Y o,Ê Ç-4-oz The root lesion nematod e, Pratylenchus neglectus, in field crops in South Australia Sharyn Patricia Taylor B.Sc. (Hons) University of Adelaide Thesis submitted for the Degree of Doctor of Philosophy The University of Adelaide (Faculty of Agricultural and Natural Resource Sciences) Department of Applied and Molecular Ecology December 2000 Table of Contents THE Roor LEStoN NEMAToDE rPnerw.rNcnus NEGLECTU*T IN FTELD cRoPS lt¡ Sourg AusrRnua TABLE oF CONTENTS ll Sunlulnv vlt DECLARATIoN x PUBLICATIONS ARISING FROM THIS TFIESIS xi ACrcNOWI-PDGMENTS xii AeeREvIRTIoNS USED xiv Cn¡.r'rnn I INTRoDUcrtoN I Cunprrn2 LITERATUREREVIEW 4 2.0 INTRODUCTION 4 2.I Pn.¿ryrc¡'Icuus NEGLECruSANDP. MORNEI 4 2.2 LIFE CYCLE 4 2.3 Svuprovrs 5 2.4 INTERACTIONS WITH OTHERPATHOGENS 6 2.5 HoST RANGE AND GEOGRAPHIC DISTRIBUTON 7 2.6 ECoNoMIC IMPORTANCE 9 2.6.1 Pratylenchus thornei l0 2.6.2 Pratylenchus neglectus 11 2.7 EFFEC.TS OF EDAPHIC FACTORS ON NEMATODE DENSITY AND YIELD LOSS l1 2.7.1 Tillage t2 2.7.2 Nutntion 12 2.7.3 Wateravailability l3 2.8 MpRSURII.IC YIELD LOSS 13 2.9 POpuI-¡TION DYNAMICS 15 2.9.1 Evaluation ofpopulation dynamics l5 2.9.2 Resistance l8 2.9.3 Tolerance 20 2. 1 O VInu ISNCE, AGGRESSIVENESS AND PATHOGENICIry 2l 2. I 1 POpuI-ATION VARIABILIry 2l 2.11.1 Morphology 22 2.11.2 Host range shrdies 22 2.1I.3 Moleculartechniques 23 2. I 2 DISTRIBUTION, SAMPLING AND EXTRACTION 24 2.12.1 Sampling 24 2.12.2 Field expenments - desigrr and samphng pattems 25 2.12.3 Honzontal distribution 26 2.12.4 Vertical distribution 27 2.12.5 Extaction and storage 28 2.I3 IvponTANCE oF THIS RESEARCH 29 Crnrren3 Gnnenq,lTccFINIQUES 30 3.I EXTRACTION OF NEMATODES FROM PLANT MATERIAL 30 3.1.1 Consfuction of the mistrng chamber-the double funnel method 30 3.1.2 Calibration of the mrsting chamber 3l 3.1.2.1 Materials and methods 31 3 1.2.2 Results and discussion 31 3. I .3 Extaction of nematodes from plant roots - misting 32 3.1.4 Extaction of nematodes from plant roots - staining then maceration 33 3.2 ExrReCrIO¡I OF NEMATODES FROM SOIL 33 3.2.1 The modified Whitehead tay method JJ 3.2.2 Assessment of soil moisture 34 3.3 COUNINC AND IDENTIFICATION OF NEMATODES 34 3.4 PRepIReION OFNEMATODE INOCULA 34 3.4.1 Carrot culture inoculum 34 3.4.2 Soil inoculum 35 3.5 GRowrs RooM ExPERIMENTS 36 3.5.1 Germination of seed 36 3.5.2 Tube tests 36 3.6 PorrrunIS ON OUTDOORTERRACES 36 3.7 FIELD SITES 37 3.7 .l Site selection 37 3.7.2 Field site establishment and maintenance 38 3.7.3 Application of nematicide 38 3.7.4 Soil sampling 38 3.7.4.I Undisturbed soil core 38 3.7 4.2 Soil sampler 39 3.8 HARVESTTNG AND MEASUREMENTS 39 3.9 ATIOzyITIE ELECTROPHORESIS 39 3. I O EXPERIMENTAL DESIGN AND STATISTICAL ANALYSES 40 3.10.1 Expenmental desiglt 40 3.10.2 Data analysis and transformation 40 Cnngrrn4 ETpICINNCY OF EXTRACTION AND SEPARATION OF PNITWENCHUS NEGLECTUS FROM PLANT ROOTS AI\ID SOIL 42 4.0 TNTRODUCTION 42 4.I MRTSTRLS AND METHODS M 4.1.1 Elimination of lint and soil contamination from samples M 4.l.t.l Dataanalysis 44 4.1.2 Separation efficiency of nematodes using a modifìed Whitehead tray method 44 4 I .2.I Data analysis 45 4.1.3 Recovery of P. neglectr;s from stored soil 45 4.1.3.1 Experiment I 45 4.1.3.2 Experiment 2 45 4 1.3.3 Data analysis 46 4.1.4 Comparisonoftwomethodsforextraction of P.neglectus fromwheatroots 46 4.I .4. t Data analYsis 46 4.1.5 Efficiency of separation of P. neglectøs from different crops 46 4.1.5.t Pratylenchusneglectus inoculumandplantestablishment 47 4.1 .5.2 Experimental design and assessment 47 4. I .5.3 Data analysis 48 lll 4.2 Resulrs 48 4.2.1 Elimination of fibre and soil contamrnation from samples 48 4.2.2 Separation efficiarcy ofnematodes using a modifìed Whitehead ftay method 49 4.2.3 Recovery of P. neglectrzs from stored soil 50 4.2.3.1 Experiment I 50 4.2,3.2 Experiment 2 52 4.2.4 Comparison of two methods for exfaction of P. neglectus from wheat roots 55 4.2.5 Efficiency of separatron of P. neglect¡zs from different crops 57 4.2.5.1 Hawest time I (6 weel<s) 57 4.2.5.2 Hantest time 2 (16 weeks) 59 4.3 DISCUSSIoN 63 4.3.1 Extaction of P. neglectus from soil 63 4.3.2 Extactton of P. neglectu,s from root samples 65 Cu¡.prnn5 Sp¡rul DISTRTBUTIoN oF, AND SAMnLING FoR, PRATvLENCHUS NEGLECTUSAND P. THORNEI IN SOUTH AUSTRALIA 68 5.0 INTRODUCTIoN 68 5.I MATER]ALS ANDMETHODS 69 5. I . I Horizontal distribution - plant row effects 69 5.1.1.1 Data analysß 70 5.1.2 Verticaldistribution 70 5.I .2.1 Data analysis 70 5.1.3 Companson of sampling methods 70 5.1 .3.1 Data analysis 71 5.2 Rrsulrs 73 5.2.1 Horizontal disfibution -plant row effects IJ 5.2.2 Verticaldisfibution 74 5.2.3 Comparison of samphng methods 78 5.3 DrscussroN 83 Crnprnn6 POPULATION DYNAMICSOF PRATYLENCHUS NEGLECTUS IN WHEAT IN A POT STUDY 86 6.0 INTRODUCTIoN 86 6.I MRTEruRIS AND METHODS 87 6.1.1 Pratylenchus neglectusinoculum 87 6.I.2 Experimental desigrr and assessment 88 6.1.3 Data analysis 88 6.2 RESULTS 89 6.2.1 Pratylenchus neglectusinoculum 89 6.2.2 Nematode densities and multiplication rates 89 6.2.3 Plant dry mattsr responses 95 6.2.4 Regressionanalyses 97 6.2.5 Rainfall, soil temperatwe and au temperature 100 6.3 DISCUSSION t02 lv CurrrnnT RES|STANCE oF CEREAL, PULSE, olLsEED AND PASTURE vARIETIES ro PRATYLENCHUS NEGLECTUS IN FIELD EXPERIMENTS 107 7.0 INTRoDUCTION 107 7.1 MATERIALS ANDMETHODS 108 7.1.1 Field trial establishment 108 7.1.2 Experimentaldesign 109 109 7 .1.3 Pratylenchus neglectus and plant assessment 7 .l.4 Statistical analyses 112 7.1.4.1 Field trials at Sandilands and Paskeville 112 7.1.4.2 Field trial at Condada 113 7.2 RESULTS lt3 7.2.1 Sandilands, 1996 113 7.2.I .1 Rainfall il3 7.2.1 .2 Initial tlensity, final density and multiplication rate ofP ' neglectus I14 122 7 .2 . I . 3 Pratylenchus neglectus density in canola plots 7.2.2 Paskeville, 1997 t23 7.2.2.1 Rainfall t23 7.2.2 2 lnitial density, final density and multiplication rate ofP ' neglectus 124 7.2.2.3 Pratylenchus neglechs density in canola plots 132 7 .2.3 Comparison between varieties using final density or multiplication rate t33 7.2.4 Combined fìeld trial results for Sandilands and Paskeville t34 7.2.5 Condada cereal variety trial, 1996 t37 7.3 DISCUSSION 140 CHAPTERS POpI.T¡T¡<TN DYNAMICS OF, ¡¡P YIELD LOSS CAUSED BY , PRATWENCHUS NEGLECTTS ÂND P. THORNEI IN FIELD EXPERJMENTS I45 8.0 INtRoouclor 145 8.I M¡TSRIRLS ANI) METHODS r46 8.1.1 Fielcl srtcs 146 8. 1. L l,4klicarb rate trial 146 8.1.1.: l'urieq¡ tials 146 8.I .l .3 ktution trials 147 8.1.2 Prun lenclns and plant assessment 148 8.1.3 Søtisticalanalyses 148 8.2 RESULTS 149 8.2.1 Aldrcarb rate tnal 149 8.2.2 Vanery tnals 152 8.2 2 I Sandilands variety trial, 1996 152 8.2.2 2 Paskeville variety trial, 1997 t56 8.2.2 3 Condada vaiety tial, 1996 160 8.2.3 Rotation tnals 168 8.2.3 I Minnipa rotation tia| I996 and 1997 168 I 2.3.2 Condada rotation trial, 1996 and 1997 177 8.3 DISCUSSION 188 8.3.1 Use of aldicarb to measure yield loss 188 8.3.2 Comparison between aldicarb and regression analyses to calculate yield loss 188 8.3.3 Compansons between varietal tolerance and resistance to P. neglectus and P. thornei 190 8.3.4 Modelling yield loss 191 8.3.5 Determrning relationships between either initral or final nematode density and yield t92 8.3 .6 Populatron dynamics of P. neglectus and P. thornei in field fials 194 CH¡,prnn9 VARIATION WITHIN A]\D BETWETI,I AUSTR,AI.IAN POPIJLATIONS OF PRATYLENCHUS NEGLECTUS AND P. THORNEI 198 9.0 INTRODUCTION 198 9 .I CoI-IecrION ¡,NO MNN'¡TENANCE OF P RATY LENCHUS POPL]LATIONS 199 9.1.1 Matenals and methods r99 9.1.1.1 Populatiors r99 9.1.1.2 Clonal lines r99 9.2 IDENTIFICATION OF DIFFERENCES WITHIN AND BETWEEN PRATYLENCHUS 199 POPULATIONS USING ALLOZYME ELECTROPHORESIS 200 9.2.1 Materials and methods 200 9.2.1.1 Experiment I 200 9.2.1.2 Experiment 2 201 9.2.2 Results 201 9.2.2.1 Experiment I 201 9.2.2.2 Experiment 2 201 9.3 DI FFERENÏAT REPRODUCTON OF PÀ,4 TlL ENCHUS POPULATIONS IN CEREALS 207 9.3.1 Materials and methods 207 9.3.1.1 Experiment I 207 9.3.1.2 Experiment 2 207 9.3.1.3 Experiment 3 208 9.3.2 Results 208 9.3.2.1 Experiment I 208 9.3.2.2 Experiment 2 2lt 9.3.2.3 Experimenl3 212 9.4 DrscussroN 216 9.4.1 Alloqfmeelecftophoresis 216 9.4.2 Companson between reproductive fibress of nematode populations 217 9.5 CoNcr-usroNs 220 Cu¡,ylnn IO GENERAL CONCLUSIONS AND DISCUSSION i), IO.I S¡.upI-INc AND EXTRACTION 222 IO.2 DeTenvINING RESISTANCE RATINGS IN FIELD EXPEzuMENTS 224 IO.3 RESISTANCE WTHIN FIELD CROPS TO P.