I o, l2z-ot ú c) A STUDY OF THE INTERACTION BET\ryEEN SUSCEPTIBLE AND RESISTANT GRAPEVINES AND PHYLLOXERA A thesis submitted in fulfilment of the degree of Doctor of Philosophy (Natural and Agricultural Resource Sciences) June, 2000 by Alison Valerie Kellow Department of Horticulture, Viticulture and Oenology, The University of Adelaide. TABLE OF CONTENTS ACKNOWLEDGEMENTS................ vtr PUBLICATIONS ARISING FROM THIS RESEARCH......... ............X DEFINITIONS OF TERMS USED IN THIS THESIS ...........................X LIST OF ABBREVIATIONS........ XI Ll.1 Biology of phylloxera I 1.1.2 History of the spread of phylloxera 3 LI.3 The use of rootstocl<s for control ofphylloxera 5 1.1 .4 Susceptibility, tolerance, resistance and immunity .6 ,7 1 .2 GRAPEVN\E ROOT-PHYLLOXERA INTERACTIONS .,,.,.,.. 1.2.1 Response of susceptible vines to phylloxera attack 7 L2.2 Response of resistant grapevines to phylloxera attack.'..'.'...- I 0 1.3 PLANT INTERACTIONS V/ITH SUCKING PESTS 1 z 1.3. I Gall-forming aphids I 2 1.3.2 Plant-parasitic nemøtodes I3 p e s ts I5 l . 3. 3 R e sistanc e mechanisms to p lant- paras itic "'.... "'.'. 1.4 Gnepnvnrm DEFENCE MECHANISMS 22 1.4.1 Grapevine deþnce against other pathogens and pests """"""'22 1.4.2 Resistance of grapevines to phylloxera.'. """"""'25 1.5 CoNcI-usIoNS.........,. ."."."21 2. DEVELOPMENT OF METHODS FOR COCULTIVATION OF POTTED OR MICROPROPAGATED GRAPEVINES WITH PHYLLOXERA............... ..,.,........29 2.1 INrRoDUcrIoN ...... .29 2.2 SELECTION OF GRAPEVINE VARIETIES FOR COCULTIVATION .30 2.3 Py'nEDVINE-BASED cocuLTrvATIoN TRIAI-s ...........'.'31 2.3.1 Pot trial one - summer 1996-1997 32 2.3.2 Pot trial two - summer 1996-1997 ... "...'."..".".'.'-13 2.3.3 Pot trial three - summer 1996-1997.... ."'."..""""33 2.3.4 Pot trialfour - summer 1997-1998 .34 2.3.5 Pot trialfive - summer 1997-1998 .36 2. 3. 6 D is cus sion of p ot c o cultiv ation trials.....'...'.. .36 2.4 Trssue cULTURE-BASED cocuLTrvATIoN TRIALS .5 I 2.4. I Introduction........... 37 2.4.2 Establishment of grapevines in tissue culture '-.'..... """""""""38 2.4.3 Sloped medium cultures (PhytatraysrM) .......,,.....,..........38 2.4.4 P erlite-based medium cultures .42 2.4.5 Hairy root cultures .44 2.4.6 Excis ed ro ot cultur¿,s...............,.. .46 2.4.7 Catlus cultures """""'49 2.4.8 Phylloxera egg sterilisation """"""56 2.5 CoNcr-usIoNS........... ......'..57 3. DEVELOPMENT OF METHODS FOR ANALYSIS OF ROOT SAMPLES.........................59 3.1 INrRoDUcrIoN........,. ...."'.59 3.2 ANALYSIS OF FREE PHENOLIC COMPOUNDS IN GRAPEVINE ROOTS USING HPLC .59 II 3.2.1 Triats of methods for extraction of free phenolics from grapevine roots...".....".....'..'.....'.60 3.2.2 Trials of HPLC chromatography conditions ......'.62 3.2.3 Assessment of the reproducibility of subsampling..'...'.....'.. '...'.65 3.2.4 Discussion of HPLC methods 68 3.3 Lrcnr vrrcRoscopy AND HIsrocHEMIsrRY ......,..........68 3.j.1 Sample preparation ."."...'.,".'..'..'.'.69 3.i.2 Trials of microscopy ønd staining methods........ ....'.....'...'.'.'....70 3.3.3 Discussion of microscopy methods 7l 3.4 EXTRACTION OFRNA FROMPRMARY ROOTS,...... .,,,...72 3.4.1 RNA extraction method one ........ 72 3.4.2 RNA extraction method two 73 3.4,3 RNA extraction rnethod three...... 3.4.4 RNA extraction method four.. .'.."'...74 3.4.5 Discussion of RNA extraction methods 75 3.5 SuppEMENTARYMETHODS..... '.".'........'76 '16 4. THE INTERACTION BETWEEN Y. VINIFERA CV. SHIRAZ AND A BIOTYPE B.LIKE STRAIN OF PHYLLOXERÄ......... ...............77 4.1 INTRoDUcrIoN .....,.... 4.2 MATERIALS AND METHoDS 4.3 RESULTS 4.3.1 Location of feeding site within primøry roots............ 4. 3.2 N o do s ity anatomy and dev e\opment................ 4.3.3 Root responses to phylloxerafeeding observedby histochentistry....-.. 4.3.4 Starch qccumulation in nodosities 4.3.5 Free phenolic content of uninfested roots and nodosities .". 83 4.3.6 Free amino acid and amide content of uninfested roots and nodosities 85 4.3.7 Gene expression in uninfested roots and nodosities 87 92 4.4.1 Location ofthefeedíng site and localised root response .92 4.4.2 Changes in the endodermis and possibLe implications for photosynthate unloading .......,.93 4.4.i Starch accumulation in nodosities 97 4.4.4 Free phenolic content of uninfested roots ønd nodosities 100 4.4.5 Changes infree amino acid and amide profile ,.....,',,'..,''...'... ] 0 ] 4.4.6 Gene expression in uninfested roots and nodosities 103 4.4.7 Summary ................. ".....'."'........'.108 5. COMPARISON OF THE INTERÁ.CTION BET\ilEEN SUSCEPTIBLE AND RESISTANT GRAPEYINE SPECIES OR ROOTSTOCKS AND A BIOTYPE B.LIKE STRAIN OF PHYLLOXERA............... ..........L10 5,1 INrnooucTIoN.......... ......110 5,2 TISSUE CULTURE-BASED BIOASSAYS OFVWL-1 PHYLLOXERA COCULTTVATED WITH AND RANGE ÔF GRAPEVINE VARIETIES .. r12 5.2.1 Perlite-basedmediumbioassays..... .....'..,.....'...'l12 5.2.2 Excised root culture bioassøys 115 5.2. 3 Díscussion of bioassay,r...... 119 5.3 CoNSTNUTTVE OR PREFORMED DIFFE,RENCES BETIWEEN RESISTANT VARIETIES ..,,....................,I22 5.3,1 Anøtomy of primary roots............ 122 5. 3.2 Histo chemistry of p r imary ro ots............. 126 5.3.3 Free phenolic content of primary roots. 129 5.4 DFFERENCES IN INDUCED RESPONSE TO PTryLLOXERA ATTACK t39 5.4.1 Root response to phylloxera attqck observedby histochemistry......". .".'.'.'139 5.4.2 Changes infree phenolics in response to phylloxera attack ..146 5,5 DISCUSSIoN r53 6. INTERACTION Oß V, VINIFßN¿, AND SCHWARZMANN WITH A NOVEL BIOTYPE oF PHYLLOXERA,, STRAIN SRU-1......... .................,...158 6.1 INTRoDUcrroN.....,..... .....158 III 6.2 TIssUE CULTURE-BASED BIoASSAYS oF SRU-I PTryLLOXERA COCULTTVATED wlTH A RANGE OF GRAPEVINE VARIETIES..,.... 159 6.2.1 Materials and methods r59 6, 2.2 P hytatray and p erlite -b as ed medium bioas s ays .......... 160 6.2.3 Excised root culture bioassøys 163 6.2.4 D is cus sion of b ioøs.tdy.t...............,....... 166 6.3 RoornBsPoNsEToSRU-IPHYLLOXERAATIACKOBSERVEDBYHISTOCHEMISTRY,.,.,.....,....,167 6.3.1 Materials and methods 167 6.3.2 Root response to SRU-I phylloxera... ...........'....168 6.3.3 Discussion of histochemistry results 169 6.4 CTIANGES IN FREE P}IENOUCS IN RBSPONSE TO SRU-1 PHYLI,OXERA ATTACK ......170 6.4.1 HPLC materials and methods 170 6.4.2 Free phenolics in SRU-I phylloxera induced nodosities ........ 170 6.4.3 Discussion of HPLC analyses of free phenolics 171 6,5 CH,qNcss IN GENE ExPREssIoN IN LEAVES IN RESPONSE TO SRU-1 PHYLLOXERA ATTACK ON 6. 5. I Mctterials and methods........ 173 6.5.2 Gene expression in leaves... 173 6.5.3 Discussion of gene expression analyses....... 174 6.6 DISCUSSION t75 7. CONCLUSTONS AND FUTURE DIRECTIONS...,....... ..................178 APPENDTX ONE - MAJOR REACTIONS IN THE STRESS-INDUCED PHENYLPROPANOID PATHWAY INPLANTS ...........41 APPENDIX TWO - BIOCHEMICAL AND MOLECULAR METHODS ........................................Ä.2 p-glucosidase treqtment of HPLC uctracts ........ A2 Soluble protein extraction and SDS-PAGE A2 Extraction of free amino acids............ A2 RNA extraction and analysis........ A3 Enzymatic Starch As say ........... A7 Preparation of samples for GMA embedding qnd semi-thin sectioning A8 Preparation of samples for øraldite resin embedding and ultrathin sectioning for TÐM......,..,.. A8 Light Microscopy A8 Stains and staining schedales........ A9 Trans mis s io n ele cff on micro s c opy All APPENDD( FOUR . COCTILTIVATION OF VIVL-I PHYLLOXERA WITH EXCISED ROOT .ULTURES ..........412 APPENDIX FTVE . RETENTION TIMES AND SPECTRAL CHARACTERISTICS OF HPLC STANDARDS AND RESVERATROL DERTVATIVES...... ..................414 APPENDD( SIX - MASS SPECTROMETRY DATA FOR IDENTIFICATION OF HPLC PEAKS 2,3 AND 4.................. ................4I.7 APPENDIX SEVEN - ABSORPTION SPECTRA OF HPLC PEAKS 1-11 .............,.,,.........,........A.22 APPENDIX EIGHT . IDENTIFICATION OF MAJOR COMPOI-INDS IN GRAPEVINE ROOT EXTRACTS .........434 LIST OF REFERENCES Ä.3s IV ABSTRACT This thesis comprises a study of the interaction between grapevine roots and the gall- forming pest grape phylloxera (Daktulosphaira vitifoliae Fitch)' A study of the interaction with the susceptible Vitis vinifera cv. Shiraz has shown that on primary roots phylloxera feed within the root cortex. Nodosity formation is accompanied by inhibition of endodermis differentiation and accumulation of starch to a concentration approximately ten times that in uninfested roots. Comparisons of nodosities with uninfested roots by SDS-PAGE analysis of proteins revealed higher concentrations in nodosities but no significant differences in composition; HPLC analysis of free amino acids showed more than double the amino nitrogen content in nodosities; Northern blot analysis, targeting various genes including those proposed to be defence-related, revealed no significant change in gene expression with the exception of the VvTLZ gene which was upregulated in leaves of SRU-I phylloxera infested vines. The interaction of phylloxera with several rootstocks or resistant vine species (Schwarzmann, Ramsey, V. riparia, Börner, V. rotundiþlia) was compared with the interaction with V. vinifera. Tissue culture-based cocultivations allowed assessment of resistance to VWl-strain phylloxera, and allowed biotype comparisons of the strains VWL- 1 and SRU-I. VWL-I phylloxera established successful nodosities on V. vinifura and Ramsey, with poorer performance on Ramsey. VWL-I phylloxera could not survive on the other varieties, and did not even attempt to feed onV. rotundiþlia.