 Modes of resistance to ALs-inhibiting herbicides 53 f .3.6 Resistance in weed species to ALS inhibiting herbicides 54 t.4 Herbicide cross-resistance in Lolium ngtdum 55 1.5 Objectives of this ProJect 56 iii Chapter 2 Materials and Methode 2.L Materials 2.1.L Plant material 58 2.1.2 Chemicals 59 2.2 Plant culture 2.2.1 Germination 60 2.2.2 Pot culture 61 2.2.3 HydroPonics culture 6r 2.3 Herbicide treatment 63 Chapter 3 Il'ffre¡encæ ln tJle res¡nnse to he¡btctdes of two Lrígñfurm btotypes suggest two reslstance mech¡nlsms' 3. t Introduction 64 3.2 Materials and methods 9.2.L Sulfonylurea a¡rd imidazolinone herbicides 69 3.2.2 Herbicides which do not inhiblt ALS 69 3.3 Spectra of herbicide reslstance g.g. f Crop-selective sulfonylurea and lmidazolinone herbicides 70 5.g.2 Non-selective sulfonylurea and imidazoli¡rone herbicides 75 3.3.3 Response to other groups of herbicides 80 3.4 Discussion 88 iv Chapter 4 Chlo¡sqlfrrron reslstance ln Lrigidtutt can lnvolve lncreased metabollsm 4.1 Introduction 90 4.2 Materials and methods 4.2.I 1r4ç¡Chtorsulfuron metabolism studies: Method A 91 4.2.2 tl4ClCfrtorsulfuron metabolism studies: Method B 92 4.2.3 Acetolactate synthase assays 93 4.3 ll4Clchlorsulfuron metabolism in excised shoots 93 of biotype SLR31 using method A 4.4 Il4Clchlorsulfuron metabolism in excised shoots 99 of biotype SLR3I using method B 4.5 Il4Clchlorsulfuron metabolism in excised shoots to4 of biotype WLRI using method B 4.6 Discussion t07 Chapter 5 He¡blctde reslstancc ln Lrígúdum can invoþe herbictde-lnsengltlve ALSI 5.r Introduction r09 5.2 Materials and methods 5.2. L Enryme PreParatlon rro 5.2.2 Enz5rme AssaY conditions rlo 5.3 Acetolactate s]¡nthase activitv llt v Chapter 6 Uptake, t¡anslocatlon and met¡bollsm of chlorsulfuron tn intact plants. 6.r Introduction tt7 6.2 Materials and methods 6.2.1 Root uptake of chlorsulfuron rr8 6.2.2 Leaf uptake of chlorsulfuron lr8 6.4 Root uptake and whole plant response to chlorsulfuron L20 6.5 Leaf uptake and of chlorsulfuron in i¡rtact plants 124 6.6 Discussion t27 Chapter 7 Are rnlxed fr¡nctton oddases lnvoþed ln chlo¡sulfr¡ron metabollsm ln Lrigrtfutfit ? 7.L Introductlon L28 7.2 Materials and methods 7.2.1 Mixed function oxidase inhibitors 13r 7.2.2 Crop safeners L32 7.3 Effectors of mixed function o>ddases 7.3.1 MFO inhibitors (herbictde synergists) r33 7.3.2 Crop safeners r40 7.4 Discussion r43 vi Chapter I ffiect olttght pertod followtng he¡btctde a¡rpllcatlon on chlorsulft¡ron reslstance 8.1 Introduction r46 8.2 Materials and methods 8.2.1 [laClchtorsulfuron metabolism by excised seedlings L48 in response to darkness or light 8.2.2 Response of whole plants to liglrt period following t49 chlorsulfuron aPPlication 8.3 llaClchlorsulfuron metabolism in response to darkness or light r50 8.4 Response of whole plants to light period following r53 chlorsulfuron application 8.5 Response of whole plants to light pertod following r55 diclofop-methyl application 8.6 Discussion 158 Chapter I DoGs sclectlon wttl dlctofopmettryl necessarlly lead to chlorsr¡lfr¡¡on reslgtance ? 9. f Introduction r60 9.2 Materials and methods 9.2.L Selection Procedure r6r 9.2.2 Testing of ProgenY t62 9.3 Effect of diclofop selection upon chlorsuHuron-response 163 of biotv?e WR6. 9.4 Discussion r68 vll Chapter 10 rnheritanrce of sulfonylurea resistance with herbtclde-sensitlve AIS. 10.1 Introduction L70 ro.2 Materials and methods lO.2.l Crossing procedure L72 LO.2.2 Selection of resistant parents and t73 fìrst generatlon crosses. fO.2.3 Second generation crosses (F2) 175 LO.2.4 Experiments ustreg indlvidual families t76 ro.3 Inheritance of resistance from SLRSI parents which t77 survive chlorsulfuron ro.4 Inheritance of resistance from SLR3I parents which 186 sun¡ive diclofop-methyl ro.5 Discussion r95 Chapter 11 Inherttance of sulfonylurea reslstance lnvolvlng herbtctde-lnsensltlve ALSi I l. t Introduction L97 lL.2 Materials and methods r98 11.3 Inheritance of chlorsulfuron from blotJ4¡e WLR1 200 LL.4 Inherttance of sulfometuron-methyl resistance 204 from blotype WLRI. f f .5 Inheritance of resistance in individual backcross families 208 f I.6 Discussion 2tL viii Chepter L2 Comparlson of herbtctde reslþnse of L.rigÍd;um btotypes wtth dlfrerent he¡ülclde t¡eatment hlstory. 12.L Introduction 2L5 12.2 Materials and methods 2r6 L2.3 Response to herbicides 217 L2.4 Discussion 224 Chapter 13 Concluslons and ft¡¡ther research needs f 3. t Conclusions 226 L3.2 Chlorsulfuron. diclofop-methyl cross resistance 23L I3.3 Further research 233 Llteratr¡re clted 238 IX Abstract There are at least two mechanisms of resistance to ALS inhibitors in L.ngidum populations. Biotype SLR3I is cross-resistant to some crop-selective ALS inhibitors but is not cross-resistant to non- selective ALS inhibitors. The population has herbicide-sensitive ALS but excised seedlings of SLR3I metabolised Ii 4C]chlorsulfuron at approximately twice the rate of a susceptible biotype in the tissue closest to the meristems. Population WLRI is resistant to crop-selective and non-selective ALS inhibitors and has ALS which is less-sensitive to inhibition by both crop-selective and non-selective herbicides. Thus, herbicide-insensitive ALS may cause resistance to both selective and non- selective sulfonylurea and imidazolinone herbicides in L.rigidum. A second resistance mechanism exists, however, which dose not cause resistance to non-selective ALS inhibitors and involves increased chlorsulfuron metabolism. The major metabolite of [l4c]chorsulfuron in L.rtgidum coeluted with that of wheat suggesting that the major pathway of metabolism in L.rigidum may be the same as that in wheat. Similar HPLC elution proflles for resistant and susceptible L.rtgi.dum biotypes suggest that the pathway of metabolism is the same in all biotypes. There were no differences in root or leaf uptake between intact resistant and susceptible plants which could explain resistance. Some mixed function oxidase inhibitors slightly increase chlorsulfuron phytotoxicity toward biotype SLR3I but combined treatment caused less damage to SLRSI than chlorsulfuorn alone applied to a susceptible biotype. Conversly, the crop safener NAA reduced the phytotoxic effect of chlorsulfuron toward wheat but not toward L.rigtdum biotype SLR3t. Darkness, after commencement of Il4Clchlorsulfuron treatment, caused metabolism in excised seedlings to slow dramatically. Whote SLR3I plants placed in darkness immediately following chlorsulfuorn treatment suffered increased phytotoxic effects, but damage X was less than that suffered by a susceptible biotype. These results suggest that chlorsulfuron metabolism in L.rigidum does not involve mixed function oxidase enzyrnes and/or that other resistance mechanisms may exist in population SLR3l. In population SLR3l, diclofop-methyl and chlorsulfuron resistance are both nuclear encoded dominant or semi dominant characters and at least one gene controlling diclofop-methyl resistance in the absence of chlorsulfuron resistance is present. In population WLRI, sulfometuron-methyl and chlorsulfuron resistance are both nuclear encoded and, probably, dominant traits. At least one gene controlling chlorsulfuron resistance in the absence of sulfometuron-methyl resistance is present. Different herbicide treatments in the field may lead to resistance to ALS inhibitors in populations of L.ri,gidum. Four diclofop- methyl applications may cause cross-resistance to chlorsulfuorn but diclofop-methyl treatment is unlikely to select for herbicide-insensitive ALS. Three applications of chlorsulfuron may cause chlorsulfuron resistance while five applications may select for herbicide-insensitive AI^S. Chlorsulfuron appears less likely than diclofop-methyl, to select for cross- resistance to herbicides from different chemical groups with different modes of action. xl Declaratlon This work contains no material which has been accepted for the award of any other deglree or diploma in any university or ottrer tertiary institution and, to the best of my knowledge and belief, contalns no materlal previously published or written by another person, except where due reference has been made. I give my consent that thls work may be photocopied or loaned from the universit¡r library. Signed Date: rltltl J.T.Christopher xtr Acknowledgments I would like to thank my proJect supervisors Dr. S.B. Powles, Dr. J.A.M. Holtum and Dr. D.R. Liljegren for their help and support throughout the course of this study. I also wish to thank all of the members of the Weed Science Research team of the Department of Crop Protection at the Waite Institute for their help, encouragement and cheerful companionship' I would also like to thank Ms M.