zz-z-0L The Effects of Acetolactate Synthase (ALS) lnh¡b¡t¡ng Herbicides on the Growth, Yield, Nodulation and Nitrogen Fixation of Selected Legumes Annette Anderson B.Sc. (Hons). James Cook University A thesis submitted for the degree of Doctor of Philosophy in the Faculty of Agricultural and Natural Resource Sciences at Adelaide University. Depafment of Agronomy and Farming Systems Roseworthy Campus Adelaide University September 2001 TABLE OF CONTENTS ABSTRACT \rI DECLARATION CHAPTER 1 : GENERAL INTRODUCTION .......... 1 1.1 Legumes, Nítrogen Fixøtion and ALS-Inhíbiting Herbicides........... I 1.2 Aims ønd Structure of the Thesis 3 1.2.1 Aim of this thesis, 3 1.2.2 Structure of thesis J CHAPTER 2: LITERATLIRE REVIE}V................ 5 2.1 Introduction 5 2.2 AcetolactøteSynthøseInhibitingHerbicides............ 7 2.2.1 InEoduction 7 2.2.2 Mode of action of AlS-inhibiting herbicides................. 9 2.3 Degradalion and Persistence Of ALS-Inhibiting Herbicides in the 5oi1...............'......... 11 2.4 Microbial Transþrmation ønd Degradation.......... ................. 16 2.5 Elfects of AlS-Inhibiting Herbícídes on Non-Legume Crops..... .......,....... 17 2.6 Effects of ALS-Inhibitíng Herbícíde Residues on Legumes and Symbíotic Nitrogen Fkation t, 2.6.1 Introduction 22 2.6.2 Noduleformation.. 23 2.6.3 Benefits of legumes and nitogen fixation to the farming system 24 2.6.4 Herbicides and nitrogen fixation 27 2.6.5 Effects of AlS-inhibiting herbicides on the host plant 28 2.6.6 Effects of AlS-inhibiting herbicides on survival and growth of rhizobia...... 34 2.6.7 Effects of AlS-inhibiting herbicides on the nodulation process .......... ..........37 2.6.8 The effect of AlS-inhibiting herbicides on niEogen fixation 40 CHAPTER 3: THE EFFECTS OF F.LT]METSULAM ON THE GROWTH OF CHICKPEAS - A GLASSHOUSE EXPERIMENT 43 3.1 Introduction 43 3.2 Materials and 44 3.2.1 Experimental set up 44 3.2.2 Treatrnents 45 3.2.3 Experimental design 45 3.2.4 Plant dry matter and nodulation measruements 46 3.3 Þ ost ltr 47 3.3.1 Shoot biomass. 47 3.3.2 Root biomass 49 3.3.3 Relative growth rates ............. 5l 3.3.4 Nitrogen content of chicþea shoots and roots 52 3.3.5 3.4 Discussion... 57 3.5 Summøry and Key Findings...... 61 CHAPTER 4: THE EFFECTS OF ALS-N\HIBITING HERBICIDES ON GROWTH AND PRODUCTION OF MEDICAGO RUGOSA.... 62 4.1 Introduction 62 4.2 Materids and Methods 65 4.2.1 Site selection and soil characteristics................... 65 4.2.2 Treatrnents 66 4.2.3 Experimentaldesign 67 4.2.4 Sampling and data collection..... 70 4.3 Results...,.,... 72 4.3.1 Biomass and yield for wheat (year one) 72 4.3.2 Shoot biomass of M rugosa from second year of experiment. 72 4.3.3 Relative growth rate of medic. 't3 4.3.4 Seed Yield 15 4.3.5 Total nitrogen 75 4.3.6 Nuhient Analysrs 75 4.4 Dìscussion... 77 4.5 Sammary and Key Findings...... 80 CHAPTER 5: EFFECTS OF,ALS.INHIBITING HERBICIDES ON CHICKPEA GROWTH AND PRODUCTION IN THE F'IELD .................81 5.1 filroduction 8t 5.2 Materials and Methods 83 5.2.1 Site selection and soil characteristics................... 83 5.2.2 Treafrnents 84 5.2.3 Experimentaldesign 85 5.2.4 Sampling and measurements 88 5.2.5 Data analysis and interpretation. 90 .91 5.3.2 Leaf A¡ea Index .94 5.3.3 Grain yield.. .96 5.3.4 Absolute and relative growth rates .97 5.3.5 Plant Nitrogen or Total Nitrogen...... 101 5.3.6 Nutrientanalysrs 107 109 5.4 Discussion... 110 CHAPTER 6: DEVf,LOPMENT OF METHODOLOGIES FOR A¡[ EXPERIMENT INVESTIGATING THE EFFECTS OF ALS.INHIBITING HERBICIDES ON NODULATION OF CITICKPEA ..............120 6.1 Introduction 120 6.2 Methods ønd Materiøls.................. 122 6.2.1 Selection of growth media 122 6.2.2 Determination of most appropriate nutrient solution....... 124 6.2.3 Selection of appropriate pot si2e...... 125 6.3 Results......... 126 6.3.1 Selection of growth media........... 126 6.3.2 Determination of most appropriate nutrient solution....... 127 6.3.3 Selection of appropriate pot si2e...... t27 6.4 Dìscussion and Choice of Experimental Protocols 128 6.5 Summøry..... 129 lll CHAPTER 7: THE EFFECTS OF CHLORSULFURON ON CHICKPEA GROIVTH, NODT]LATION A}ID RHIZOBIUM ......... 131 7.1 7' 7.2.2 Experimentaldesign 134 7.2.3 Sampling and Measurements........... 135 7.2.4 Datainterpretation 137 139 7.3.1 Shoot biomass and total shoot area.... 139 7.3.2 Root biomass and root length density 145 7.3.3 Number and weight of nodules 150 7.3.4 Plant Nitrogen or Total Nitrogen. 157 7.3.5 Plant nitogen and nodule weight 162 7.4 Díscussìon... 165 7 .4.1 Effects of chlorsulfiuon on plant biomass, shoot area and root length density 165 7.4.2 Chlorsulñron effects on nodulation and nitrogen ................... 169 7.5 Summary and Key Findings...... ... 175 CHAPTER 8: THE EFFECTS OF IMAZETHAPYR ON CHICKPEA GROWTH, NODIILATION AÌ:ID RH12O81UM.............. .......................177 8.1 fnfioducîion 177 8.2 Materíals and Methods.... 178 8.3 179 8.3.1 Shoot biomass and total shoot area..... 179 8.3.2 Root biomass and root length density. t82 8.3.3 Number and weight of nodules.... 188 8.3.4 Total nitogen or plant nitogen 194 8.3.5 Relationship between plant nitrogen and nodule weight 196 8.4.1 Effects of imazethapyr on plant biomass, shoot area and root length density of chicþeas grown under controlled envi¡onmental conditions...... 199 8.4.2 Effects of imazethapyr on nodulation and plant nitrogen of chicþeas grown under controlled environment conditions .....................202 8.4.3 Comparison of effects of chlorsulfruon (Chapter 7) andimazethapyr (Chapter 8) on growth and biomass, of chicþea plants grown in pots, under controlled conditions. 204 8.4.4 Comparison of the effects of chlorsulfi,uon (Chapter 7) andimazethapyr (Chapter 8) on nodulation and nibogen of chicþeas grown in pots, under controlled conditions 205 8.4.5 Implication of results 207 8.4.6 Summary of Chapters 7 and 8 210 CHAPTER 9: POSSIBLE MECHANISMS RESPONSIBLE FOR THE REDUCTION IN THE NT]MBER OF NODTJLES ON CHICKPEA PLAI\TS FOLLOWING PRE- EXPOSURE OF CHICKPEA RHIZOBIA (CC1192) TO ALS-INHIBITING H8R8ICIDES.................. .....211 9.1 Introductìon 211 9.2 Elfects of ALS-Inhibiting Herbicides on the Growth of Chickpea RhÍ2obia................ 213 9.2.1 Introduction 213 9.2.2 Materials and Methods 213 9.2.2.1 Treatments and experimental setup......... .........214 9.2.2.2 Experimentaldesign. 21s 9.2.3 Results 217 9.3 Effects of Chlorsulfuton on Chickpea Rhizobiø Grown in a Deftned Media............... 220 lv 9.3.1 Introduction 220 ..... 9 .3.2 Materials and Methods ' '........... 220 9.3.2.1 Treatments and experimental setup......'.' .......'.220 9.3.2.2 Experimental design and sampling 221 9.3.3 Results 222 9.4 Does RinsÍng lØiîh % Ringer's Solution Remove all the Herbìcidefrom Rhizobíøl Cells? ............... 223 9.4.1 Introduction 223 9.4.2 Materials and methods. 223 9.4.3 Results 228 9.5 Discussion... 229 CHAPTER 10: GENERÄL DISCUSSION................. ....---.232 10.1 Dìscussíon of Results.... 232 l0.l.l Key furdings 239 10.2 Implications of Results.... 240 10.3 Future Ílork 247 10.3.1 Nodule formation and infection. 247 10.3.2 Suggested further work............ 248 10.4 Summøry..... 249 v Abstract Acetolactate synthase (ALS) inhibiting herbicides are widely used in the cereal growing regions of South Australia and include sulfonylureas, sulfonamides and imidazolinones. Sulfonylureas, such as chlorsulfuron, are used in cereal crops of southern Australia to control broadleaf weeds. Sulfonylurea residues have been found to inhibit the growth of some legume crops and pastures in seasons following application. Sulfonamides (e.g. flumetsulam) and imidazolinones (e.g. imazethapyr) are recommended for weed control in legume crops and pastures. Yellowing and stunting of growth of legumes has been observed in the field following applications of some sulfonamides and imidazolinones. It is possible that these herbicides are impacting on symbiotic nitrogen fixation of legume crops or pastures. This study investigated the effects of AlS-inhibiting herbicides on the growth and grain/seed production of chickpea and medic and the symbiotic nitrogen fixation of chickpeas. The presence of triasulfuron or chlorsulfuron residues reduced the shoot biomass of Medicago rugosa, by 31 - 60%. Flumetsulam alone had no effect on M. rugosa shoot biomass. Seed yield of M. rugosd was not affected by any of the herbicides. A second field trial investigated the effects of four application rates of chlorsulfuron in combination with 'in-crop' applications of flumetsulam or imazethapyr on Cicer arietinum (chickpea) growth, yield and nitrogen fixation. Chlorsulfuron at 0.75, 1.5 and 3 g active ingredient (ai) ha-l (the recommended application rate is l5 g ai ha-r) reduced shoot biomass by 22o/o,36%o and 49o/o respectively. Imazethapyr (29g ai ha-r) reduced chickpea shoot biomass by 52Yo,whilst flumetsulam had no significant effect on shoot biomass. Chickpea yield was affected by an interaction between VI chlorsulfuron and imazethapyr. Chlorsulfuron (at 1.5 and 3.0 g ai ha-r) reduced nitrogen fixation (kg N fixed ha-r) by 40% and 57Yo respectively. Imazethapyr reduced nitrogen fixation by 52%. The results for nitrogen fixation reflected those of shoot biomass, so the results were converted to amount of nitrogen fixed per unit of shoot biomass. Only imazethapyr reduced nitrogen fixation after this conversion. Investigations into the effects of chlorsulfuron andimazethapyr on nodulation were conducted in a pot experiment. Imazethapyr or chlorsulfuron were present or absent, (i) during the growth of rhizobia prior to inoculation, (ii) pre-germination of chickpea seeds, and (iii) in the soil in which the plants were grown.