Annual Progress Report: Final Report: First Name. S}'.dne}'. School of Biological Sciences A 12, The University of Svdnev PO Box.

Transcription

1 REPORT FORMAT!REPORT TYPE: r " Annual Progress Report: Fnal Report: D [XJ Project Ttle: (< 15 words) CRDC Project Number: US32C.. ~.... lp&:~ ~ r.ruq&: Admn Contact: nformaton Offcer Ttle (Mr/Mrs/etc) Frst Name p rgansaton The Unversty of Sydney (name of organsaton that wll be admnsterng the fundng) f [Postal Address: Offce of Research and Scholarshps A14, The Unverstv of Svdnev PO Box S}.dne}. NSW. 1 Town {2} (2) Phone Fax State Last Name 26 Postcode research@reschols.usvd.edu.au Emal -. ~ ( ~rncpal Researcher: o Organsaton Dr Bruce Ttle (Mr/Mrs/etc) Frst Name The Unversty of Svdnev Lyon Last Name Postal Address:! School of Bologcal Scences A 12, The Unversty of Svdnev PO Box Sydney NSW Town State {2} (2} Phone Fax 26 Postcode brucel@bo.usvd.edu.au Emal..Project Supervsor: Organsaton Dr Bruce Ttle (Mr/Mrs/etc) Frst Name The Unversty of Sydney Lyon Last Name :!Postal Address: : School of Boloocal Scences A 12 The Unverstv of Sydney PO Box Sydney NSW Town State (2) (2) Phone Fax 26 Postcode brucel@bo.usyd.edu.au Emal

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3 CRDC Project US32C solaton of pathogencty genes from Vertcllum wlt fung nfectng cotton July 1996-June 2 1. What was the background of the project? 1 j, Vertcllum wlt s a fungal dsease of numerous plant speces that s charactersed by symptoms of wltng, chloross, necross and defolaton. The causal agent, Vertcllum dahlae can nflct sgnfcant losses on cotton crops and, f not adequately controlled, could serously affect the long-term sustanablty of the cotton ndustry. The best defence aganst crop losses from such pathogens s to breed resstant crop varetes, and the ntroducton of Vertcllum wlt-tolerant Gossypum hrsutum varetes such as Scala V-1 and ts successor Skala V-2 has sgnfcantly reduced the mpact of V. dahlae on the Australan cotton crop. However, epdemologcal surveys that we conducted n the early 199s revealed that a hgh proporton of V. dahlae strans are capable of causng severe dsease even on these mproved varetes (Table 1; Ramsay et al., 1996; Zhu, Multan and Lyon, unpublshed). Such strans are prevalent n cotton producton regons n both NSW and Queensland. Table 1. Otgn, colour on Potato Dextrose Agar (W, whte; B, black) and host pathogencty of eght selected solates of V. dahlse from cotton. Response of named cultvar to nfecton: R, resstant; M, moderately resstant; S, susceptble. Dsease severty ndex (S) s presented n parentheses l. c solate Qcgo Colour Qublt!a[ c~::1212ole Sle sae Yeac ema s-z Ar;calaB. S!dlla ~-l oeso Sl12lmH 18 MerahNorth NSW 1984 W/B M (.27} M (.37) s (.51) M (.41) s (.7) 111 Trange NSW 1992 B R (.2) M (.5) s (.52) s (.64) s (.72) 112 Dalby Qld 1992 B R (.24) M (.48) M (.33) s (.6) s (.54) 113 Boggablla NSW 1992 B M (.4) s (.6) s (.6} s (.64) s (.76) 117 Bourke NSW 1984 w R (.2) M (.3) R (.2) A (.2) s (.56) 12 Dalby Old 1992 W/B R (.25) M (.36) M (.28) s (.6) s (.68) 128 Bowen vlle Qld 1993 B M (.34) M (.4) s (.51) s (.6) s (.7) 135 Cecl Plans Old 1993 B R (.22) A (.24) M (.32) s (.7) s (.68) n order to further enhance host plant resstance to Vertcllum wlt, cotton breedng programs wll need to be able to rapdly ncorporate new resstant cotton germplasm whch s known to be effectve aganst strans of the V. dahlae pathogen that are predomnant n the feld at any gven tme. Durng the course of our research on fungal pathogens of cotton, we have developed molecular genetc technques such as RAPD-PCR for the dentfcaton of dfferent solates of the fungal pathogen V. dahlae. n order to understand better the epdemology of Vertcllum wlt dsease n cotton, we have examned over one hundred ndependent solates of ths fungus from a number of cotton producton regons. Locatons nclude the Gwydr, Macntyre, Maquare and Namo valleys and! around Bourke n NSW, ~d the Darlng Downs and around St George and Theodore n. Queensland. We have allocated these solates to 15 RAPD groups (RGs) of closely related strans based on the possesson of 8% or more DNA markers n common (Zhu, Multan and Lyon, unpublshed). CRDC Project US32C Fnal Report September 2

4 n addton to the sgnfcant dversty observed for fung from dfferent cottonproducng regons, ntensve samplng of sngle felds n two dstrcts revealed a hgh level of genetc dversty even wthn localsed V. dnhlae populatons. nterestngly, use of cotton cultvars that are generally more tolerant to Vertcllum wlt does not seem to cause any declne n ths level of genetc dversty. However, we have no nformaton on whether such changes n host cultvar result n the selecton of more pathogenc strans from the background populaton. Although we have successfully dfferentated solates of V. dahlae usng RAPD-PCR, at present pathogencty tests reman the only means of determnng the pathogenc classfcaton, or races, of the fungal strans present n dseased cotton plants. We have employed an artfcal noculaton technque for the nducton of Vertcllum wlt dsease n a slecton of cotton cultvars, and have tested 3 ndependent V. dahlae solates on fve cotton cultvars. These were Sokra 1-4, Deltapne 9, Skala V-1, Acala Royale and Pma S7. As expected, all but one of the V. dahlae solates was able to cause sgnfcant dsease on Sokra 1-4. More alarmngly, over half of the solates (17) were capable of causng sgnfcant dsease on the wlt-tolerant varety Scala V-1, and three were able to cause sgnfcant dsease on the more tolerant Acala Royale varety (Zhu, Multan and Lyon, unpublshed). C We have attempted to correlate genetc fngerprnts as determned by RAPD-PCR wth the pathogencty characterstcs of these solates, but have yet to observe any clear trend. The results reveal that some hghly pathogenc strans are apparently closely related to some less pathogenc solates, whle the hghly pathogenc strans appear to be genetcally unrelated even though they may exhbt the same host specfcty. As a result of these fndngs, we turned to the dentfcaton and charactersaton of potental fungal pathogencty genes as a means of more drectly dentfyng genetc markers assocated wth V. dahlae pathogencty. Ths approach nvolved the clonng of putatve plant and fungal genes that are hghly expressed wthn roots durng nfecton of cotton plants by V. dahlae. The research utlsed the cdna lbrary prepared by Ms. Melssa Hll as part of her Ph.D. project funded by the CRC for Sustanable Cotton Producton (SU251; Hll et al., 1999). Ths research, ntated by Ms. Catherne Kenry durng her Honours year n 1995, successfully dentfed 3 genes for whch the DNA sequences were partally determned. The dentfed genes dd not appear to correspond to any prevously charactersed pathogencty genes from other fungal speces, but they mght nonetheless represent unque genes assocated wth V. dahlae pathogencty. Ms. Kenry successfully completed her Honours degree, fnshng n the top 1% of students n the School, and was awarded an Australan Postgraduate Award to contnue ths research. O As ntally conceved, the research project (US32C) was to have been carred out by Ms. Kenry, who had been awarded an Australan Postgraduate Award to contnue the research project begun durng her Honours year. A part-tme salary (one day per week) was requested from the CRDC to rase the value of Ms. Kenrys stpend to the same level as an ndustry scholarshp. n md-1996, Ms. Kenry was awarded a scholarshp to undertake a Ph.D. at Cambrdge Unversty n England, and at the end of September 1996 she dscontnued her studes at the Unversty of Sydney, and concluded her nvolvement n US32C. Completon of the most mportant facets of the project subsequently requred the employment of a research assstant for 2.5 days per week, wth a consequent ncrease n the requested budget. Due to the altered crcumstances, proposed attempts to determne the CRDC Project US32C Fnal Report September 2 2

5 functonal role of putatve pathogencty genes by usng RNA hybrdsaton, or DNA transformaton wth altered genes, were deemed to be beyond the scope of the project. The ncdence of Vertcllum wlt dsease n cotton s ncreasng and has the potental to cost the ndustry mllons of dollars annually n lost yeld. Although the CSRO has been successful n breedng cultvars of cotton wth enhanced dsease tolerance, the exstence of more pathogenc strans of V. dahlae threatens to negate ther effectveness. The clonng of V. dahlae genes assocated wth pathogencty could play an mportant role n the control of ths dsease by provdng nsght nto the characterstcs that make some fungal strans more pathogenc on partcular cotton cultvars. The development of sutable gene probes would enable the rapd dentfcaton of pathogenc organsms and could provde nformaton on ther feld survvat persstence and mode of spread. 2. What were the project objectves and to what extent were these acheved? Q () Complete charactersaton of putatve fungal genes solated from the cdna lbrary prepared from Scala V-1 plants nfected wth V. dahlae. The frst objectve of ths project was to complete the charactersaton of putatve fungal genes solated from a cdna (gene) lbrary produced from Scala V-1 plants nfected wth a stran of Vertcllum dahlae that causes severe wlt dsease n some cotton varetes. A total of 116 cloned genes were selected from the lbrary on the bass that ther actvty s ncreased n cotton plants nfected wth V. dahlae. All of the 116 clones were charactersed by partal DNA sequencng, and the majorty of the clones accordngly appear to be of cotton plant orgn. However, 18 canddate genes wth putatve functons suggestve of fungal genes, or that could not be assgned a putatve functon at the tme, were selected for further charactersaton. The 18 canddate genes have now been charactersed by DNA hybrdsaton, PCR amplfcaton and/ or DNA sequence examnaton and database comparson to determne ther lkely orgn. Eght of the genes have been shown by actvty analyss (Northern blottng), or PCR amplfcaton usng specfc amplfcaton prmers, to be of plant orgn. The DNA sequences of the remanng ten genes are now recognsed as beng closely related to gene sequences of plant or hgher eukaryote orgn n the DNA sequence database. All 18 clones charactersed n ths study are thus beleved to be of plant rather than fungal orgn, and are no longer consdered as potental canddates for fungal pathogencty genes. () Analyse the functonal role of potental fungal pathogencty genes and conduct surveys to dentfy genetc polymorphsms n other V. dahlae solates that mght be assocated wth ncreased pathogencty. The second project objectve could not be acheved as proposed snce the research conducted for Objectve () revealed that none of the canddate genes solated from the cdna lbrary prepared from Scala V-1 plants nfected wth V. dahlae were actually of fungal orgn. t s probable that the solaton of putatve fungal pathogencty genes wll only be possble followng the constructon of a new cdna lbrary from nfected cotton plant tssue harvested at a longer tme nterval after fungal noculaton. However, there was nsuffcent tme or resources avalable to construct and screen a cdna lbrary of ths knd. () Conduct survey of V. dahlae solates usng AFLP fngerprntng to dentfy genetc polymorphsms that mght be assocated wth ncreased pathogencty. CRDC Project US32C Fnal Report September 2 3

6 The AFLP technque can screen for polymorphsm more effcently than RAPDs, and once found, polymorphc markers are more reproducble, and so more relable as stran-specfc markers. Ths technque may therefore acheve the overall am of ths project, whch s to dfferentate pathogenc and nonvpathogenc races of v. dahlae. The DNA of 22 selected V. dahlae solates has been analysed usng a set of 16 AFLP prmer combnatons (EAA, EAC, EAG and EAT wth MA, MC, MG and MT). Usng these prmers, a large number of AFLP bands have been detected and, mportantly, a hgh percentage of the AFLP bands are polymorphc between the V. dahlae solates. Ths makes t possble to dstngush between the solates on the bass of ther DNA fngerprnts. Analyss of the full AFLP data set has not yet been completed, so t remans unclear f any assocatons can be found b~tween specfc AFLP polymorphsms and stran pathogencty characterstcs. Further work wll nclude phylogenetc analyss to determne genetc relatedness of the dfferent V. dahlae solates, and mult-dmensonal analyss to compare genetc and pathogenc attrbutes. (v) Develop a molecular assay for the dfferentaton of pathogenc strans n feld materal. Development of a molecular assay for the detecton of specfc V. dahlae strans s postponed untl potentally useful AFLP polymorphsms can be dentfed that show assocaton wth specfc pathogencty trats. 3. What methodology was used, and a justfcaton for the use of ths methodology? The prncpal methodology employed n ths research utlsed genes from a cdna lbrary produced from the roots of cotton seedlngs 24 hrs after nfecton wth V. dahlae. An assumpton was made that the nvadng fungus would encode a proporton of the genes beng expressed n such tssues, and that some of these genes would be essental for the pathogencty of the organsm on the cotton host. The lbrary was ntally screened to dentfy genes that were up-regulated n ths tssue (e. not present n unnfected cotton tssue). Such genes mght reasonably be expected to be ether fungal or plant genes expressed n response to the pathogen-host nteracton. DNA fngerprntng usng the AFLP technque was nndertaken to dentfy correlatons between the DNA fngerprnts and pathogencty characterstcs of the V. dahlae solates. The AFLP technque can screen for polymorphsm more effcently than RAPDs, and once found, polymorphc markers are more reproducble, and so more relable as stran-specfc markers. 4. Detaled results ncludng statstcal analyss of results? n earler research (CRDC project US8C), we attempted to correlate RAPD-PCR fngerprnts of solates of Vertcllum dahlae wth ther pathogencty characterstcs, but an obvous trend dd not emerge. As a result of these fndngs, we turned to the charactersaton of potental pathogencty genes as a means of more drectly dentfyng genetc markers assocated wth V. dahlae pathogencty. Ths research utlsed a cdna lbrary, prepared by Ms. Melssa Hll as part of the CRCSCP project SU251, whch was expected to nclude cloned fungal and cotton genes that are hghly expressed durng V. dahlae nfecton of cotton plants. CROC Project US32C Fnal Report September 2 4

7 ! The ntal research, conducted by Ms. Catherne Kenry as an Honours project n 1995, successfully dentfed 3 genes that were expressed n a manner suggestve of mportant fungal nfecton-assocated genes. These genes were further charactersed by partal DNA sequencng, and although they dd not appear to correspond wth any prevously charactersed pathogencty genes from other fungal speces, they may nevertheless represent unque genes assocated wth V. dahlae pathogencty. Ms. Kenry contnued work on the research program n 1996, concludng the DNA sequencng of the remanng clones and conductng further molecular genetc tests n efforts to dentfy the 3 canddate fungal genes. C.A. Kenry, M.K. Hll, K.P. Rdgway and B.R. Lyon presented ths work at the 8th Australan Cotton Conference, n a poster enttled "Molecular genetc analyss of the cotton-vertcllum nteracton". d ;,, Four of the canddate clones were ntally analysed n detal usng Southern and Northern blottng (see Table 2). These results reveal the presence of homologous sequences to clones 6t and 7h n both V. dahlae and cotton, and detect DNA homology wth clones 7n and 8z n ~otton alone. Such results are unexpected, as these four clones were orgnally selected on the bass that they were expressed n fungal mycela and were up-regulated followng fungal nfecton of cotton plants. Only clones 7n and 8z were prevously found to exhbt some cross-homology wth cotton, a fndng that mght suggest that these genes confer some knd of housekeepng functon n both speces. A larger sample of the canddate clones therefore needed to be examned usng the technque of Southern blottng to resolve ths current dffculty n defnng the host source of each of the clones. Table 2. Expresson pattern, homology wth publshed DNA sequences and Southern and Northern hybrdsaton results of four cdna clones. Level of hybrdsaton sgnal s ndcated by the number of plus sgns. For the Northerns, tme of peak gene expresson s ndcated n hours post nfecton Clone Q«erental Gene EKprassoa DNA Sequence Plant Fungal Northern Fung nfecton Plant Homology southern Southern <peak) ; t:.: l. l. : l:. l. :! ;: 6t none h,48h 7h + ++ none n none ++ 12h az none ++ Ms. Kenry also worked towards ensurng that the laboratorys consderable collecton of V. dahlae cultures was correctly catalogued and safely consgned to long-term storage. Ths culture collecton, comprsng over 1 ndependent solates of the Vertcllum wlt pathogen from all major cotton producton regons n NSW and Queensland, s an mportant resource for current and future research ~to ths dsease n Australan cotton crops.. 1 ; The frst objectve of the project was to complete the charactersaton of putatve fungal, genes solated from a gene (cdna) lbrary produced from Skala V-1 plants nfected wth a stran of V. dahlae whch causes severe wlt dsease n some cotton cultvars. A total of 116 cloned genes were selected from the lbrary on the followng bass. () Clones that are up-regulated n cotton plants nfected wth V. dahlae and are expressed n fungal mycela (up-regulated Vertcllum), or () Clones that are up-regulated n cotton plants nfected wth V. dahlae but are not expressed n fungal mycela or unnfected plants (up-regulated Vertcllum or cotton). CRDC Project US32C Fnal Report September 2 5

8 ;. [. l : l, All of the 116 clones were charactersed by partal DNA sequencng (approxmately 85 of the clones were sequenced as part of CRCSCP student projects SU326 and SU327). The majorty of the clones appear to be of cotton plant orgn, however 18 canddate genes wth expresson profles or putatve functons whch are suggestve of fungal genes were selected for further charactersaton (Table 3). Some of the genes that orgnally could not be matched wth sequences n the DNA database were subsequently foun4 to be closely related to genes of plant orgn. As a result of these fndngs, eght of the 18 clones (6a, 6t, 7d, 7n, 7p, 8z, 9b and 9c) are now presumed to be of plant orgn and can no longer be consdered as potental canddates for fungal pathogencty genes (Table 3). Table 3. Canddate fungal genes selected from the cdna llbrary, putatve functon suggested by sequence comparsons, and proposed orgn as determned by DNA sequence or DNA hybrdsaton analyses Clone canddate gene Putatve functon Orgn of clone! r! t r Sq up-regulated Vertcallum or cotton arablnosdase Vertcllum or cotton 6a up-regulated VertcHlum metalothonen-lke proten cotton 6q up-regulated Vertcllum?? 61 up-regulated Vertcllum? cotton 7d up-regulated Vertcllum regulatory proten cotton 7e up-regulated Vertcllum?? 7h up-regulated Vertclllum glycne-rch cell wall proten Vertcllum or cotton 7n up-regulated Vertcllum or cotton pathogeness-related proten cotton 7p up-regulated Vertcllum? plant 7z up-regulated Vertcllum or cotton?? Sc up-regulated Vertcllum or cotton?? Bu up-regulated Vertcllum or cotton?? az up-regulated Vertcllum or cotton? cotton 9a up-regulated Vertlclllum or cotton?? 9b up-regulated Vertlclllum? plant 9c up-regulated Vertcllum photosystem SKD proten cotton 9r up-regulated Vertcllum or cotton?? 9u up-regulated VertcHum chtn synthase? The fungal or plant orgns of the remanng ten clones (Sq, 6q, 7e, 7h, 7z, 8c, Bu, 9a, 9r a1ld 9u) were the subjects of further research. These genes have been charactersed by DNA hybrdsaton, PCR amplfcaton and/or DNA sequence examnaton and database comparson to determne ther lkely orgn. The remanng genes have been shown by actvty analyss (Northern blottng), or PCR amplfcaton usng specfcdesgned amplfcaton prmers, to be of plant orgn. Clone pvghlol (Sq), whch has hgh sequence smlarty wth an arabnosdase gene from the bacterum Bacterodes ovatus. The known role of arabnosdase (xylanase) enzymes n plant cell wall degradaton (breakdown of xylan cell wall macromolecules), suggested that clone pvghl Ol mght represent a pathogencty gene derved from V. dahlae, as cell wall degradaton would be essental for fungal nvason of plant tssue... :!. l Ths gene has been further charactersed n an attempt to confrm the fungal orgn of the done, wth specfc PCR prmers beng desgned to enable amplfcaton of the gene from genomc DNA. Although DNA fragments were amplfed from both fungal and plant DNA, only the fragment from the plant DNA hybrdsed wth the cloned DNA, thereby provng that the gene s derved from the cotton plant and not V. dahlae. t s possble that V. dahlae also possesses a gene that s closely related to the cotton gene, b:lt ths would need to be cloned from the fungal DNA before further charactersaton could be carred out. Our research has therefore determned that all of the 116 clones selected from the Scala V-1 - V. dahlae cdna lbrary appear to be of plant orgn. Ths unantcpated result CRDC Project US32C Fnal Report September 2 6

9 . suggests that the presence of fnngal gene actvty (mrna expresson) n nfected cotton plant root tssue, at such a short tme (24 hr) after noculaton wth V. dahlae, s mmmal f not non-exstent. t was therefore deemed unwse to re-screen the exstng gene lbrary for more lkely fnngal gene canddates. t s lkely that the solaton of putatve fungal pathogencty genes wll only be possble followng the constructon of a new cdna lbrary from nfected cotton plant tssue harvested at a longer tme nterval after fungal noculaton (48-96 hr). However, there was nsuffcent tme avalable to construct and screen a cdna lbrary of ths knd.. As an addtonal aspect.of ths project, we surveyed a selecton of V. dahlae solates usng a new DNA fngerprntng technque known as amplfed fragment length polymorphsm (AFLP). We have used AFLPs wth sgnfcant success n the genotypng (DNA fngerprntng) of a number of cotton cultvars. Experments by other researchers ndcate that the basc AFLP technque can be employed to obtan DNA fngerprnts for the dfferentaton of fung. Prevous experence n the use of AFLPs for plant analyss has enabled us to optmse the AFLP technque for use wth V. dahlae. Ths requred the desgn and manufacture of unque AFLP prmers wth fewer selectve nucleotdes, due to the sgnfcantly smaller genome sze of the Vertcllum fungus. We have determned that the optmum prmer pars for V. dahlae DNA comprse EcoR prmers wth two selectve nucleotdes and Msel prmers wth one selectve nucleotde. The DNA of 22 V. dahlae solates has now been analysed usng a set of 16 AFLP prmer combnatons (EAA, EAC, EAG and EAT wth MA, MC, MG and MT). Usng these prmers, a large number of AFLP bands have been detected and, mportantly, a hgh percentage of the AFLP bands are polymorphc between the V. dahlae solates. Ths makes t possble to dstngush between the solates on the bass of ther DNA fngerprnts. Snce a large proporton of the genome s beng scanned wth these AFLP prmers (approx. 25% of potental EcoR restrcton stes), the assocaton of a DNA marker wth stran pathogencty wll more lkely be detected. 1. The DNA fngerprntng results (AFLP and RAPD) reman to be correlated wth the. cotton cultvar nfecton data n an attempt to dentfy whether the possesson of partcular DNA markers s assocated wth degree of stran pathogencty on selected cotton cultvars. Analyss wll nclude phylogenetc analyss to determne genetc relatedness of the dfferent V. dahlae solates, and mult-dmensonal analyss to compare genetc and pathogenc attrbutes. 5. A dscusson of the results, ncludng an analyss of research outcomes compared wth the objectves Our research determned that all of the 116 clones selected from the Scala V-1 - V. dahlae cdna lbrary appear to be of plant orgn. Ths unantcpated result suggests that the presence of fnngal gene actvty (mrna expresson) n nfected cotton plant root tssue, at such a short tme (24 hr) after noculaton wth V. dahlae, s mnmal f not nonexstent. t was therefore deemed unwse to re-screen the exstng gene lbrary for more lkely fungal gene canddates. We beleve that the solaton of potental fungal pathogencty genes wll only be possble followng the constructon of a new cdna lbrary from nfected co~on plant tssue harvested at a longer tme nterval after fungal noculaton. However, there was nsuffcent tme or resources avalable to construct and screen a cdna lbrary of ths knd. CRDC Project US32C Fnal Report September 2 7

10 Our surveys have revealed that some strans of V. dahlae prevalent n Australan cotton producton regons are capable of causng severe dsease even on wlt-resstant G. hrsutum cultvars such as Scala V-1. The am of ths project was to dentfy DNA markers assocated wth enhanced or dfferental host pathogencty n these fung. A successful research outcome would enable the rapd dentfcaton of pathogenc organsms and could provde nformaton on ther feld survval, persstence and mode of spread. Such nformaton could be of use durng trals of new dsease-resstant cultvars and n the desgn of dsease control strateges. We have mantaned nformal communcaton lnks wth cotton plant pathologsts such as Stephen Allen and Davd Nehl at ACR and Joe Kochman at DPQ regardng the dentfcaton DNA markers that may be assocated wth ncreased pathogencty n V. dahlae. We have also communcated research fndngs as approprate n ndustry forums such as the Australan Cotton Conference and The Australan Cottongrower. Detaled results wll be publshed n a promnent nternatonal plant pathology journal when the research s completed. 6. An assessment of the lkely mpact of the results and conclusons of the Research project for the cotton ndustry and, where possble, a statement of the costs and potental benefts to the Australan. cotton ndustry and future research needs Surveys of strans of V. dahlae prevalent n Australan cotton producton regons have revealed that some are capable of causng severe dsease even on wlt-resstant G. hrsutum cultvars such as Scala V-1. As an adjunct to ths project, addtonal cotton plants n the Namo regon were sampled n March 1997 n order to obtan examples of V. dahlae currently nfectng crops n that dstrct. Although the sampled cotton plants exhbted the symptoms of mld Vertcllum wlt, fung cultured from the vascular tssues of these plants were not dentfed as V. dahlae, but nstead comprsed at least fve dstnct speces, ncludng solates of Fusarum, Phomopss and Cladosporum whch are potental pathogens of cotton. These fndngs suggest that not all dseased plants n a feld sufferng wlt symptoms may be nfected wth V. dahlae, and that a range of prevously undetected pathogen speces may be prevalent n a partcular cotton feld at a gven tme. t s clear that the development of molecular dagnostc tools, such as those used n ths analyss, wll have mportant mplcatons for a thorough understandng of dsease epdemology n cotton crops. Q 7. A descrpton of the project technology (e.g comrnercally sgnfcant developments, patents appled for or granted, lcences etc) Not applcable. 8. A techncal summary of any other nformaton developed as a part of the Research Project ncludng dscoveres n methodology, equpment desgn, etc Prevous experence n the use of AFLPs for plant analyss has enabled us to optmse the AFLP technque for use wth V. dahlae. Ths requred the desgn and manufacture of unque AFLP prmers wth fewer selectve nucleotdes, due to the sgnfcantly smaller genome sze of the Vertcllum fungus. We have determned that the optmum prmer CADC Project US32C Fnal Report September 2 8

11 pars for V. dahlae DNA comprse EcoR prmers wth two selectve nucleotdes and Mse prmers wth one selectve nucleotde. A total of eght EcoR prmers have now been desgned and manufactured (EAA, EAC, EAG, EAT, ECA, ECC, F;CG and ECT). When used n conjuncton wth the four Msel prmers (MA, MC, MG, MT), they ncrease the potental for AFLP analyss to a total of 32 prmer combnatons. These 32 prmer combnatons can be used on the exstng DNA templates to ncrease the amount of the V. dahlae genome surveyed for DNA polymorphsm to 5%. 9. Recommendatons on. the actvtes or the steps that may be taken to further develop, dssemnate or explot the project technology Our research has determned that all of the nearly 12 clones selected from the Scala V- 1 - V. dahlae cdna lbrary appear to be of plant orgn. Ths unantcpated result suggests that the presence of fungal gene actvty (mrna expresson) n nfected cotton plant root tssue, at such a short tme (24 hr) after noculaton wth V. dahlae, s mnmal f not non-exstent. Ths s despte our clear evdence that expresson patterns of plant genes undergo dramatc qualtatve and quanttatve changes durng ths tme perod. t s suggested from our results that the solaton of potental fungal pathogencty genes usng smlar procedures wll only be possble followng the constructon of a new cdna lbrary from nfected cotton plant tssue harvested at a longer tme nterval after fungal noculaton. Our evdence suggests that the expresson of some cotton genes begns to declne at hr, and ths may be n response to hgh growth rates of the nfectng fungus. nfected plant tssue sampled at these tmes mght therefore be predcted to contan hgher traces of fungal gene expresson, leadng to the solaton of fungal genes nvolved n pathogencty. 1. A lst of publcatons arsng from the research project. Ramsay JR, DS Multan, and BR Lyon (1996). RAPD-PCR dentfcaton of Vertcllum dahlae solates wth dfferental pathogencty on cotton. Aust. J. Agrc. Res. 47: Lyon BR, Y Zhu, Y-Y Wang, and DS Multan (1996). Vertcllum wlt of cotton: Epdemology of Vertcllum dahlae. pp , Proceedngs of the 8th Australan Cotton Conference, Broadbeach, Australa. Kenry CA, MK Hlt KP Rdgway, and BR Lyon (1996). Molecular genetc analyss of the cotton-vertcllum nteracton. Poster sesson, 8th Australan Cotton Conference, Broadbeach, Australa. Hll MK, KJ Lyon, and. BR Lyon (1999). dentfcaton of dsease response genes expressed n Gossypum hrsutum upon nfecton wth the wlt pathogen Vertcllum dahlae. Plant Mol. Bol. 4: Ho, K, Y Zhu, DS Multan, and BR Lyon. DNA polymorphsm and pathogencty relatonshps of Vertcllum dahlae solates from cotton plants (n preparaton). CAOC Project US32C Fnal Report September 2 9

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