Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. Genetic Studies of Pathogenicity • In Botrytis cinerea (Botryotinia fuckeliana) A thesis presented in partial fulfilment of the requirenlents for the degree of Doctor of Philosophy in Plant Science Massey University Pauline Weeds 1997 11 Abstract Botryti.r, cinerea is a common pleomorphic fungus causing 'grey mould' disease on a wide range of crops resulting in serious losses both pre- and post-harvest. Traditional control measures rely heavily on frequent fungicide applications. A greater understanding of the infection process and more information on d1e factors determining successful and unsuccessful host/pathogen interactions is important for the development of new control strategies. Although widely studied, relatively litde is known about its genetics and the factors that determine its pathogenic ability. This study examines the genetics and pathogenicity of B. cinerea through mutation and selection. T�o new genetic markers were developed based on resistance to the toxic analogues sodium selenate and potassium chlorate. These markers ,vere then utilised in sexual crosses and competition studies in planta. Selenate resistant (SelR) mutants of Botrytis cinerea were selected by plating conidia or • mycelial plugs onto minimal medium amended with selenate and taurine. Mutants could be divided into three classes based on growth in d1e presence of selenate or chromate and on improved growth in response to taurine in minimal media. Some mutants grew poorly on minimal media but were responsive to taurine, indicating they were defective in sulphate reduction. Strains showing the SelR phenotype may result from mutations in different genes; d1e genetic symbol Sell was allocated to one. Nitrate non-utilising (Nit) mutants, generated as spontaneous sectors on minimal media amended with chlorate, behaved as nit1 mutants in growth tests (putatively defective in nitrate reductase apoenzyme) and the genetic symbol nit! was allocated to one of these mutants. \lVThen nit1 mutants were paired on medium wid1 nitrate as sole nitrogen source, some pamngs complemented, behaviour attributed to intragenic complementation. Selected crosses of SelR and nit1 mutants with wild type strains gave 1: 1 segregation of both phenotypes and no evidence of linkage to either Mbc1 (benzimidazole resistance) or 111 Dqf! (clicarboximide resistance) markers; loose linkage was confirmed between Mbcl and Daj1. Both Sel1R and nit! mutants were stable following subculture and retained pathogenicity in a French bean leaf assay. Complementation was demonstrated between a taurine responsive SelR mutant and a nit! mutant selected from the same parent. Non-aggressive mutants were isolated from a single-ascospore strain of B. cznerea following mutagenic treatment (ultraviolet and 4-nitroquinoline-1-oxide) and screening on French bean leaves. Crosses "\vith reference strains SASS6 or SAS40S revealed one u.v. mutant (Mp97) in which the non-aggressive phenotype segregated 1:1; indicating a single gene of major effect on pathogenicity to which the genotypic symbol Pat! was allocated. No evidence of linkage was found between Pat! and either Mbcl , Daj1, nit! or Sell . Further characterisation of this gene in stuclies invol"ving Pat! and \vild-type strains revealed various host and temperature responses. Pat! strains produced small, restricted lesions on French bean and soybean leaves and slowly spreading lesions on rose flowers. on tomato stems at 20 and 25°C the mutant was essentially non-pathogenic, although a reduced number of invasive infections were produced at 10 and 15°C. Pat! re\c.:\·VQ\,> strains grow/normally, are inclistinguishable from wild-type in gross morphology, and grow well o ' n minimal medium inclicating no unusual nutrient requirements, and it was concluded from comparison of physiological characteristics that the non-aggressive character is unlikely to be due to gross unfitness. No difference was found between Patl and wild-type strains in total polygalacturonase .. II\V,tfO activityj andclifferences in polygalacturonase isozyme profiles were not correlated with the presence of the Pat! gene. Pat! was found to correlate v,rith low acid production indicating a role for organic acid in pathogenesis of B. cinerea. Microscopic examination of 4-day-old lesions showed a clistincdy stained ring of mesophyll cells surrounding lesions of Mp97 but not it;; parent (A4) , suggesting a clifference in host response. Differences in phytoalexin induction in soybean were not found. It is possible that Patt strains may be deficient in the ability to tolerate or metabolise defence compounds. Two hypotheses are presented for further investigation. The first that Patl strains may have reduced toxicity due to low production of organic acids, and the second that these strains are non-aggressive due to a reduced ability to metabolise defence compounds. In competition experiments aggressive and non-aggressive strains were found to co­ exist in the same lesion when inoculated at the same time but when challenge inoculations were delayed 6 hours or more the initial inoculation was found to dominate, suggesting non-aggressive strains may be useful as biocontrol agents. IV Acknowledgments This thesis is dedicated to the memory of my brother Ian (Loopy) Weeds whose busy productive life and too early death (October 1987) inspired me to get on with life: this work being one result. I thank my supervisors Ross Beever and Peter Long for their help and patient encouragement. I am also very grateful to the following people for their important contribution; Keith Sharrock for advice and assistance '.vith the polygalacturonase activity and phytoalexin induction work and his contribution of the PG isozyme studies, Shiva Ganeshanandam for advice and assistance with data analysis, Daryl Cook for help with the monoclonal antibody work, Lorraine Davis & Hugh Nielson for trouble shooting and technical stuff at Massey, Martin Heffer for the photography and for always being there when the fungus was photogenic, Ian Hallett and Paul Sutherland for help with the microscopy, Henry Pak for translating all those German texts, Patrick Connolly & Rod • Ball for the logit analysis, Michael Eden for the tomato plants, pots and timely advice, Christoph Janisch & Willie Cornett, for all that computing help, Ken Young for the donation of copious glasshouse roses, Bob Fullerton for supplying pre-conditioned S clerotinia .rclerotiorum sclerotia and culturing advice, Molly Dewey for the gift of the BC­ KH4 monoclonal antibody, the staff at the Mt Albert library for finding all those urgent references so fast, HortResearch for the use of facilities, Crop and Food for the use of a glass house, AGMARDT for the funds, and Landcare Research Mt Albert for a friendly and supportive working environment, my air conditioned office and the generous use of facilities. Thanks to the people at Mt Albert Research Centre and Massey University who were always generous with their advice and time including; Kim Plummer, Matt Templeton, Jo Bowen, Eric Rikkerink, Brian Hawthorne, J ohn Young, Peter Johnston, Eric McKenzie, Helen Lindsay, Morag MacLean, Rosie Bradshaw, and Ken Milne. v For my friends, work-mates, and fellow students - thanks for being there and making it fun; Jessica Beever for the sympathetic ear and all those lunchtime walks, Duckchul Park for all the computer things, Kerry Everett, for chats and lunchtime walks, Robyn Howitt, Carolyn Moore, Joanne Patterson, Tala Aliifaalogo, Michelle Napier, Tia Ifopo, Daryl & Kirstin Cook, Pam Howell, Mark Levick, Martin Ball, Paula Wilkie, Allison Gianotti, and Jane Frohlich. A special thanks to Stephanie Parkes for always being interested and making the lab a great place to work. Last but never least thanks to my friend Heather Samson and my family including; Joy & Arthur, Sandy, Ian, Sally, Janine, Jo and my daughter Lily for your support, understanding and encouragement. Victory to the truth. Triumph to the invincible sun. VI Contents Abstract .�. . ........................................................................................................ ii Acknowledgments ................................................................................................ iv Contents ......................................................................................................... vi Tables ......................................................................................................... ix Figures ......................................................................................................... x Preface ......................................................................................................... xiv Chapter 1 Introduction ........................................................................................ 1 Botryti.r cinerea ............................. ..................................................... .................................... ... ...... 1 Taxonomy ............................................................................................................................................ 1 I\forphology ......................................................................................................................................... 2 Patholo!!)! ........................................................................................................................................ 5 Infection ............................................................................................................................................... 7 Disease development. ........................................................................................................................ 8 TIle se�-ual stage ................................................... .................................................... . ....................... 1 0 Host pathogen interactions ........................................................................................................... 12 Noble rot ........................................................................................................................................... 14 Control ............................................................................................................................................... 14 Genetic ,rtudieJ ofB. cinerea ..................................................................................... ..................... 15 Phenotypic variation ....................................................................................................................... 15 Cytology ............................................................................................................................................. 17 Tools for genetic analysis .............................................................................................................. 18 Mating-type ....................................................................................................................................... 21 Fungicide Resistance ....................................................................................................................... 21 ObjcctivCJ of thiJ .rtuc!y ................................ .................. ................................................................ 23 Chapter 2 General Methodology ......................................................................... 24 Storage ................................................................................................ ......................................... 24 Media and growth conditionJ ........................................................................................................ 24 Botryti.r .rtrainJ .............................................. ............................................................................... 24 Growth reJtricting medium ..................................................................................................... ...... 25 MutageneJiJ ............ ..... .. .............................. ..... ....................... .................... ..... ................. .. .... ..... 25 Spontaneous mutations .................................................................................................................. 25 Ultraviolet radiation ........................................................................................................................ 26 4-nitroquinoline-1-oxide (NQO) ................................................................................................. 26 Pathogenici!J a-rJqJJ ........................................................... ........................................................... 29 French bean ...................................................................................................................................... 29 Modifications to French bean assay for spore inoculations .................................................. 32 CroHingprocedure ........................................................................................................................ 33 AJcopore iJolation ......................................................... ....................................................... ....... 33 Vll Testingfor fungicide resistance ...................................................................................................... 3 7 Testingfor llryce!ia! incompatibili!Y ............................................................................................... 3 7 Genetic terminology ....................................................................................................................... 3 7 Data ana!Jisis ............................................................................................................................... 37 Chapter 3 New genetic markers for B. cinerea ................................................. 39 Introduction .................................................................................................................................. 39 Materials and Methodr ................................................................................................................ 40 Selection of selenate resistant mutants ....................................................................................... 40 Selection of nitrate non-utilising mutants .................................................................................. 40 Selenate response test ..................................................................................................................... 40 Complementation tests .................................................................................................................. 40 Pathogenicity assay .......................................................................................................................... 41 Restdts .......................................................................................................................................... 4 1 sodium selenate resistant mutants ............................................................................................... 41 Nitrate non-utilising mutants ........................................................................................................ 46 Pathogenicity .................................................................................................................................... 47 Linkage relationships ...................................................................................................................... 47 I\farker complementation .............................................................................................................. 47 Dircu.rsion ...................................................................................... .............................................. 50 Chapter 4 Isolation and characterisation of non-aggressive mutants ............... 53 Introduction .................................................................................................................................. 53 Materials and Methods ................................................................................................................ 53 I\futagenesis ...................................................................................................................................... 54 Screening of putative non-aggressive strains ............................................................................ 54 Growth on minimal medium ........................................................................................................ 54 Recognition by monoclonal antibodies ...................................................................................... 56 Re.rult.r ..... : ....... ..... ... . . . ........ . . . ... ...... ......... ...... ............. . . ..... ............ .... ..... ......... . . . . . . . . . . . . . . . . . . . ..... ..... 56 Generation of non-aggressive mutants ...................................................................................... 56 Gro\\Tth properties of the mutants .............................................................................................. 57 Recognition by monoclonal antibodies ...................................................................................... 57 Segregation of pathogenicity ......................................................................................................... 5 7 Linkage relationships ...................................................................................................................... 6 0 S/\.S405 - a homothallic single-ascospore strain ....................................................................... 64 Discu.rsion .................................................................................................................................... 64 Chapter 5 A major gene controlling pathogenicity in Botrytis cinerea ............. 69 Introduction ................. . . . ...... . . . .. ..... . . . . . . . . .. .. . . . ........... .. . . . . ............................. . . . . . . . ................ . . . . .. . . .. 69 Materials and ltdethodr ................................................................................................................ 70 Pathogenesis assays ......................................................................................................................... 70 ?vllcroscopic studies of lesions ...................................................................................................... 71 Physiological characterisation ....................................................................................................... 7 2 Polygalacturonase activity and lesion extraction ...................................................................... 7 2 Addition of cell free lesion extract t o inoculum plugs on French bean leaves ................. 7 4 Phytoalexin induction ..................................................................................................................... 74 Competition ...................................................................................................................................... 75 Results .......................................................................................................................................... 76 Characterising of Patl on French bean ...................................................................................... 76 Physiological and morphological characterisation ................................................................... 79 Characterisation of Patl on other hosts ..................................................................................... 88 Competition ............................................................................................................. ......................... 88 Discussion .................................................................................................................................... 92 Chapter 6 General Discussion ............................................................................ 97 Future work ....................................... .... ................................................................................... 100 References ......................................................................................................... 104 Index ......................................................................................................... 117 Vlll Appendix 1 Abbreviations .................................................................................. 119 Appendix 2 Media and solutions ........................................................................ 120 Fungal growth media ................................................................................................................. 120 CM (Complete medium) .............................................................................................................. 120 CM+Triton (Triton medium) .................................................................................................... 120 11EA (Malt extract agar, Oxoid) ............................................................................................... 120 11EA+A (Malt extract agar, Oxoid no. 2) ............................................................................... 120 11EA+carbendazim (Benzimidazole) ..................................... ................................................. 120 11EA+NaCl (Sodium chloride medium) ............................................ ................................ . ... 121 11EA +vinclozolin (Dicarboximide) ........................................................................................ 121 MM (Minimal medium) .............................................................................................................. 121 MM+Cl03 (Chlorate medium) ................................................................................................. 121 1-fM+Cr04 (Chromate medium) .............................................................................................. 121 MM+Hx (Hypoxanthine medium) ......................................................................................... 121 MM+N� (Ammonium medium) .......................................................................................... 121 MI1+N02 (Nitrite medium) ..................................................................................................... 122 MM+N03 (Nitrate medium) .................................................................................................... 122 MM+Se04 (Selenate medium) ................................................................................................... 122 MM+Se04B (Selenate medium B) ............................................................................................ 122 MM+taurine (Taurine medium) ............ ..................... ............................................... ............... 122 Modified-Richardson's-medium A ....................................................................................... ...... 122 Modified-Richardson's-mediumB ........................................................................... ................... 123 PDA (potato dextrose agar) ........................................................................................................ 123 PDA+bromophenol. .................................................................................................................... 123 PDA-V2 (Half-potato dextrose agar) ............ .. .......................................................................... 123 Vogel's .................................................................. ............................................................................ 123 Vogel's medium N ........................................................................................................................ 124 Vogel's medium N-....................................................................................................................... 124 Vogel's+sucrose ............................................................................................................................. 124 Btiffers ....................................................................................................................................... 125 Potassium phosphate buffer (0.1 M) ........................................................................................ 125 Others ....................................................................................................................................... 125 Aniline blue clearing/ staining solution .............. . ....................... ........ .......... . ............................ 125 QO (4-Nitro-1-quinaline oxide) ............................................................................................ 125 Polygalacturonase cup plate assay substrate ............................................................................ 125 Sodium thiosulphate solution ..................................................................................................... 125 PBS ................................................................................................................................................... 126 PBST ................................................................................................................................................ 126 Appendix 3 Botrytis strains ................................................................................ 127 Appendix 4 Glossary of selected terms .............................................................. 129 Appendix 5 Summary of genetic nomenclature ................................................ 134 Genorype designation ................................................................................................................. 134 Phenorype designation ................................................................................................................ 135 Mating-rype designation ............................................................................................................. 135 Appendix 6 Summary of genetic symbols .......................................................... 136 Appendix 7 Pathogenicity related fungal attack chemicals .............................. 137 Appendix 8 Polygalacturonase standard curves ................................................ 139 Appendix 9 Segregation of polygalacturonase isozymes .................................. 141 Abstract .................................................................................................................................... 141 Table 1 Table 2 Table 3 Table 4 Table 5 Table 6 Table7 Table 8 Table 9 Table 10 Table 11 Table 12 Tables Growth of selenate resistant mutants on minimal medium amended ,vith sodium selenate, potassium chlorate or taurine. Control strains (Sel1 S) were Al, AS, IX REB678-1, SAS56, and SAS405 . ................................................................................................. 42 Segregation of selenate (SelR) or nitrate non-utilising (nit 1) mutants in Botryti.r cinerea crosses involving known fungicide resistance markers . .............................................. 45 Segregation of class B selenate resistant (Sell), nitrate non-utilising (nif1), benzimidazole resistant (Mbc1), and dicarboximide resistant (Dafl) markers in four-point crosses of Botryti.r cinerea ............................................................................................. 48 Progeny analysis of seA'Ual crosses, 10 and 11, reciprocal crosses of mutant (Mp97, Patt) with the reference strain (SAS56) . ...................................................................... 61 ProgeJ:?y analysis of sexual cross involving five genetic markers selenate (Se!l), ,rinclozolin (Dqfl), carbendazim resistance (Mbc1), mil (nitrate non-utilising), and Patl (pathogenesis) . ........................................................................................................................ 63 Pattern of fungicide resistance in progeny from SAS405 'fertilised' with SAS405 microconidia or with water . .......................................................................................................... 65 Assessment of progeny from SAS405 'fertilised' with SAS405 microconidia or 'N1th water for consistency with segregation of fungicide resistance markers as a diploid with the resistance allele dominant over the sensitive allele ..................................... 66 Comparison of physiological characteristics for 10 aggressive and 10 non­ aggressive progeny of the cross Mp97 / SAS56 and of the parental strains Mp97 and SAS56 . ....................................................................................................................................... 77 Growth of Mp97 and the parental strain A4 on detached leaves of French bean and soybean seedlings, and rose flowers .................................................................................... 88 Sources and genotypes of Botryti.r cinerea strains . ..................................................................... 127 Genetic symbols used for genes of B. cinerea ........................................................................... 136 Summary of references investigating the importance of B. (7nerea attack chemicals to d1eir aggressiveness on a particular host (correlation 'yes' indicates positive correlation unless otherwise stated) . ......................................................................................... 137 Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figures Spore forms of Botrytis dnerea. (A) Conidia from lO-day-old cultures grown on MEA at 200C . (B) Microconidia from 1-month-old cultures grown on :MEA in x d1e dark at 1SoC. (C) Ascospores isolated from apod1ecia ....................................................... 6 Growth stages of apod1ecia after 4 mond1s incubation at 100C follo'vving standard mating procedure. (A) Apothecial stipes developing from a sclerotium. (B) Young apod1ecia. (C) I'vlature apothecia . ................................................................................................... 6 Examples of grey mould disease on various hosts. (A) Bunch rot of grapes (B) Petal fleck on glasshouse grown rose flowers (C) Soft rot of kiwifruit and infection spread during storage resulting in 'nesting'. (D) Stem canker caused by infection of lateral shoot removal wounds in glasshouse tomatoes. (photos from the collection of the Plant Science Dept. Massey University) ................................................. 9 Apoth.ecia gruwing on a peach 'mummy' found by G. Tate and co-workers in a commercial peach orchard ('Golden Queen') Hawkes Bay (New Zealand) in ................. 11 " .,,,� Apothecia of Botrytis dnerea (produced in d1e laboratory as described p33) and Sderotinia sc!erotiorum (produced in d1e laboratory from sclerotia pre-conditioned by burial in soil for 6 months and then incubated on moist paper towels under 12-hours-on/12-hours-off cycled white and long-wave ultraviolet radiation for 10 days). . ............................................................................................................................................ 11 Percentage of conidia surviving after exposure to ultra"violet radiation. Assessed as percent spore germination on CM after 8 hours incubation at 20oC .............................. 28 Percentage of conidia surviving after incubation in 4-nitroquinoline-l-oxide for 1 hour. Assessed as colonies counted using a dissecting microscope following 2- days incubation on CM + Triton ................................................................................................... 28 Average lesion radii on French bean leaves following inoculation with mycelial plugs from the margin of different aged colonies of A4 grown on MEA at 20oC. Bars indicate standard error of means from 32 replications . ................................................. 31 Average lesion radii on French bean leaves following inoculation with mycelial plugs from d1e margin of different aged colonies of A4 grown on :MEA in tissue culture erC) plates at 10°C. Bars indicate standard error of means from 24 replications . ...................................................................................................................................... 31 Lesion size and percent infection of Botrytis dnerea on French bean leaves following inoculation of conidial suspensions (strain A4) amended with phosphate glucose solution or Vogel's +sucrose. Bars indicate standard error of means from 16 replications ........................................................................................................... 34 Figure 11 Agar plate with sclerotia ready for harvesting and mating Oeft), McCartney vial containing sclerotia and apothecia (right rear), and sclerotia with apothecia (right Xl front). . ............................................................................................................................................ 35 Figure 12 Test for fungicide resistance: SAS56 (Mbc1 5 Dcif1 S) upper and SAS405 (Mbc1 HR Daf1 LR) lower. (A) IvffiA .. (B) MEA +carbendazim. (C) ]\.![RI\ +vinclozolin . .................... 36 Figure 13 Test for mycelial incompatibility. (A) SAS56 paired with SAS56. (B) SAS56 paired \vith SAS405. (C) SAS405 paired with SAS405 . ....................................................................... 36 Figure 14 Growth responses of selected selenate resistant mutants of Botrytir cinen:a on minimal medium amended ,,;'th selenate plus taurine (MM +Se04, upper row) and on minimal medium amended \vith taurine (MM +taurine, lower row) after 6 days: Strains are; (A) P2-011 (SelR class A), (B) P5-026 (SeJ1R class B), and (C) Al (v.i.ld-type) . ................................................................................................................................. 43 Figure 15 Growth responses of a nitl mutant of BotrytiJ cinerea on medium with nitrate as the nitrogen source (MM+N03). Strains are; P6-00S (nit1 mutant) - upper, thin sparse mycelial growth spreading across the plate and abutting the thick dense grO\vtb of A5 (v.i.ld-type) - lower . ............................................................................................... 43 Figure 16 Germination response of selected selenate-resistant mutants of BotrytiJ cinerea on media amended with a range of selenate concentrations . ...................................................... 44 Figure 17 Mean lesion sizes caused by selected strains of Botrytzj cinerea on French bean leaves after 3-days incubation at 20 - 2SoC. Strains are; wild-type strains (AI and AS), selenate mutants from class i\, class B, and class C (MsOIO, MsOIS, and Ms024 respectively), selenate resistant progeny from crosses of MsOIO (P2-00S) and Ms01S (PS-006), nitl mutants (MnOOI and Mn012), one nit1 progeny from cross of MnOOl (P6-00S), one progeny carrying both nit1 and SeJ1R markers (p16-025). Bars indicate standard error of means from 9 replications . ............................... 44 Figure 18 Complementation pattern of selected nitl mutants derived from strain AS. (A) Score matrix for mutants paired on tv1]v1+N03 medium. (B) Complementation map derived from data in A. Mutants in non-overlapping groups complement, those within one group or in overlapping groups do not . ..................................................... 47 Figure 19 Complementation between nitl mutants derived as spontaneous sectors from the parent strain AS (MnOOl, Mn004 and Mn005) or REB689-I (MnOI2). Tested using method A on MM+N03. A 1:1 mix of spore suspensions was placed in the centre of each plate. (A) MnOOS paired with I:vinOO1. (B) MnOOS paired with Mn004. (C) Mn004 paired wid1 Mn012 . .................................................................................... 49 Figure 20 Complementation between SelR (class A) and nitl mutants on MM+N03 (method A). (A) Ms033 (parent A5) paired \vid1 Mn012 (parent REB689-1). (B) Ms033 (parent AS) paired with 11nOOI (parent A5). (C) Ms010 (parent AI) paired wid1 MnOOl (parent AS) . ............................................................................................................... 49 Figure 21 Schematic outline of screening method for non-aggressive mutants on French bean leaves ........................................................................................................................................ 55 Figure 22 Germinated conidia 1n1muno-stained with BC-KH4 and anti-mouse MAb conjugate FITC viewed under ultraviolet. light (3S0 - 390 nm) . (A) A4 parental v.i.ld-type strain. (B) MpI23 (non-aggressive 4-nitroquinoline-I-oxide mutant from A4). (C) Sclerotinia JcJerotiorum . ............................................................................................. 58 Figure 23 Figure 24 Figure 25 Figure 26 �igure 27 Figure 28 Figme 29 Figure 30 Figure 31 Figme 32 Figme 33 Figure 34 Figure 35 Lesions on French bean leaves inoculated with 5-mm plugs of BotrytZ:r cinerea. Strains include Mp97 (non-aggressive), SAS56 (aggressive) and 8 progeny of the cross (Mp97/ SAS56). Four progeny show a non-aggressive phenotype and four xu show an aggressive phenotype . .................................................................................................... 58 Distribution of lesion sizes in progeny of non-aggressive mutants crossed with aggressive single-ascospore reference strains. (A) Control cross A4 (sclerotial parent) with SAS56 (microconidial parent). (B) Cross SAS56 (sclerotial parent) \,,1.th Mp68 (microconidial parent). Mean lesion size of parental strains in parentheses ....................................................................................................................................... 59 Distribution of lesion sizes in progeny of Mp97 crossed with aggressive single­ ascospore reference strain. (A) Cross Mp97 (sclerotial parent) \V1.th SAS56 (microconidial parent). (B) Reciprocal cross SAS56 (sclerotial parent) "\",1.th Mp97 (microconidial parent). Mean lesion size in parentheses . ....................................................... 62 A typical restricted lesion produced on a French bean leaf inoculated with a 5- mm agar plug taken from the actively growing edge of a non-aggressive strain (P21-47) of Botrytis cinerea and incubated for 3 days at 20 - 250C under conditions of high hmnidity. Arrows indicate the characteristic dark brown ring enclosing the lesion ........................................................................................................................................... 78 I\fultilobed appressoria of Botrytis cinerea isolate Mp97 on infected French bean leaves incubated for 22 or 24 hours at 20 250C and stained with aniline blue . .............. 81 Multilobed appressoria of Bottytis cinerea isolate A4 on infected French bean leaves incubated for 22 or 24 hours at 20 25°C and stained "\ .. 1.th aniline blue . .......................... 81 Hyphae of A4 penetrating through successive layers of French bean tissue after 20 homs incubation at 20 - 25°C. (A) Upper epidermis. (B) Palisade parenchyma. (C) Spongy parenchyma . ............................................................................................................... 82 Mycelium of Bottytis cinerea in lesions on adaxial surface of French bean leaves incubated for 24 hours and cleared and stained with aniline blue. M = mycelium, S = darkly stained mesophyll cells. (A and C) A4 aggressive. (B and D) Mp97 non-aggressive . ................................................................................................................................ 83 Daily growth rate on :MEA of Mp97, A4, and SAS56 averaged over 8 days (lOoC), 6 days (15oC), and 4 days (20 and 250C). Means with the same letter are not significantly different (Duncan's multiple range test, alpha - 0.05). Bars indicate standard error of means from 5 replicate plates . ...................................................... 84 Infection of tomato stem-pieces recorded as a percentage of 22 replications after 14-days incubation . ......................................................................................................................... 84 Total polygalacturonase acti'vity of progeny (P-strains), parents (Mp97 and SAS56) from the cross Mp97 x SAS56 and A4 (expressed as activity units of tl1e Aspet:gi!!u.r standard). (A) 8-hours incubation. (B) 24-homs incubation. Bars indicate standard error of means from 4 replications . ................................................... 85 Polygalacturonase (pG) isozymes in l-day-culture filtrates following isoelectric focusing and incubation for 4.5 hours at 37°C (overlays stained witl1 mtl1enium red). A letter after the strain number distinguishes replicated cultures. (photo Dr Sharrock) ........................................................................................................................................... 87 Polygalacturonase isozymes in extracts of 4-day-old lesions of bean leaves inoculated with B. cinerea. Lanes loaded with 10 fll of extract from lesions homogenised in 10 volumes of buffer. SAS56 lesion extract diluted 50 and 100- fold respectively. :MP97 lesion extract undiluted. A4 lesion extract diluted 100 and Xlll 50-fold respectively (overlay incubated for 15 hours). (photo Dr Sharrock) ..................... 87 Figure 36 Lesion radii on French bean leaves inoculated with either SAS56 (aggressive) or P33-146 (a non-aggressive single-ascospore strain) and then challenged after 0,6, 12 or 24-hours incubation with either P33-146 or SAS56 respectively (incubated for 4 days at 20 - 25°C). Bars indicate standard error of means of 4 replications in each of 4 blocks ............................................................................................................................... 90 Figure 37 Percent germination on selenate (MM+Se04B) and vinclozolin (MEA +vinclozolin) differential media, of spore suspensions made from whole leaf-lesions inoculated with either Ms044 (selenate resistant, SelR) or SAS405 (vinclozolin resistant, DqflLR) and then challenged (Ms044 challenged with SAS40S, SAS40S challenged with Ms044) after 0, 6, 12 or 24-hours incubation (incubated for 7 days at 20 - 25oC). Bars indicate standard error of means of 3 replications of 300 spores . ............................................................................................................ 90 Figure 38 Interaction zone on 11RA+NaCl. (A) P33-146 paired with P33-146. (B) P33-146 paired with SAS56. (C) SAS56 paired with SAS56 . ................................................................. 91 Figure 39 Interaction zone on MEA+NaCl. (A) SAS405 paired with SAS405. (B) SAS405 paired with Ms044. (C) Ms044 paired \'lith Ms044 .................................................................. 91 Figure 40 Polygalacturonase standard curves generated from zone diameters produced on substrate gel by a series of dilutions of the A. nl�"-r PG standard. R2 values indicate the appropriateness of the fit (the closer to 1 the better the fit) .......................... 140 XlV Preface This thesis consists of an introduction and literature review (Chapter 1), a description of the techniques developed and general methodology (Chapter 2), followed by the main experimental work grouped into three subject areas (Chapters 3 5) and written as discrete units to facilitate publication, and ending with a general discussion and outline of possible future ,vork (Chapter 6). To keep the text uncluttered some information has been placed in the appendices; a list of abbreviations (Appendix 1), components, instmctions, and references for media and solutions (Appendix 2), source and genotype of B. cinerea strains (Appendix 3), a glossary of selected terms (Appendix 4), a summary of genetic nomenclature (Appendix 5) , a summary of genetic symbols (Appendix 6), a summary of enzymes related to pathogenicity with relevant references (Appendix 7), and experimental details of the polygalacturonase (pG) cup-plate-assay (Appendix 8) Chapter 3 has been accepted for publication in Mycological Research co-authored . with supervisors Drs Beever (Landcare Research, Mt Albert) and Long (I'v1assey University) . Preliminary results and conclusions from the work presented in Chapter 3 were presented at the Xl th International Botrytis Symposium held in Wageningen (the Netherlands) in June 1996. Weeds PL, Beever RE, Long PG. 1996. New genetic markers for Botrytis cinerea. In xrh International Botrytis Symposium Programme and book if abstracts. Wageningen, the Netherlands. Pp12. Weeds PL, Beever RE, Long PG. 1997. New genetic markers for Botrytis cinerea (Bot?J!otinia fuckeliana) . lvlycological Research. In press. Chapter 5 is being prepared for submission to Physiological and Molecular Plant Pathology, co-authored with supervisors Drs Beever and Long, and Dr Sharrock CHort Research, Ruakura) who advised on PG enzyme and phytoalexin assays and carried out isoelectric focusing experiments on mutant and wild-type strains. Results and conclusions from the PG isoelectric focusing experiments are included within the text (boxed) for xv completeness. An extension of these studies, showing segregatlon patterns of PG isozymes, was presented as a poster at the Australasian Plant Pathology Society 11 th Biennial Conference held in Perth (Australia) in September 1997 (Appendix 9). Weeds PL, Beever RE, Sharrock KR, Long PG. 1997. A major gene controlling pathogenicit:'j in Botrytis cinerea (Botryotinia jucke/iana) . Pl?Jsiological and Molecular Plant Pathology. In preparation. Sharrock KR, Weeds PL, Beever RE. 1997. Segregation of polygalacturonase isozymes in sexual progeny of B. cinerea (Botryotinia juckeliana) . Proceedings of the 11 th Biennial Conference of the Australasian Plant Pathology Society, 29 Sept. - 2 October, Perth, Australia. p. 142. Clarification of terms Some terms are used inconsistently through this thesis. Selenate resistant (SelR and Sel1R) mutants are sometimes referred to as selenate mutants. NaSe04 and Se04 and selenate are used interchangeably. The term 'parent' refers to both the wild-type progenitor of mutants and both the sclerotial and fertilising strain in sexual crosses. Chapter 1 Introduction 1 Chapter 1 Introduction Botrytis cinerea B. cinerea is a common pleomorphic fungus that causes 'grey mould' disease on a "Wide range of plants in temperate regions U arvis, 1977]. Taxonomy Botryotinia fucke/iana (de Bary) Whetzel (anamorph Botrytis cinerea Pers.) is classified in the Ascomycota (Ascomycetes), order Leotiales and family Sderotiniaceae [Hawksworth et ai. , 1995]. :Micheli established the genus Botrytis in 1729. Botrytis cinerea, one of :Micheli's species, was named by von Haller in 1771 and included by Persoon in 1801 as one of five species of Botrytis. In 1 822 Persoon increased the number of species to 27, by 1 886 this had grown to 128 and was later expanded to 380 species. The genus has since been redefined three times by \X'hetzel, Buchwald, and Hennebert and 22 species are now recognised [Hennebert, 1973; Jarvis, 1977; Coley-Smith et aI., 1980]. For a long time B. cinerea was viewed by many as a group of related species, due to the "Wide variation in vegetative forms and the lack of an established sexual stage, and individuals were commonly referred to 'a Botrytis species of the cinerea type' [Whetzel, 1945] . De Bary first proposed a connection between the anamorph B. cznerea and its teleomorph which he named Pe=d:<:fl Jucke/ialla de Bary [de Bary, 1887]. The teleomolph was later transferred to the genus S clerotillia by Fuckel in 1869 - S clerotinia Jucke/iana (de Bary) Fuckel 1869 [Gregory, 1949], and to Botryotinia by \X'hetzel in 1945 - Botryotinia Juckeliana (de Bary) Whetzel. The question of a perfect stage connection, which had been debated for many years, was largely resolved when Groves & Drayton [1939] obtained apothecia from pure cultures of 'Botrytis of the cinerea type' in the laboratory. Unfortunately they were unable to name their fungus because of doubts as to d1e exact identity of de Bary's Pe:dza jucke/iana. Chapter 1 Introduction 2 The connection was not fully accepted until Gregory [1 949] located a contemporary description of Pe�za fuckeliana and Botrytis cinerea and re-examined de Bary's original slides of Pe=(!za fuckeliana. Gregory also later identified apothecia produced from B. cinerea as identical to those of Pe'{jza fuckeliana as described by de Bary [Groves & Loveland, 1 953] . This has since been confirmed by the work of Faretra and coworkers [Faretra et aL, 1 988b; Faretra & Pollastro, 1 99 1 , and 1 993a] who carried out crosses of some 300 field-isolates from various host plants and countries and showed that they could produce viable progeny when crossed with B. cinerea reference strains. In a less comprehensive study of New Zealand field-isolates Beever & Parkes [1 993] found that some failed to produce apothecia when crossed with reference strains, possibly due to the use of suboptimal growing conditions or alternatively to the inability of some isolates to participate in apothecial production. Theoretically such isolates could represent a distinct species but more information would be needed to demonstrate this. A recent study of field-isolates /I identified two non-interbreeding sympatric populations (vacuma and transposa), differing slightly in conidial size, co-existing in the Champagne region. The grouping is supported by the presence of two transposable elements, Boty and Flipper, in only one group and by differences in allelic frequency [Giraud et aL, 1 997] . However, investigations of the two populations are continuing with studies of crosses between vacuma and transposa suggesting the conclusion, of two populations, may have been premature [T Giraud, pers. com.] . Until more information is available the question of speciation within B. cinerea will . remain unanswered. According to nomenclatural conventions the scientific name of the sexual stage Botryotinia fuckeliana should take precedence over the asexual stage Botrytis cinerea [Hawksworth et aL, 1 99 5; Yoder et aL, 1 986] . However, in the case of B. cinerea a pragmatic approach has been taken (as with Aspergillus nidulans, the asexual stage of Emericella nidulans) and the familiar binomial, which is recognised world-wide, has been retained although it is considered appropriate to include both names in publications concerning genetics [Faretra & Grindle, 1 992] . Therefore in accordance with common usage the anamorph name Botrytis cinerea will be used throughout this thesis. Morphology Hyphae of B. cinerea are septate with septa perforated by a single pore, hyaline and indistinguishable from the hyphae of many Ascomycotina [Hennebert, 1 973; Coley-Smith et aL, 1 980] . Two asexual spore forms are commonly found; conidia (strictly macro conidia) and microconidia (Figure 1 p6). The main prop�es for dispersal are 1 Introduction 3 conidia, which are produced in large numbers on darkly pigmented straight conidiophores comprising a swollen basal cell and alternate branches at the apex. These short dark, septate branches each have a terminal ampulla on which conidia develop synchronously on short fine dentides. Conidia are 9 - 1 2 x 7 - 1 0 J.lm [Harrison, 1 988], smooth, hydrophobic, globose, obovate or elliptical shaped, single celled spores with between 3 and 1 8 nuclei [Coley-Smith et aI , 1 980]. The genus Botrytis, from the Greek �OTel)r:;; (bunch of grapes), is named after the appearance of the conidia and conidiophores, which resemble a bunch of grapes. Germination is usually by one or two (sometimes up to five) germ-tubes and occurs more readily when supplied with exogenous nutrients (Infection p 7). Grey mould, the common name for plant diseases caused by B. cinerea, comes from the grey appearance of masses of conidia on plant tissue [Coley-Smith et aI , 1 9 80] . Microconidia, (Figure 1 p6) are hyaline, hydrophilic, globose spores 2 - 4 J.lffi m diameter [Urbasch, 1 985] produced in chains from short phialides inflated at the base and tapering to the apex [Coley-Smith et aI, 1 980]. The phialides are produced from germ­ tubes, mature hyphae, sclerotia, and also �rithin empty hyphal cells [Brierley, 1 9 1 8] . Microconidia develop t o full maturity after release and when mature contain a single sickle-shaped nudeus and one or two large lipid bodies. I\1icroconidia are believed to function as spermatia in the sexual cycle [Drayton, 1 932; Coley-Smith et aI , 1 980] although a survival function has also been proposed with specific conditions (as yet unknown) necessary to 'break dormancy' [Urbasch, 1 985J. Germination and growth of microconidia on artificial medium and successful infection of wounded plant tissue was reported by Brierley [1 9 1 8] . Grindle [1 979] also reported successful germination but other researchers have been unable to repeat this work. Urbasch [1985] found that microconidia occasionally developed a short germ-tube (1 J.lm) but never observed hyphal formation. I\1icroconidia of B. fabae were found to germinate following extended exposure to cold temperatures [Harrison et aI , 1 977]. However, similar treatment of B. cinerea microconidia was unsuccessful in inducing germination [Urbasch, 1 985]. In response to adverse conditions the fungus is reported to produce structures made up of aggregations of 1 ,000 to 3,000 microconidia (30 to 90 J.lm diam). These are initially held together by mucilage but later a sac forms around them [Urbasch, 1 984] . Microconidial aggregates have not been reported by other workers, although structures similar to those pictured by Urbasch were observed in some microconidial suspensions prepared for mating experiments during the course of this study. 1 Introduction Other structures associated with B. cinerea include chlamydospores, sclerotia, and apothecia. Chlamydospores of B. cinerea have been reported in only a few studies; on dried stems of an Umbellifer [price, 1 9 1 1] , in nutrient enriched soil solutions [park, 1 954] , on malt agar (under stressed growing conditions), on greenhouse tomatoes [Urbasch, 1 983] , and on greenhouse and outdoor plants of Fuchsia �ybrida [Urbasch, 1 986] . They are described as large (60 - 70 �Lm), thick walled structures produced as terminal or intercalary cells under adverse conditions and liberated by hyphal disintegration [price, 19 1 1 ; Urbasch, 1 983] . Mature chlamydospores are resistant to drought, nutrient deficiency, and bacterial invasion and tolerate a wide pH range suggesting a role as survival structures [park, 1 954; Urbasch, 1 986] . They appear to be well suited to short term survival of adverse conditions and could have a role in over-summering on crops such as grapes, or as a component in latent infections, but little information is available (Disease development p8) . Chlamydospores germinate only when conditions are favourable, to produce either mycelium or microconidia [U rbasch, 1 986J . Sclerotia are flat or concave on the attachment surface with a pigmented rind (1 or 2 cells thick) , a cortex of loosely packed cells, and a medulla of interwoven cells [Backhouse & Willetts, 1 984] . The size varies between strains but is typically between 1 and 6 mm diam. [Harrison, 1 988) . Although commonly regarded as important for nutrient storage and survival during adverse conditions there is little quantitative data on the survival of sclerotia in the field. Some reports suggest they can survive long periods on tl1e soil . surface but much less if they are buried, especially if the soil is wet [Harrison, 1 988] . Sclerotia can germinate in four ways; to produce mycelium (common on agar media at temperatures above 1 5°C) , to produce conidiophores (more common in the field) , to produce microconidia, or under special conditions to produce apothecia [Coley-Smith et aI., 1 980; Lorenz & Eichhorn, 1 983; Backhouse & Willetts, 1 985] . Apothecia are produced from sclerotia following fertilisation, possibly with microconidia as the spermatia. Stipes grow from the sclerotial surface developing a cap at about 4 - 5 mm which differentiates into a light brown cupulate disk (1 6 mm diam.) becoming reflexed and dark brown to black with age (Figure 2 p6) . Final height is typically from 3 - 1 0 mm with a maximum of 25 mm. Long clavate asci with an apical pore are produced on the upper surface each containing eight hyaline, unicellular, ovoid ascospores measuring about 13 .5 x 6.7 �m interspersed with sterile paraphyses (Figure 1 p6) [Coley­ Smith et al., 1 980; Lorenz & Eichhorn, 1 983; Jarvis, 1 977; Faretra & Antonacci, 1 987] . The ascus initial is binucleate; during development the two nuclei fuse leaving one nucleus in 1 Introduction 5 the young ascus, which then divides to produce eight uninucleate ascospores . The nucleus in each ascospore undergoes further division willie still in the ascus to produce multinucleate ascospores (typically with 4 nuclei) [Lorenz & Eichhorn, 1 983; Coley-Smid1 et aI., 1 980; Faretra & Antonacci, 1 987J . Apothecia are formed only in light and are positively phototropic [Coley-Smith et ai., 1 980J . Pathology B. cinerea is a necrotrophic fungus with an extremely wide host range causing grey mould disease on a large number of economically important vegetable, flower, and fruit crops in temperate regions [Sinclair et ai., 1 987; Agrios, 1 988] . Infection may develop during cultivation in both field-grown and protected crops, and post-harvest in storage or transit. Data on the possible existence of host specificity are conflicting. Field-isolates of B. cinerea taken from one host were shown to vary in aggres sion when inoculated onto another [van den Heuvel, 1 976J , although no evidence was presented to show that the interactions conform to the 'quadratic check' indicative of a gene-for-gene system [Day, 1 974; Elad & Evensen, 1 995] . In contrast, a recent analysis of RAPD markers (8 field-isolates from different regions and a variety of hosts) found no evidence for host specialisation and strains did not group according to host or geographical origin [van der V1ugt-Bergmans et ai. , 1 993] . This supports the generally accepted view d1at host specialisation does not occur in B. cinerea. Chapter 1 Introduction 6 B � c lO um Figure 1 Spore forms of Botrytis cinerea. (A) Conidia from 10-day-old cultures grown on MEA at 20°C . (B) Microconidia from l -month-old cultures grown on MEA in the dark at 1 5°C. (C) Ascospores isolated from apothecia. A B c 5 mm Figure 2 Growth stages of apothecia after 4 months incubation at 1 0°C following standard mating procedure. (A) Apothecial stipes developing from a sclerotium. (B) Young apothecia. (C) Mature apothecia. Chapter 1 Introduction 7 Infection Grey mould disease is most damaging as a soft rot, blossom blight, or stem canker, but mycelium can also spread internally in stems (e.g. tomatoes, grapes), or grow saprophytically on senescing or dead tissue [Sinclair et aI., 1 987; Coley-Smith et aI. , 1 980] . In addition B. cinerea can cause a pre-emergence damping-off disease under cool wet conditions [Coley-Smith et aI., 1980; Agrios, 1988] and is known to be seed transmitted by surface contamination in some crops [Burgess et aI., 1997] . Lesions can appear as brown areas under dry conditions, but rapidly become covered in whitish, grey-brown, 'furry mould', consisting of masses of conidia and conidiophores when humidity is high. Infected fruit and soft tissues (e.g. strawberries and lettuce) become soft and watery, while lesions on stems (e.g. tomato) can develop into sunken brown cankers (Figure 3 A-D p9). B. cinerea requires a nutrient supply for infection of most tissues [Heale, 1 992] . Artificial inoculations of conidia usually require a supply of exogenous nutrients to achieve high infection rates [Rossall & Mansfield, 1 981 ; Harper & Strange, 1 981] and this requirement may be supplied by leaf or wound exudates under natural infection conditions [Heale, 1 992] . Infection occurs, either from germinated conidia through plant cuticles and wounds, or when mycelium invades healthy tissue from an adjacent infection such as saprophytic growth on senescing flower parts or from one grape berry to another in a bunch [Coley­ Smith et aI., 1 980] . Conidia germinate in conditions of high humidity and penetrate the host either directly through the epidermis, as with dry inoculation of rose flowers [\X'illiamson et aI., 1 995] , or via an infection peg in association with single or multi-lobed appres soria [Garcia-Arenal & Sagasta, 1 980] . Penetration through stomata has also been observed [Garcia-Arenal & Sagasta, 1980] . The type of penetration (ie. direct or via appressoria) has been shown to be influenced by the concentration of the spore suspension with higher concentrations of conidia giving more direct penetrations [van den Heuvel & Waterreus, 1 983] . In addition Garcia-Arenal & Sagasta [1 980] found more complex penetration structures with higher nutrient levels in tl1e inoculum. Light and electron microscopy studies of tl1e infection site suggested that both mechanical and enzymatic factors are involved in penetration [van den Heuvel & Waterreus, 1 983; Garcia­ Arenal & Sagasta, 1 980; McKeen, 1 974] . In addition conidia inoculated with exogenous nutrients produced a matrix material enclosing the germ-tubes and providing attachment to the leaf surface [McKeen, 1 974; Cole et aI., 1 996] . While no matrix material has been observed with dry inoculated spores, traces were detected at the site of penetration using 1 Introduction 8 immunolabelling techniques and the monoclonal antibody BC-I

rapevine in 1930 [\Vhetzel, 1945) . A more recent report is given by Polach and co-workers [polach &;. Abawi, 1974; Polach & Molin, 1975] who found B. cinerea apothecia in bean fields (Phaseo/us vulgan's) and apple orchards at different locations in New York State in two subsequent years. Identification was confirmed morphologically and by pathogenicity tests. In New Zealand the only report of B. cinerea apothecia occurring in the field was from a peach mummy in the Hawkes Bay in 1993 [Beever et aI., 1997] (Figure 4 p 11). It has been a popular belief among researchers that the sexual stage occurs very infrequendy and is generally unimportant in disease epidemiology. The scarcity of reported findings would seem to support this. However, reports from studies that have actively loo�ed for, and failed to find, apothecia are even scarcer [Braun & Sutton, 1987] . The reports available show that environmental conditions suitable for apod1ecial formation are found in a number of different locations around the world and studies of field-isolates from many different countries have demonstrated the widespread occurrence . of both mating-types (MATt-1 , MAT1-2, Mating-type p21) [Faretra et al. , 1988b; Faretra & Pollastro, 1991, and 1993a; Beever & Parkes 1993; Akutsu et aI., 1996; Delean & Melgarejo, 1996]. It may be that apothecia are more common but have been missed due to their small size and ephemeral nature. Or, as apothecia of B. cinerea closely resemble apothecia from other fungal species such as Sclerotinia sclerotiorum (Figure 5 p11), they may have been wrongly identified. Indirect evidence has been found from a study of 259 field-isolates using a variety of genetic markers, that the sexual stage occurs frequendy although a parasexual explanation is also possible [Giraud et aI., 1997] . Chapter 1 Introduction 1 1 Figure 4 Apothecia growing on a peach 'mummy' found by G. Tate and co-workers in a commercial peach orchard ('Golden Queen,), Hawkes Bay (New Zealand), in September 1 993. (photo R. E. Beever) . The specimen has been lodged in the Landcare Fungal Herbarium (Herb. PDD 65768). lO mm Botrytis cinerea ScJerotinia scJerotiorllm Figure 5 Apothecia of Botrytis cinerea (produced in the laboratory as described p33) and Sclerotinia sclerotiorum (produced in the laboratory from sclerotia pre-conditioned by burial in soil for 6 months and then incubated on moist paper towels under 1 2-hours­ on/ 1 2-hours-off cycled white and long-wave ultraviolet radiation for 1 0 days). 1 Host pathogen interactions Fungal attack compounds Introduction 12 As B. cinerea hyphae grow in the host tissue a large number of fungal attack compounds (enzymes and toxins) are secreted which macerate the tissue ahead of the advancing hyphae (expansion zone). Compounds associated with cell death in the expansion zone include; phenol degrading enzymes such as laccase, cell wall degrading enzymes such as polygalacturonase (pG), pectin-lyase (PL), pectin-methyl-esterase (PME), acid-proteinase, cellulase, and phospholipases, and toxins such as organic acids (citric and oxalic acid) and glucans [reviewed Kamoen, 1992]. In addition, multiple isoforms have been identified for many enzymes for example polygalacturonase, pectin-esterase, and pectin-lyase, and the number of isoforms varies both between isolates and in the same isolate under different growing conditions or infection stages [Leone et al., 1990; Leone & van den Heuvel, 1987; Magro et al., 1980; Di Lenna & Fielding, 1983; Marcus & Schejter, 1983] . 1bis suggests either that these enzymes may have more than one function, or that enzyme redundancy is the norm. Redundancy could reflect environmental heterogeneity with specifi� isozymes conferring an adaptation to a particular niche. As many attack compounds are secreted early in the infection process (spore germination and penetration) they have been extensively shldied as determinants of pathogenicity in B. cinerea (summarised in Appendix 7 p137). Most of these works have • proposed a pathogenicity-determining role for the enzyme of interest from a correlation of high enzyme activity with aggressiveness on a specific host. Studies using this approach found no evidence of such a role for PG enzymes (4 studies) or PME (4 studies) although one reported a negative correlation for the latter. Examples where a correlation between enzyme activity and aggressiveness was found include; polymethylgalacturonase in 2 of 3 studies, PL (2 studies), protease (3 studies) , laccase (2 of 3 studies), and active oxygen species (3 studies). Results obtained by this method are frequently contradictory and definitive evidence of the importance of any one enzyme has been difficult to establish, probably due to differences between isolates and/or hosts and numerous other factors still poorly understood. The situation would be further complicated if enzymes and toxins acted together. Recent studies seeking to clarify tl1e role of attack compounds have used a gene disruption approach. Van Kan et aJ. [1997] showed that aggressiveness of B. cinerea on gerbera and tomato remained unaltered in mutants deficient in the cutinase-A gene. In 1 Introduction 13 contrast disruption of a polygalacturonase gene reduced aggressiveness of B . cznerea on tomato leaves [Have et aI., 1 997] . Host defence compounds Host plants respond to fungal invasion by forming various defence compounds such as phytoalexins, enzyme inhibitors, and physical barriers, for example lignin. Phytoalexins known to be induced by B. cinerea include; resveratrol and pterostilbene in grape [Hoos & Blaich, 1 990; Pezet & Pont, 1 990] , wyerone acid in broad bean [Harrison, 1 988] , phaseollin, phaseollidin, and phaseollinisoflavan in French bean [van Den Heuvel & Grootveld, 1 978, and 1 980; Fralle et aI., 1 980] , and 6-methoxymellein, p-hydroxybenzoic acid and polyacetylene in carrot [Defago & Kern, 1 983; Mercier et ai. , 1 993] . The effectiveness of these chemicals in plant defence depends on the ability of the plant to respond to infection by rapidly producing high concentrations at the site of infection. The importance of defence chemicals in host/pathogen interactions is supported by their connection with resistance, for example studies on grape showed a strong correlation between resistance to B. cinerea infection and the ability to synthesise phytoalexins [pezet & Pont, 1 990] . Also, non-aggressiveness of some field-strains of B. cinerea on French bean has been correlated with their inability to break down phytoalexins [van den Heuvel & Grootveld, 1 978] . In addition to phytoalexins and fungitoxic substances, compounds inhibitory to fungal attack enzymes are also found. Inhibitors of B. cz'nerea polygalacturonase cell wall degrading enzymes (pGIP's) have been identified in tomato [Stotz et aI., 1994] , apple, marrow, and grape [Fielding, 1 98 1 ; Yao et aI., 1 995] , pear [Sharrock & Labavitch, 1 994], and raspberry Dohnston et aI., 1 994] . There is considerable interest in cloning PGIP genes for use in developing crop varieties with increased resistance to infection by B. cinerea [Labavitch et aI., 1 996; Ramanathan et aI., 1996) . Physical barriers to fungal invasion involving phenolic substances and lignification have been shown to be overcome by laccase enzymes produced by B. cinerea [Viterbo et aI., 1 992] . Substances that inhibit laccase were found to protect the host plant from infection. Cucumber plants treated with cucurbitacins, which repress the formation of laccase in B. cinerea, developed a lignified layer at the infection site preventing fungal infection [Viterbo et aI., 1 993] . A greater understanding of plant defence mechanisms "rill assist in developing new strategies for increased plant resistance. 1 Introduction 1 4 Noble rot B. cinerea is a serious disease of grapes often causing severe losses and requiring expensive control measures. However, if infection occurs when the grapes are mature and environmental conditions are suitable, the grapes decay slowly and can be used to make sweet dessert wines (e.g. Sauternes and Trockenbeerenauslese) Oarvis, 1 977]. For this beneficial B. cinerea infection, known as noble rot (pourriture noble in France) , to develop the grapes must be at full maturity and healthy with intact skins when infected. Humid (or foggy) nights ensure growth of the fungus and dry sunny days evaporate water from the berries resulting in high sugar concentrations 0 arvis, 1 977; Coley-Smith et aI., 1 980] . Producing wine by this method is risky as a wet season may result in total loss and even when conditions are favourable the yield is greatly reduced, but the final product is highly prized and can give excellent financial returns. Attempts to artificially induce infection on harvested grapes have only been moderately successful [Coley-Smith et aI., 1 980] . Control Control of B. cinerea is hampered by its wide host range and ability to infect plants both during gro�th and in storage [Coley-Smith et aI., 1980] . The specific environmental conditions of temperature and humidity required for germination provide a focus for cultural control measures. Cultural practices that reduce humidity and the presence of free water on fruit, leaves, and flowers, such as increased ventilation and leaf removal, have . been shown to reduce infection [Hammer & Marois, 1 989; English et aI., 1 993] . In addition, disease pressure from conidia and overwintering sclerotia can be reduced by sanitation practices, and natural host resistance can be enhanced by curing resulting in a significant reduction in storage rots caused by B. cinerea) for example in kiwifruit [pennycook & Manning, 1 992; Ippolito et aI., 1 994] . Despite advances in crop management and storage practices, control o f B. cmerea diseases still relies heavily on the use of synthetic chemicals. The main systemic chemicals used against B. cinerea are the benzimidazole and dicarboximide fungicides, although the rapid development of resistance in fungal populations has limited their efficacy (Fungicide Resistance p21) . Fungicide-use strategies recommend the use of mixtures or rotation of fungicides (including protectants such as Euparen which are less prone to resistance problems) to reduce the build up of resistant populations [\"Xlalton et ai., 1 995J , and monitoring disease levels to ensure better timing of fungicide applications [Nair et aI., 1 997] . New approaches to resistance monitoring are also being developed to assist in the implementation of fungicide-use strategies. For example the use of polymerase chain Chapter 1 Introduction 1 5 reaction (PCR) to glVe rapid identification o f benomyl resistant strains to assist ill monitoring field populations [Luck & Gillings, 1 995] . Increasing public and market concerns about the safety o f control chemicals has led to residue limits for many export crops [Sas, 1 997] and a growing interest in developing alternative control methods for B. cinerea diseases . Numerous studies have shown effective reduction of B. cinerea infection using biological control agents such as yeasts, filamentous fungi, or bacteria, although this work is still largely in the developmental stages [reviewed Elad et ai., 1 996] . A preparation of Trichoderma ha1"'{janum (brand name Trichodex) is ,. registered for use in greenhouses in Israel, Australia and some other countries, but is not yet available in New Zealand. Biological control organisms have been selected and trialed for use on greenhouse crops in New Zealand [Eden et ai., 1 996a] , but registration is hampered by the high cost of toxicology testing. Genetic studies of B. cinerea Phenotypic variation Cultural co�ditions have been shown to modify some morphological features of B. cinerea making them of uncertain value in taxonomy and possibly explaining some of the confusion in this genus (Taxonomy p 1 ) [paul, 1 929; Jarvis, 1 977]. Studies of different field-isolates have also shown high levels of variability in morphology, pathogenicity, and . quantitative and qualitative production of pathogenicity related enzymes [Hansen & Smith, 1 932; Di Lenna et ai., 1 9 8 1 ; Leone, 1 990; Grindle, 1 979] . The most widely accepted explanation for variation in field-isolates of B. cinerea (and many other fungi) is heterokaryosis. Heterokaryosis exists when two or more genetically different nuclei inhabit the same cell or mycelium, and was first proposed by Hansen & Smith [ 1 932] to explain the variation found in isolates of B. cinerea following repeated single-spore isolations. Hansen [1 938] s tudied the phenomenon in 900 isolates of 30 genera of imperfect fungi including B. cinerea and concluded that various characteristics of these fungi such as culturing variation, sectoring, reversions and changes in virulence, could occur through heterokaryosis maintained in multinucleate conidia. The work of Summers et ai., ( 1 9 84] which showed that 0.5% of single-conidial progeny from a single­ spored dicarboximide sensitive strain were dicarboximide resistant and 0.2% were dicarboximide sensitive from a dicarboximide resistant strain strongly supports an explanation of heterokaryosis. Heterokaryosis may arise in two ways, via somatic mutation (which in multinucleate vegetative fungi could result in significant differences between 1 Introduction 16 nuclei residing in the same mycelium), and VIa the transfer of nuclei between strains through anastomosis (hyphal fusion). Since Hansen's study [1938] considerable progress has been made in understanding vegetative incompatibility systems and conseguently the factors limiting anastomosis [reviewed by Leslie, 1993]. Little is known about the process in B. cinerea although evidence for a vegetative incompatibility system has been demonstrated [Beever & Parkes, 1993] . Beever and Parkes found pigmented interaction zones between isolates paired on NaCl amended malt extract agar indicating mycelial incompatibility, one of several events " associated with vegetative incompatibility [I c (\I 20 QJ ::g 1 0 0 � � " '\. '\ \ 1\ \ '\ '" "'-., ....... � � o 0.5 1 .5 2 2.5 3 3.5 4 4.5 5 Exposure time (min) Figure 6 Percentage of conidia survIVIng after exposure to ultraviolet radiation. Assessed as percent spore germination on CM after 8 hours incubation at 20°C. 1 00 90 80 -; 70 > .� ::l 60 '" 50 � 0 c:: (\I 40 QJ 30 � 20 1 0 0 o r Q�� � � Preliminary I experiment 2 3 4 5 Concentration NQO (lIg / ml) 6 7 Figure 7 Percentage of conidia surviving after incubation in 4-nitroquinoline- l -oxide for 1 hour. Assessed as colonies counted using a dissecting microscope following 2-days incubation on CM+Triton. Chapter 2 General Methodology 29 Pathogenicity assays Assays for pathogenicity were used to screen putative non-aggressIve mutants and progeny from crosses, for analysis of aggression, and to investigate competition at the infection site by different strains of B. cinerea. The host material chosen needed to be readily available, uniform in cultivation and disease susceptibility, uncontaminated by systemic fungicides, compact (to enable experiments to be carried out in a small space) and give a fast turn around enabling results to be obtained in 3 - 4 d. French bean (Phaseo/us vulgaris) met the above criteria, i IS" use in pathogenicity tests with B. cinerea i s well documented and so � Wc,S" chosen as the main host plant. French bean The method followed van den Heuvel [1976] . French bean seedlings (Phaseo/us vulgaris cv. Top crop) were grown to the two-primary-leaf stage in either washc:d potting mix grade pumice, or vermiculite, in an unheated glasshouse without added nutrients. The roots were excised and the stem and leaves suspended on a plastic rack over a tray of water with the cut ends immersed. Mycelial plugs of B. cinerea were inverted onto the upper leaf surface. A light misting of RO water was applied to the leaves prior to inoculation and although preliminary experiments showed this did not · significantly affect lesion development it was helpful in preventing the plugs from falling off the leaves: Inoculated plants were incubated in one of two ways. First, plastic racks of bean seedlings were placed in large metal trays (50 x 8 10 x 430 ffiffi, 3 racks per tray) which were sealed in plastic bags to maintain a high humidity. This method was suitable for screening large � jf/,"rf ,H, pT, numbers and was used for bulk screening of putative mutants For all other purposes racks were placed in individual plastic trays (50 x 30 x 1 5 cm) and enclosed in a plastic bag. Trays were incubated for 3 d at 20 - 25°C on the laboratory bench under a d., ,�"l 11\\'.- 01",'1 combination of daylight and white fluorescent light! Disease lesions were recorded as radial spread from the plug to the lesion margin. Three aspects of the assay protocol were investigated; the effect of wounding the leaf at the site of inoculation, the importance of the age of the inoculum, and the variability of the assay. Effect of wounding The effect of wounding was tested by stabbing beneath the inoculum plug with a needle. Leaves were inoculated with plugs cut from colonies of field-isolate REB658-1 grown on MEA. Wounded and unwounded leaves were arranged in 4 blocks (different trays) of 1 2 replicate plugs each (one plug per leaf, 2 leaves per plant) . The average lesion Chapter 2 General Methodology 30 radius was 8.29 mm (SE 0.30) and 8.58 mm (SE 0.28) in wounded and unwounded treatments respectively. There was no significant difference between treatments (P = 0.46) so wounding was not used in subsequent experiments. Age of inoculum Two protocols were used for experiments involving plug inoculations on French bean. When relatively small numbers of strains were tested or large numbers of replicate plugs were needed, the inoculum was grown on full sized petri dishes of MEA (8.5 cm) where colony growth typically reached the plate margin after 3 - 4 d. When large numbers of < F,'t! " rt -< ', � S-S) strains were testecVcultures were grown in 1 .5 cm wells in 24 well tissue culture plates (IC plates, Geiner No. 6621 60: each well contained 0.5 ml of MEA amended with 4 g r-I additional agar, MEA+A). As colony growth covered the 1 .5-cm wells in 1 .5 to 2 d at 20°C these plates were grown at 1 0°C to allow the maximum time window for fast and slow growing strains to produce sufficient mycelium for inoculation onto the bean leaves. At this temperature the well was covered with mycelium in 3 - 4 d wh�n plates were inoculated at the margin and two plugs were taken from the opposite edge of the well for inoculum. The effect of colony age on lesion size was tested with both protocols using strain A4. On full sized plates at 20°C lesion size was greatest with 2-d-old colonies (8.80 mm SE 0.23) , medium for 4-d-old colonies [7.36 mm SE 0. 1 8) , but poor for 6-d-old colonies (3 . 14 mm SE 0.42) (Figure 8 p31) . To ensure sufficient inoculation material, plugs were taken after 3 - 4 d as a compromise between optimum lesion size and colony growth. The mean lesion size of 9 . 1 mm (SE 0.28) caused by 4-d-old plugs from TC plates incubated at l OoC was adequate for measurements, and plugs from this age of colony were used subsequently (Figure 9 p31) . Chapter 2 1 4 ",", 1 2 e 5 10 :.a 8 .: .. I: .S G '" � I: 4 .: (1J � 2 0 2 d 4 d General Methodology G d 8 el Age of colony (days) 31 1 1 d 1 4 d I C,I Figure 8 Average lesion radii on French bean leaves Ifollowing inoculation with mycelial plugs from the margin of different aged colonies of A4 grown on MEA at 20°C Bars indicate standard error of means from 32 replications. 14 1 2 S 5 10 .� :.a 8 � = 0 6 ·00 � = 4 .: (1J :=E 2 0 .- 1 d 2 d 4 d G d 8 d lO d 13 d Age of colony (days) ! Pi' 301 Figure 9 Average lesion radii on French bean leaves Ifollowing inoculation with mycelial plugs from the margin of different aged colonies of A4 grown on MEA in tissue culture erC) plates at 1 0°C Bars indicate standard error of means from 24 replications. Chapter 2 General Methodology 32 Variability of the assay While the assay method as described consistently gave 1 00% infection, a moderate amount of variation in lesion size was observed between replicates of the same strain in some experiments. To assess this variation the field-isolate REB658-1 and strain A4 were tested in a fully nested design using 3 blocks (treatments randomised on different racks in one tray) of eight plants, two plugs per leaf to give a total of :lZ plugs per block. No significant difference was found between strains or plants (P = 0.76 and 0 . 1 3 respectively) however, there was a significant difference between blocks (racks) and between leaves (P = 0.004 and 0 .0 14 respectively) showing both the position in the tray and differences between leaves affected the lesion size. This variation and the difference in the maximum lesion size observed between the two age experiments described above (Figure 8 p31 , Figure 9 p31) , highlighted the need to tl10roughly test putative mutants in repeated, well replicated assays. Modifications to French bean assay for spore inoculations In preliminary experiments inoculation of B. cinerea conidia suspended in water or in (<. con 0.01 % Tween 80 gave a low rate of infection even on wounded leaveJIt has been shown that the addition of exogenous nutrients to spore suspensi(;ms can markedly improve infection rates [Harper & Strange, 1 981 ; van den Heuvcl & Waterreus, 1 983] . Therefore, two nutrient solutions were tested for tl1eir ability to assist infection by strain A4: phosphate+glucose solution containing 9 . 12 g r-1 of potassium dihydrogen phosphate (I<.H2P04) with 1 9.8 g 1- 1 glucose, and Vogel's+sucrose containing 20 ml t l Vogel's N medium and 1 5 g t1 sucrose. French bean leaves (not misted) were inoculated with washed spores resuspended in either nutrient solution to give concentrations of 1x 103, lx l04, l x 10s, and lx l0G spores ml-1 (5 fJ.l in the centre of each leaf, 1 6 leaves per treatment) . Inoculations of the nutrient solutions alone were included as controls. The .' tray was sealed in plastic bags and incubated for 4 d as above. Infection was recorded as percent infection and as mean radial lesion size of successful infections. In an overall comparison of the two nutrient solutions (lesion radii data) the average lesion size for the Vogel's+sucrose solution was significantly larger than for the phosphate+glucose solution (12 . l8 mm, SE 3.72 and 8.75, SE 2. 1 8 respectively; P = 0.03 1 5) and the overall rate of infection was also higher (75% and 62% respectively) . At lower spore concentrations (1x103 and lx104 spores ml-1) there was no significant difference bern"een the rn"o solutions * Lights were arranged as a bank of six 340 mm long 30 W cool-white fluorescent tubes (Philips) with two 340 mm long black light tubes (Esellese F40T12) and one 60 W tungsten double-life-plus bulb (Sylvania) suspended approximately 800 mm above the vials. Chapter 2 General Methodology 33 but at 1x10s and 1 x106 spores ml-1 the Vogel's+sucrose solution significantly increased infection (Figure 1 0 p34) . Vogel's+sucrose at 1x106 spores ml-1 was used in all subsequent applications of the assay. Problems were experienced in these trials with the inoculum droplet rolling off the leaf and in subsequent experiments 4-mm diam. plastic rings (cut from the barrel of 1 ml Beral disposable plastic transfer pipettes) were used to position the inoculum droplet. Crossing procedure The procedure for crossing and ascospore isolation was based on Faretra et aL [1988a] and Beever & Parkes [1993] . Cultures were grown on 'MEA in petri dishes at 1 5°C in the dark for 4 weeks then transferred to O°C (in the dark) for a further 4 weeks. Crosses were made by transferring 8 - 1 5 sclerotia to narrow-necked glass McCartney vials (30 ml), each containing 2 ml sterile water, and adding 1 ml of a suspension of microconidia, conidia , • 0-1- ''''" q�\;\OS\""1 (lCI(IV\, and hyphal fragments in sterile water prepared from the dishes in which the sclerotial were grown. Sclerotia fertilised with suspensions prepared from their own petri dishes were included as controls. The vials were incubated on their sides with loosely fastened caps under a mixture of white fluorescent, long-wave ultraviolet, aq.d tungsten radiation (12-h­ on/12-h-off{ at l OoC for 3 - 5 months until apothecia formed (Figure 1 1 p35) . Mating­ types (MATI- l or MATI-2) were determined by successful production of apothecia with another strain of known mating-type. Single-ascospore strains that produced apothecia with strains of both mating-types were labelled MAT1-l /2 (Mating-type p21) . Parents for crosses were chosen to include fungicide resistance markers and segregation of these markers in equal ratios were interpreted as evidence of crossing. Ascospore isolation Single-ascospore strains ,:vere obtained by washing individual apothecia three times in sterile Tween 20 or Tween 80 (0.01 %), and dispersing the spores in 200-fJ.l sterile water by grinding with a glass rod. The resulting ascospore suspension was adjusted to a 1x 104 ml-1 concentration and 20 fJ.l was spread onto 1/2 strength PDA (PDA-V2) . After incubation for 1 6 h, germlings were transferred individually to IvIEA in screw capped tubes. Chapter 2 General Methodology 34 25 .pho sph ate I gluOlsc o V ogcl's I HI crosc --. 20 S ! ;; 1 5 CIS ... = .S '" 1 0 � = CIS .., ::E 5 0 0 l x l 0(3) 1 x l 0(4) l x l 0(5) l x l 0(6) 1 00 90 80 = 70 0 U 60 � c 50 ' 80) 17 - 26 high (> 80) 8 24 6 Class B 39 low « 35) 1 4 high (> 80) 8 - 24 7 Class C 3 mixed mixed 0 0 3 * Taurine responsive i.e. d1in sparse growth on }yfl"\I and dense growth on iVD\f+taurine The response to various levels of selenate was determined for the mutants Ms010 (class A), Ms01 5 (class B), and a selection of 'wild-type strains ( Figure 1 6 p44) . Logit analysis gave EC 50 values greater than 3.00 trlL\1 for strain MsOI0 and 0.93 mlvI (SE 0.OS7) for strain Ms015. Values for the \vild-type s trains were as follows:. AI , O.OS mM (SE 0.001) ; A5, 0.09 mM (SE 0.005) ; REB67S-1 , 0 . 10 mM (SE 0.005) ; and SAS405, 0. 1 0 mlYr (SE 0.001) . Crosses of representative SelR mutants from class A and class B with wild-type strains (Table 2 p45, crosses 1 and 2) , and one cross with class B recombinant progeny (Table 2 p45, cross 3), gave segregation ratios close to 1 : 1 with resistant progeny retaining the mutant parental phenotype. It was concluded that selenate resistance in these instances is conferred by a single gene. The genotype symbol Sell R was allocated to class B selenate resistant mutants represented by allele Ms01 5. The mutant Ms0 1 5 was crossed with class A mutants to test for allelism and although a number of combinations produced apothecia the progeny were rejected as authentic crosses because fungicide resistance markers did not segregate 1 : 1 (i.e. could not be verified as a true cross, Crossing procedure p33) . Therefore to test whether class A SelR mutants,map to the same locus as Sel1R will require further genetic studies. The different phenotypes shown by the classes of SelR mutants suggest that more than one locus may be involved. There was no evidence of linkage between SelR resistance and either lVIbcl or Daf1 fungicide markers in any cross (Table 2 p45) . Chapter 3 New genetic markers for B. cinerea 43 Figure 14 Growth responses of selected selenate resistant mutants of Botrytis cinerea on minimal medium amended with selenate plus taurine (MM+Se04• upper row) and on minimal medium amended with taurine (MM + taurine, lower row) after 6 days. Strains are; (A) P2-01 1 (SelR class A), (B) PS-026 (SeI1R class B), and (C) A1 (wild-type) . Figure 15 Growth responses of a nit1 mutant of Botrytis cinerea on medium with nitrate as the nitrogen source (MM+NO,) . Strains are; P6-00S (nit1 mutant) - upper, thin sparse mycelial growth spreading across the plate and abutting the thick dense growth of AS (wild-type) - lower. Chapter 3 1 00 90 = 0 80 ·c os = 70 .§ 60 .. (Jl Chapter 3 New genetic markers for B . cinerea 46 Nitrate non-utilising mutants Spontaneous sectors arose as dense outgrO\vths at the plug margin on TvIM +Cl03 from strain AS. A total of 8 chlorate resistant sectors that grew as thin sparse colonies on 1YIM+N03 were classified as nitrate-non-utilising mutants and retained for further study. All grew normally on hypoxanthine (11M+Hx) and nitrite medium (l'v1lv1+NO:z) and it was concluded, by comparison with mutants in other fungi [Correll et aL, 1 987] , that they were defective in the nitrate reductase apoenzyme and the genotypic symbol nit1 was allocated, represented by the allele Mn001 . These mutants were routinely scored by growth on :rvIM+N03 (Figure 1 5 p43) . They remained vegetatively stable after storage on silica gel. Eight chlorate resistant mutants that were recovered from NQO treated spores all showed wild-type growth on :rvIM+N03, and were classified as Crn (chlorate resistant, nitrogen normal) mutants. fo, WMp\� ""!0-.\""\'.o ,"", I Eight nitl mutants, derived from strain AS, were tested lin all combinations (method B) and all grew as thin colonies except in 5 combinations (Figure 1 81\ p47) where relatively thick dense mycelial growth was restored along the line of contact. All complementing pairs produced medium to weak growth compared to \vild-type s trains on the same medium. This dense growth (shown in a mixed spore test, Figure 1 9 p49) is interpreted as reflecting intragenic complementation bet\vec;:n the strains and can be represented as a complementation map \vith 4 groups (Figure 1 8B p47) [Fincham, 1 966] . Mn012, a nit1 mutant derived from REB689-1 (Appendix 3 p127) did not complement any of the A5 derived mutants. When the nit1 mutants were crossed, the nit1 phenotype segregated as a single gene with no evidence of linkage to either Mbcl or Daf1 (Table 2 p4S, crosses 4, S and 6) . The fungicide resistance markers Mbcl and Def1 showed loose linkage (29.6 - 35.3%) in the same crosses. Chapter 3 New genetic markers for B . cinerea 47 Figure 18 Complementation pattern of selected nit1 mutants derived from strain AS. (A) Score matrix for mutants paired on 1v1M+NO} medium. (B) Complementation map derived from data in A. Mutants in non-overlapping groups complement, those within one group or in overlapping groups do not. A B nitl mutants MnOO 1 to MnOO8 8 7 6 5 4 3 2 1 1 - - - - - - - - 2 3, 4 5 2 - - - + + + - 3 - - - + - - 4 - - - + - 1, 6, 7, 8 5 - - - - 6 - - - 7 - - 8 - Pathogenicity Selected vv-ild-type s trains, pnmary mutants, and single-ascospore progeny (non­ auxotrophs SelR strains) were tested for pathogenicity in a randomised design with nine replications. All were highly aggressive (Figure 1 7) . Linkage relationships Crosses of three different Se!1 (class B) mutants to nit1 mutants gave 1 : 1 ratios for segregation of nit1 , Sell , Albc1 and Daf1 with no evidence of linkage between the two new markers, nit1 and Sell (fable 3 p48) . Crosses 8 and 9 (fable 3 p48) provided evidence for loose linkage between Nlbc1 and Daf1 similar to that found in crosses 4, S and 6 (fable 1 p42) . However cross 7 (fable 3 p48) gave a recombination percentage between these genes of 75 .2% for which no simple explanation is apparent. Marker complementation As some SelR mutants behave as sulphate non-utilisers (demonstrated by increased growth in response to taurine) , and nit1 mutants behave as nitrate non-utilisers, it is feasible to test for "forced" complementation between these mutants on 1vllvI+ NO} In a complementation test (method A) the mutants Ms033 and MnOOI (both from the parent AS) produced relatively dense growth compared to the thin sparse growth observed with spore mixtures of mutants generated from different parents (AS with mutants from Al or REB689-I) (Figure 20 p49) . The dense growth was interpreted as a result of complementation between the two mutant types. Table 3 Segregation of class B selenate resistant (Sell), nitrate non-utilising (nitl) , benzimidazole resistant (Mbcl) , and dicarboximide resistant (Daft) markers in four-point crosses of Bottytis cinerea Parents Progeny Recombination (%) between pairs of genes (y} for independent segregation*) Cross No. nitl:Sel1 nitl:Mbc1 nitl:Dafl Sel1:Mbc1 Sel1:Dafl Mbc1:Dafl No. Sclerotial strain Fertilising Strain 7 P6-022 (Mbcl HR-DaflLR- SeilS-nitl) P5-006 (MbclLR-DaflS-SellR) ascospores 10 1 8 P5-029 ( A1M LR-Daf? J-Je/1 R) P6-037 (Mbcl HR- Daf1LR-JelfJ-nztl) 1 00 9 :'-- ', P6-005 (MbcI HR-DaflLR-SelIS-nitl) P5-026 (MbcILR-Daf1S-SellR) 105 * X2 values greater than 2.71 (3.84) indicate differences significant a t P = 0.1 (0.05). 5 1 .5 (0. 1 0) 53.5 (0.5 1 ) 55.4 (1 .25) 43.6 (1 .75) 49.5 (0.02) 75.2 (35.26) 56.9 (1 .46) 54.0 (0.64) 52.4 (0.09) 47.6 (0.24) 48.0 (0. 1 6) 46.0 (0.64) 53.3 57.1 (2. 1 9) 54.0 (0.64) 36.0 (8.51) 55.2 (1 . 1 7) 30 .5 (1 8.89) Chapter 3 New genetic markers for B. cinerea 49 Figure 19 Complementation between nit! mutants derived as spontaneous sectors from the parent strain AS (Mn001 , Mn004 and MnOOS) or REB689- 1 (Mn01 2) . Tested using method A on MM+NO;. A 1 : 1 mix of spore suspensions was placed in the centre of each plate. (A) MnOOS paired with Mn001 . (B) MnOOS paired with Mn004. (C) Mn004 paired with Mn012. Figure 20 Complementation between SelR (class A) and nit! mutants on MM+N03 (method A). (A) Ms033 (parent AS) paired with Mn012 (parent REB689-1) . (B) Ms033 (parent AS) paired with Mn001 (parent AS) . (C) Ms010 (parent A1) paired with MnOOl (parent AS). Chapter 3 New genetic markers for B . cinerea 50 Discussion Selenate has been shown to enter the mycelia of species of Aspergillus and Penicillium via a permease system that also transports sulphate [Tweedie & Segel, 1 970; Shrift, 1 961 ] . This sulphate permease is under metabolic control and is regulated by sulphur status [Renosto et aL , 1 990]. Once inside the cell selenate can be incorporated in place of sulphate in various metabolites and it is suggested that the major mechanism of selenium toxicity is the instability of the selenium analogues in, for example, the disulphide bridges in proteins [\'{lilson & Bandurski, 1 958; Shrift, 1 972] . Although chromate is also transported into the fungal cell by the sulphate permease, it is apparently unable to form stable products when acted on by ATP sulphurylase (the first enzyme in the sulphate assimilation pathway) and therefore is not incorporated into metabolites in place of stuphate. Chromate toxicity is attributed to the intracellular accumulation of chromate causing death, possibly through its strong oxidising effects [Roberts & Marzluf, 1 971] . Mutants resistant to chromate and selenate, similar to the class A and B mutants found in this study, have been shown in other fungi to be deficient in sulphate permeases. In Aspergillus nidulans the gene 5-3 encodes a sulphate peqnease [Arst, 1 968] and in Neurospora crassa the gs-13 gene encodes a low affinity sulphate permease [Marzluf, 1 970] . Selenate resistant mutants sensitive to chromate, similar to the class C mutants described here, have been found in both A. nidulans (s- 12) and N. craSSel (gs-5 and cjs- 1 1) and identified as deficient in ATP stuphurylase [Arst, 1 968; Marzluf, 1 970] . Chlorate acts as a nitrate analogue and is apparently reduced by nitrate reductase to chlorite, which is toxic to the cell. It is suggested that chlorate resistant strains are unable to reduce chlorate to chlorite and so are able to grow on chlorate amended media [Cove, 1 976b] . Some chlorate resistant mutants recovered in this study were normal in nitrate assimilation (Crn mutants) while others were deficient (Nit mutants) resembling those reported from fungi such as Pusantlm moniliforme [I50%) may have been due to heterokaryosis following mutagenesis whereby wild-type nuclei co-exist in low numbers with the mutant nuclei [Summers et aI., 1 9 84] . When conidia from putative mutant strains were grown for storage on silica gel the relative proportions of Chapter 4 Isolation and characterisation of non-aggressive mutants 57 nuclei may have changed in favour of the wild-type, resulting in an apparent reversion to the wild-type phenotype. Growth properties of the mutants Preliminary growth tests on .MEA revealed that 34 of the 62 mutants had growth rates of less than 25% of that of the wild-type parent. All but five mutants grew well on MM. Mutants Mpl , Mp17, Mp1 8, Mp93 and Mpl l 0 produced significant growth on MEA but none on MlvI, and were tentatively designated as putative auxotrophs and were not examined further. Recognition by monoclonal antibodies A total of 2S non-aggressive mutant strains and the parental strain A4 were tested for the presence of the BC-KH4 antigen. Indirect immunofluorescence detected binding of the BC-I

. = Q.j etJ 0 ... 30 Q.. """ 0 ... Q.j 20 � 8 ::I Z 1 0 0 50 >. 40 " Isolation and characterisation of non-aggressive mutants A S.\ S5 6 I (9 5 m m ) ____ ,.-+1 , , I L l , . .. � B = �{ p97 Q.j S�\ S56 (8 .1 m m) etJ 0 Q. 30 """ 0 ... CI) 20 � 8 , I ::I z 1 0 0 I I I - - I " "" "J M ean lesion radii ( m m ) 62 Figure 25 Distribution of lesion sizes in progeny of Mp97 crossed with aggressive single-ascospore reference strain. (A) Cross Mp97 (sclerotial parent) with SAS56 (microconidial parent) . (B) Reciprocal cross SAS56 (sclerotial parent) with Mp97 (microconidial parent) . Mean lesion size in parentheses. Cl0G\ Table 5 Progeny analysis of sexual cross involving five genetic markers1 selenate (Se!1) , vinclozolin (Daj1) ,karbendazim resistance (Mbc1), nit1 (nitrate non-utilising), and Pat1 (pathogenesis). Cross No. 12 Parents Sclerotial Strain Fertilising Strain P1 6-67 (MbcIHR-Daf/LR- P21 -40 (MbclS-DaflS- nitl -Sell R) Pat 1) ;r ", • Progeny No. Ascospores 1 63 * X2 values greater than 2.71 (3 .84) indicate differences significant at P=0.1 (0.05) t I( "; 5 J:: 2. r I : Patl a s % t: 46.0 (1 .04) Recombination % between pairs of genes (y} for independent Patl:nitl Patl:Sel1 49.1 (0.06) 48.5 (0. 1 5) nitl:Mbc1 nitl:Dafl 45.4 (1 .39) 46.6 Patl:Mbc1 Patl:Dafl 44.2 (2.25) 46.6 (0.75) Sel1:Mbc1 Sell:Dafl 50.9 (0.55) 49.7 (0.01 ) Mbc1:Dafl 29.4 (33 . 14) nitl:Sel1 53.4 (0.75) Chapter 4 Isolation and characterisation of non-aggressive mutants 64 SAS405 - a homothallic single-ascospore strain In this study the single-ascospore reference strain SAS40S (Mbcl HR-Daf1 LR) sometimes produced apothecia in control crosses (i.e. strains 'fertilised' with their own microconidia or with water, Crossing procedure p33) . Analysis of fungicide resistance in the progeny of two such crosses revealed the presence of both benzimidazole and dicarboximide sensitive strains in proportions closer to 3 :1 than 1 : 1 segregation (crosses 1 3 and 1 4, Table 6 p6S) . Furthermore, analysis of Mbc and Daf phenotypes in the progeny of cross 1 4 (SAS40S fertilised with water) gave results consistent with segregation o f two genes in a diploid with resistant alleles dominant over sensitive alleles giving a ratio of 9:3:3 : 1 (Table 7 p66) . Recombination of the fungicide markers was 32% similar to that found in haploid analysis (crosses 4, 5, and 6, Table 2 p45 and crosses 8 and 9 Table 3 p48) . In cross 1 3 (SAS40S fertilised with SAS405 microconidia) , although all four classes were represented the l values were much higher due to the low score for one class (Table 7 p66) . Further crosses are needed to confirm the validity of these patterns. Discussion B. cinerea was more resistant to mutagenesis by NQO than Aspergillus nidulans [Bal et ai., 1 977] and required higher concentrations of the mutagen to achieve similar effects. In addition, approximately half the number of stable mutants were generated from NQO mutagenesis compared to U.v. mutagenesis. In general, mutant strains were fertile as both male and female parents and most mutants crossed readily with reference strains, Of the 62 mutants obtained in this study five were unable to grow on minimal medium and were identified as putative auxotrophs. Auxotrophic mutants altered in pathogenicity have been demonstrated in other plant pathogenic fungi. Biochemical mutants (induced by both U.v. and chemical mutagens) of Venturia inaequalis requiring choline, riboflavin, purines, pyrimidines, arginine, histidine, methionine or proline were found to be avirulent on apple leaves: those requiring biotin, �cotinic acid, pantothenic acid, insitol or reduced sulphur were virulent [Boone et ai., 1 956; Kline et al., 1 957] . A u.v. induced pyrimidine auxotroph of S cierotinia scierotiorum was avirulent on four of seven ,Crli dlpc �LCl\ , r qzr'llJ] susceptible hostsj Further work is needed to determine the nutritional requirements of the putative auxotrophs B. cinerea mutants generated in this study. Table 6 Pattern of fungicide resistance in progeny from SAS405 'fertilised' with SAS405 microconidia or with water. Cross No. Parents Sclerotial Strain 1 3 S"\S405 (Mbc1 HR-Da/LR) 14 S"\S405 (lVfbc1 HR-DajLR) Fertilising Strain SAS405 (Mbcl HR-Da/LR) Water Progeny % with parental phenotype (X2 for independent s egregation 1:1*) (X2 for independent s egregation 3:1*) No. Mbc as % Ascospores Daf ;" 101 86 . 1 (1 10 .47) (1 0.49) 68.3 (36.96) (2.08) 102 76.5 (39.72) (0. 1 2) 78.4 (48.74 ) (0.7 1) * X2 values greater than 2.71 (3.84) indicate differences significant at P=0. 1 (0.05) Table 7 Assessment of progeny from SAS405 'fertilised' with SAS405 microconidia or with water for consistency with segregation of fungicide resistance markers as a diploid with the resistance allele dominant over the sensitive allele. Recombination % (x2 Parents Progeny Phenotype for 10:6 s egregation *) Cross Sclerotial Strain Fertilising MhcHR / MhcHR / MbcS / MhcS / X2 for diploid (2 genes with Mbc:Daf No. Strain DafLR DafS DafLR DafS dominance 9:3:3:1*) 1 3 SAS405 (11 M HR- SAS405 (Mbe1 HR- 65 20 4 1 2 59.56 23.76 (10 .52) DajLR) DajLR) 1 4 S"-\S405 (11 M I-IR- Water 60 1 3 20 9 3 .80 32.35 (1 .23) DajLR) * X2 values greater than 2.7 1 (3.84) indicate differences significant at P=O.l (0.05) Chapter 4 Isolation and characterisation of non-aggressive mutants 6 7 The monoclonal antibody BC-I

F 1fp97 SAS56 Pr >F<1> PatllOgenicity * ( mm) 0.74 (0.22) 9.38 (0.33) O.OOOlt 1 .30 (0. 1 2) 8.0 (0.92) O.OOOlt Radial growth rate at 23-25°C (mm d·l) 1 0.83 (0.24) 14.80 (0.21) 0.0001 10.75 (0.87) 1 4.5 (0.63) 0.0001 Conidiation x 1 0.8 (no. / pet�-j dish) 1 .78 (0.33) 2.22 (0. 15) O.OOOlf 2.73 (0.34) 2.27 (0.64) 0.352M Sclerotial no. (/ perri dish) 87.26 (14.88) 36.26 (5.65) O.OOOl f 95.7 (6.01) 39.7 (5.78) 0.0003f Sclerotial weight (g) 0.67 (0.07) 1 .08 (0.07) O.OOOl f 0.62 (0.03) 1 .25 (0. 1 1) O.OOOl f Weight per sclerotium (g) 0.009 (0.001) 0.Q3 7 (0 .. 006) O.OOOl f 0.006 (0.0003) 0.033 (0.0031 ) O.OOOl f j- �. , Spore area (flm2) 52.03 (0.36) 50.63 (0.32) + 65.79 (1 . 1 5) 5 1 .24 (0.81) + * on French bean, rad.ial growth from plug t power transformed Q-= 0.7) for analysis f log transfom1ed for analysis <1> Differences were considered significant when Pr> F < 0.05 + Statistical results omitted as only a small proportion of the variation (R2 = 1 8%) was explained by the model (area = strain) indicating that add.itional factors, not accounted for in the model, are influencing spore area > a ..£:. 0 H (fq (b ::J (b (1 0 ::J R 0 E:=: ::J (Jq "'0 $» Er- 0 (fq (b e . (1 �. S· tJj "" q �. � . '" � I::. --J --J Chapter 5 A major gene controlling pathogenicity in Botrytis cinerea 78 Figure 26 A typical restricted lesion produced on a French bean leaf inoculated with a 5-mm agar plug taken from the actively growing edge of a non-aggressive strain (P21 - 47) o f Botrytis cinerea and incubated for 3 days at 20 - 25"C under conditions o f high humidity. Arrows indicate the characteristic dark brown ring enclosing the lesion. Chapter 5 A major gene controlling pathogenicity in Botrytis cinerea 79 Microscopic studies of lesions Germ-tube length did not differ significantly (P = 0.043) between strains Mp97 and A4 after 8 h incubation on French bean leaves (28.8 flm SE 0.9 and 32.2 flm SE 1 . 1 respectively) . Structures observed on leaf-portions prior to penetration included swollen germ-tube ends, simple oval appressoria, bilobed and multilobed appressoria (Figure 27 p81 , Figure 28 p81) which were similar to those described by van den Heuvel & Waterreus [1 983] and Garcia-Arenal & Sagasta [1980] . After 1 6 h incubation numerous single lobed and a few bilobed appressoria were observed in lesions from both Mp97 and A4 and after 1 8 h incubation bilobed appressoria were common in both. Multilobed appressoria were present after 20 h incubation especially in A4 induced lesions. \-\yphal penetration was evident from both Mp97 and A4 (Figure 29 p82) after 1 6 h. Few penetration hyphae were observed to originate from bilobed or multilobed appressoria, and although a few hyphae were seen penetrating through stomatal openings most penetrations occurred between epidermal cells. Groups of darkly stained mesophyll cells were frequently associated with mycelium often near the site of penetration of both strains incubated for 1 8 h. At 2 and 3 days a ring of similarly stained cells was observed surrounding the colony in both Mp97 and A4 infected leaf-portions, although in the latter the mycelium frequently appeared to eJ<\�rrl. 6eyo l\o\ this ring. After 4 d incubation the ring of darkly stained cells was visible surrounding the lesions of Mp97, but not A4, inoculated leaves (Figure 30 p83). No differences were detected in the hyphal structure of Mp97 and A4 in the lesions. Physiological and morphological characterisation Following confirmation of their aggressiveness phenotype, 20 strains selected from cross 1 0 (fable 4 p61 , 1 0 aggressive and 1 0 non-aggressive, see Characterising of Pat1 on French bean p76) were compared for a range of physiological characters. Radial growth rate was slower in non-aggressive progeny and the mutant str:;Un (l\IIp97) compared to wild-type progeny and the wild-type parent (SAS56) . Conidial production was also lower in the non-aggressive progeny although there was no significant difference between the mutant strain and the wild-type parent. Sclerotial number was higher and sclerotial weight lower in non-aggressive strains than in wild-type strains in both progeny and parents (fable 8 p77). All strains grew readily as dense spreading colonies on MM and it was concluded the non-aggressive phenotype did not reflect an auxotrophic requirement. Although small differences in spore size were found between strains these did not Chapter 5 A major gene controlling pathogenicity in Botrytis cinerea 80 correlate with differences in aggressiveness (non-aggressive range of means 45.58 - 59.30 f1m2, aggressive range of means 47.65 - 54.08) (Table 8 p77) . Differences in acid production by Mp97, its parent A4, 20 selected progeny (as above) and wild-type strains (SAS56 and SAS405) were distinctive. All aggressive strains scored 2 or higher for intensity of colour change compared with non-aggressive strains (Fat1 progeny and Mp97) which scored between 0 and 1 .5 indicating a correlation between low acid production and the Pat1 phenotype. The mutant Mp97 differed in its response to incubation temperature compared with A4 and SAS56. At 25°C Mp97 showed a significandy slower growth rate (as was shown previously) than both wild-type stains (which also differed from each other) , while at 20°C growth rates of Mp97 and A4 were indistinguishable. However, at lower temperatures (1 0 and 1 5°C) Mp97 grew significandy faster than both wild-type strains (Figure 31 p84) . Polygalacturonase expression Cup plate assay Comparison of total PG activity in filtrates of 8-h cultures showed a slighdy larger significant difference between wild-type strains (SAS56 and A4) than between non­ aggressive and aggressive progeny (Means 1 . 1 7, 1 .37 enzyme activity units P = 0.0004 and means 1 .35, 1 . 1 7 enzyme activity units P = 0.025 respectively) (Figure 33A p85). This suggests strain variation is the cause of the differences in PG activity rather than the involvement of Pat1. In contrast, in filtrates of 24-h cultures, no significant diffe�ence was found between wild-type strains (P = 0.54) or between the mutant strain (Mp97) and its parent (A4) (P = 00408) but there was a significant difference between non-aggressive and aggressive progeny (mean 1 1 .52 and 1 5 .98 enzyme activity units respectively, P = 0.0001) (Figure 33B p85) . Variances were stabilised by transforming the data prior to analysis (8-h data was square root transformed, 24-h data was power transformed, A = -0.0538) . Chapter 5 A major gene controlling pathogenicity in Botrytis cinerea 81 Figure 27 Multilobed appressoria of Botrytis cinerea isolate Mp97 on infected French bean leaves incubated for 22 or 24 hours at 20 - 25°C and stained with aniline blue. Figure 28 Multilobed appressoria of Botrytis cinerea isolate A4 on infected French bean leaves incubated for 22 or 24 hours at 20 - 25"C and stained with aniline blue. Chapter 5 A major gene controlling pathogenicity in Botrytis cinerea 82 Figure 29 Hyphae of A4 penetrating through successive layers of French bean tissue after 20 hours incubation at 20 - 25°C. (A) Upper epidermis. (B) Palisade parenchyma. (C) Spongy parenchyma. Chapter 5 A major gene controlling pathogenicity in Botrytis cinerea 83 Figure 30 Mycelium of Botrytis cinerea in lesions on adaxial surface of French bean leaves incubated for 24 hours and cleared and stained with aniline blue. M = mycelium, S = darkly stained mesophyll cells. (A and C) A4 aggressive. (B and D) Mp97 non­ aggressive. Chapter 5 A major gene controlling pathogenicity in Botrytis cinerea 84 1 4 .-.. � .Mp97 A "C 1 2 DA4 -..... e 1 0 . SAS56 e '-" � 8 ... CIS ... ..c 6 � 0 ... 4 OJ) c:: CIS 2 � � 0 10 15 20 25 Incubation temperature °c Figure 31 Daily growth rate on MEA of Mp97, A4, and SAS56 averaged over 8 days (l onC), 6 days (1 5°C), and 4 days (20 and 25°C). Means with the same letter are not significantly different (Duncan's multiple range test, alpha = 0.05) . Bars indicate standard error of means from 5 replicate plates. 100 . Mp97 90 DA4 80 1:1 70 .£ ... u 60 � .9 ':!? 50 " 1:1 40 C\I � ::s 30 20 1 0 0 10 1 5 20 25 Temperature °c Figure 32 Infection of tomato stem-pieces recorded as a percentage of 22 replications after I4-days incubation. Chapter 5 A major gene controlling pathogenicity in Botrytis cinerea 85 A 1 .4 . I'al ' 1 .2 o Wilt] - type 1 . 1 1 .� I I .R . � .;; .� I I . Ii " 0.. I T r'- 1 1. 4 ;r, 1 1 . 2 o.u II n B 1 2 • Pal' I I I o W ilJ - type I 2 I I Figure 33 Total polygalacturonase activity of progeny (p-strains), parents (Mp97 and SAS56) from the cross Mp97 x SAS56 and A4 (expressed as activity units of the Aspergillus standard) . (A) 8-hours incubation. (B) 24-hours incubation. Bars indicate standard error of means from 4 replications. Chapter 5 A major gene controlling pathogenicity in Botrytis cinerea 86 Isoelectric focusing and detection of PG isozymes Dr Sharrock found that Mp97 and A4 produced identical Isozyme profIles after separation by IEF including a prominent isozyme (PI 8) which was absent from the SAS56 profIle (Figure 34 p87) . The profiles from extracts of bean leaf-lesions (Figure 35 p87) were very different to those from culture filtrates (Figure 34 p87) . Isozymes from SAS56 lesions were predominantly of low pI « 5 .3) , with relatively little expression of the pI 9 isoenzyme that was prominent in culture filtrate profIles. A4-induced lesions appeared to contain more isozymes, covering a broader pI range (3.5 - 8.3) , than were in the culture fIltrate. Extracts of Mp97 -infected tissue revealed much less PG activity than in the necrotic lesions resulting from the two aggressive strains. The most active isoenzyme (and only one clearly visible) had a pI of about 4.8. This is partly attributable to the relatively short incubation period (105 min) which was more appropriate for the higher levels of PG activity found in extracts from larger lesions. In order to compare similar PG activities on one gel lesion extracts of SAS56 and A4 were diluted 50 and 1 00 fold (Figure 35 p87) . This revealed that the major isozymes expressed by A4 in vivo (PI range 4.2 - 5.0) were also expressed by Mp97 and that differences were restricted to the minor bands. In contrast SAS56 produced a major isozyme band at around pI 5.2 which was not evident in the in vivo extracts of tl1e other two strains. Extracts of control leaves (not inoculated) produced no detectable PG bands (data not shown) . The infections caused by s trains Mp97 and A4 followed very different cours'es, which may have resulted in the release of different inducers of PG isoform production. Total protein staining patterns of the IEF separations of Mp97 infected and control leaf extracts were almost identical, confirming that the fungus had caused minimal damage, whereas extracts of A4-infected leaf tissue contained a very different pattern of protein bands from those of the controls Chapter 5 A major gene controlling pathogenicity in Botrytis cinerea 87 +ve pI 3.5 4.2 5.3 6.0 8.3 -ve 10.0 Figure 34 Polygalacturonase (pG) isozymes in l -day-culture filtrates following isoelectric focusing and incubation for 4.5 hours at 37°C (overlays stained with ruthenium red) . A letter after the strain number distinguishes replicated cultures. (photo Dr Sharrock). +ve -ve pI 3.5 4.2 Figure 35 Polygalacturonase i sozymes in extracts of 4-day-old lesions of bean leaves inoculated with a. cinerea. Lanes loaded with 1 0 fll of extract from lesions homogenised in 10 volumes of buffer. SAS56 lesion extract diluted 50 and 100- fold respectively. MP97 lesion extract undiluted. A4 lesion extract diluted 100 and 5.3 50-fold respectively (overlay incubated for 1 5 hours) . (photo Dr Sharrock) . 6.0 8.3 10.0 Chapter 5 A major gene controlling pathogenicity in Botrytis cinerea 88 Characterisation of Patl on other hosts Pathogenesis on soybean, rose and tomato In addition to French bean the aggressiveness of mutant Mp97 and its parent A4 waS tested on soybean, rose and tomato. Responses with the soybean leaf assays were similar to that found on French bean leaves (fable 9 below). Typical Mp97 lesions were small and surrounded by a dark ring and remained restricted after 7 -d incubation, in comparison f' with rapidly expanding watery edged lesions found with A4. In the rose assay Nfn was slower to colonise than strain A4, but all flowers became fully infected by both strains after l S-d (fable 9 below). On tomato the mutant Mp97 did not infect stem-pieces at 20 and 25°C, but did produce infection at lower temperatures, albeit at a lower incidence than that of the parent strain (Figure 32 p84) . To confirm that the fungus growing at 1 5 and 1 0°C was the strain inoculated, lesions were checked by reisolating (onto MEA) from the lesion margin of all lesions produced on stem-pieces inoculated 'with Mp97 and one inoculated with A4. These isolates were re-tested on French bean leaves: all lesions produced by Mp97 cultures were consistent with the non-aggressive phenotype, while those produced by the A4 culture were typical of aggressive lesions. Table 9 Growth of Mp97 and the parental strain A4 on detached leaves of French bean and soybean seedlings, and rose flowers. French bean leaves* 1 .30 (0. 1 2) Soybean leaves t 2.85 (0. 1 7) Rose flowers (days to full infection) 1 3 .86 (0.40) * Analysis perfonned on power transfonned data (0.8) t Analysis perfOlmed on square root transfonned data A4 9.60 (0.80) 1 0.68 (0.31) 1 1 .29 (0.29) :j: Differences were considered significant when Pr>F < 0.05 Phytoalexin induction on Soybean leaves Pr 0.0001 0.0001 0.0002 There was no difference in phytoalexin induction between the mutant Mp97 and its parent A4 (absorbance 0. 1 9, SE 0.0 1 9 and 0. 1 8, SE 0.01 2 respectively) , but both differed significantly from the media control (absorbance 0.0 1 , SE 0.00 1 , Duncan's multiple range test, alpha - 0.05) . Competition The interaction of two s trains of B. emerea at the infection SIte was observed in competition experiments. The \'f ect on infection of an aggressive strain (SAS56) by a Chapter 5 A major gene controlling pathogenicity in Botrytis cinerea 89 non-aggressive mutant (p33-146) was ascertained by the size of the lesion produced. At 0- h challenge the lesion size in both combinations was part way between that caused by individual (control) inoculation of the two strains suggesting a retarding effect of the non­ aggressive strain on the growth of the aggressive strain. Lesion sizes in other challenge combinations were dominated bv the strain inoculated first, even if this was the non­ aggressive strain. When applied 12 h after the initial inoculation the second strain had no influence on the size of the resulting lesions (Figure 36 p90) . The lesion size was larger for the combination treatment involving both strain" applied at 0 h when A4 was applied first, compared with the similar treatment when Mp97 was applied first. This was attributed to settling of the conidia from the first inoculation and the lack of mixing when the second strain was added shortly after. In order to determine whether the dominating effect of the first strain inoculated was a general phenomenon, competition was examined between two genetically marked aggressive strains by estimating the composition of strains in the resulti�g lesion. The proportion of conidia from the harvested lesion carrying the marker for sodium selenate resistance was interpreted as the proportion of Ms044 biomass present in the lesion and similarly the proportion of conidia carrying vinclozolin resistance was that of SAS405. Where leaves were inoculated with spores from both strains �t 0 h the spores harvested had a 50:50 mix of botl, markers, while lesions from sites where the challenging strain was applied after 6 h contained a small percentage of spores carrying second strain markers, and at 1 2 h the spores contain no challenge markers. Analysis of lesion radii (8 replicate lesions, using Duncan's multiple range test Alpha = 0.05) revealed no significant difference between Ms044 and SAS405 (Figure 37 p90) indicating the two strains were equally aggressive on French bean. Thus the pattern whereby the first strain inoculated dominates the infection site is apparently the norm for B. cinerea infections of French bean leaves. Chapter 5 1 6 e 1 2 ,! ;a � ... 8 c 0 .;;; .£ c � ... 4 � 0 A major gene controlling pathogenicity in Botrytis cinerea .L • o ..... 6 12 Hours before challenge • SASS6 -+-- SASS6! 1'33-146 • 1'33-146 1'33-146!Si\SS6 T .L 24 90 Figure 36 Lesion radii on French bean leaves inoculated with either SASS6 (aggressive) or P33-146 (a non-aggressive single-ascospore strain) and then challenged after 0, 6, 1 2 or 24-hours incubation with either P33- 1 46 or SASS6 respectively (incubated for 4 days at 20 - 2S"C). Bars indicate standard error of means of 4 replications in each of 4 blocks . • Selenate o Vindozolin Ms044 / SAS40S SAS40S / Ms044 Hours before challenge Figure 37 Percent germination on selenate (MM+Se041l) and vinclozolin (MEA +vinclozolin) differential media, of spore suspensions made from whole leaf­ lesions inoculated with either Ms044 (selenate resistant, SelR) or SAS40S (vinclozolin resistant, DaJl LR) and then challenged (Ms044 challenged with SAS40S, SAS40S challenged with Ms044) after 0, 6, 1 2 or 24-hours incubation (incubated for 7 days at 20 - 2S"C) . Bars indicate standard error of means of 3 replications of 300 spores. Chapter 5 A major gene controlling pathogenicity in Botrytis cinerea 9 1 Figure 38 Interaction zone on MEA+NaCI. (A) P33-146 paired with P33-146. (B) P33-146 paired with SASS6. (C) SASS6 paired with SASS6. Figure 39 Interaction zone on MEA+NaCI. (A) SAS40S paired with SAS40S. (B) SAS40S paired with Ms044. (C) Ms044 paired with Ms044. Chapter 5 A major gene controlling pathogenicity in Botrytis cinerea 92 Interaction experiments were undertaken to assess the likelihood that heterokaryon formation might confound these results. Both Ms44/SAS40S and P33- 1 46/SASS6 pairings on MEA + NaCl produced distinct dark interaction zones, compared with no interaction zones when two plugs of the same strain were paired (Figure 38 p9 1 , Figure 39 p 9 1 ) . The formation of interaction zones indicates mycelial incompatibility between the strains, and probably that the strains are vegetatively incompatible and thus unable to form heterokaryons [Beever & Parkes, 1 993] . This is supported by the results of the second competition experiment where total percent germination of sodium selenate and - vinclozolin resistant strains (calculated separately) were close to 1 00% for all mixed "' , inoculation of Ms44 and SAS40S (Figure 37 p90). If a heterokaryon was formed (assuming resistance is dominant) the total percentage germination (sodium selenate + vinclozolin resistance) would be expected to exceed 1 00 if appreciable numbers of heterokaryotic conidia were formed. Discussion It is apparent that the reduced aggressiveness of Pat1 isolates is not specific to infections on French beans, indicating that the gene affects a fundamental process of B. cinerea pathogenicity. Pathogenesis of Pat1 strains on soybean closely resembles that on French bean in that limited lesions are formed. On roses spreading lesions were forme-a, but the , rate of lesion spread was slower. At higher temperatures in the tomato test the 'mutant wa� essentially non-pathogenic. Its aggressiveness on tomato at 1 0 and 1 5°C may reflect reduction in host defence responses as tomato is known to grow poorly at low temperatures [Eden et aL [1 996b] . It may also reflect the slightly faster growth of the mutant relative to wild-type strains at 1 0 - 20°C (Figure 3 1 p84) . Although Mp97 consistently produced fewer infections than A4 on tomato s tem-pieces over the range o f temperatures tested, invasive infections were sometimes established a t 1 0 and \soC albeit " less frequently than with A4. As with rose there was no evide'nce for the formation of restricted lesions. While some physiological differences were observed between Pat1 and aggreSSIve s trains (fable 8 p 77) none provides an obvious explanation for non-aggressive behaviour. Slower radial growth rate at higher temperatures and more, smaller sclerotia were correlated with non-aggressive lesion formation in parents and progeny suggesting a possible pleiotropic effect between these characteristics and Pat1 . However, these differences are relatively small and within the range found in wild-type strains [Beever et aL, 1 989] . Non-aggressive mutants of Sc!erotinia sc!erotiorum also exhibited a reduced growth I Chapter 5 A major gene controlling pathogenicity in Botryfts cinerea 93 rate on agar medium and were defective in sclerotial production [Godoy et aI., 1 990] . Phillips et aI., [1987] reported a correlation between increased spore size and increased aggressiveness in field isolates of B. cinerea taken from roses. We found no evidence to suggest a similar difference between Mp97 and A4. Two broad mechanisms can be identified whereby Patt could affect the infection process. Firstly, through an alteration to the process of host recognition and response either through induction of a faster and/ or more intense response or by greater sensitivity to defence compounds. An increased host defence response could lead to acquired resistance in the host resulting in a reduced or restricted lesion size. However, no evidence was found for enhanced induction of phytoalexins in Patt inoculated soybean leaves. It is plausible that Patt strains may have a reduced ability to metabolise phytoalexins in a similar manner to the non-aggressive mutant (BC-S) of van den Heuvel & Grootveld [1 978; van den Heuvel, 1 976] . Van den Heuvel & Grootveld [1 978] studied phytoalex0 induction in French bean leaves by aggressive and non-aggressive field-isolates of B. cinerea. Phaseollin was produced by the leaf in a zone of apparently healthy cells surrounding the lesion and was metabolised by the fungus to 6a-hydroxyphaseollin (less inhibitory) by aggressive isolates. In contrast to aggressive infections, leaves infected wi.th the non-aggressive strain (BC-S) produced restricted lesions and accumulated twice as much phaseollin by dry weight after 4 d incubation [van den Heuvel & Gro 0 tveld , 1 978] . Moreover, -BC-S was found to be severely limited in its ability to metabolise phaseollin to 6a-hydroxyphaseollin [van den Heuvel, 1 976] . In the current study microscopic examination of leaf-lesions and comparisons of phytoalexin induction suggests that Mp97 is unaffected in its ability to induce or tolerate plant defence responses during the early stages of infection. The presence of a darkly stained ring of cells surrounding Mp97 in 4-d old lesions (Figure 26 p78) is suggestive of a defence response indicating Mp97 may be defective in its ability to .- either tolerate or metabolise defence compounds at this stage� ' Increased aggression on tomato stems at lower temperatures could be due to the lower production of defence compounds at temperatures below optimum for plant growth. The second mechanism by which Pat1 could affect the infection process is through a quantitative or qualitative deficiency in the production of attack compounds. Quantitative (total activity) and qualitative (isozymes) polygalacturonase activity was investigated for Patt non-aggressive strains and wild-type aggressive strains. * Total PG activity in vivo is relatively low in Mp97 and most non-aggressIve progeny (Figure 35, p87; Appendix 9, p143). This is interpreted as an indirect effect of lesion size and development on PG activity, rather than any direct influence of Patt. ** While it is possible that the differences in the minor bands may reflect the direct influence of Patt, further work is needed to clarify the reproducibility of the results and the compounding effect of dilution of the extracts. Chapter 5 A major gene controlling pathogenicity in Botrytis cinerea 94 I" Il fT(O Comparison of total PG activity/in ��\r�e'5 of 8 h and 24 h cultures revealed no consistent differences between aggressive and non-aggressive strains. Ths is in agreement with the findings of some other workers [Leone, 1 990; Tiedemann, 1997] . It is possible that measurements of total PG activity could mask important differences in the range of PG isozymes produced by the non-aggressive strains compared with the aggressive strains. An important role in the early stages of infection has been proposed for a constitutively produced PG isozyme of B. cinerea [Leone & van den Heuvel, 1 987; Leone et aI., 1990; van der Cruyssen & I- Each new mutant allele can be assigned a unique isolation number immediately upon discovery (could include a letter) . Phenotype designation >- The phenotype of a strain should be described using a three-letter symbol for the gene controlling that phenotype. >- The symbol should be written in Roman type with the first letter uppercase and the other two lowercase. >- A plus sign should be used to indicate the wild-type phenotype and a minus to indicate the mutant phenotype. Mating-type designation >- Suggest the symbol MAT for the locus with the single locus found in many fungi called MATI and the two known alleles at that locus MATt-1 and MATI-2 (all letters in the locus symbol of both alleles are uppercase since they are co-:dominant - both are needed for activity) . Appendix 6 Summary of genetic symbols Appendix 6 Summary of genetic symbols Table 11 Genetic symbols used for genes of B. cinerea. Gene Allele Genorype/Phenorype Page Mbcl MbclHR Benzimidazole (carbendazim) 14, 20, 21 , 37, Figure 1 2,Table 2, 47, high resistance Table 3, Table 4, Table 6, 60, Table 5 MbclLR Benzimidazole (carbendazim) low 14, 20, 2 1 , Table 2, 47, Table 3 resistance MbclS Benzimidazole (carbendazim) 14, 20, 2 1 , 37, Figure 1 2, Table 2, 47, sensitive Table 3, Table 4, 60, Table 5 . Dafl DaflHR Dicarboximide (vinclozolin) high 14, 20, 21 , 37 resistance DaflLR Dicarboximide (vinclozolin) low 14, 20, 21 , 37, Figure 12, Table 2, 47, resistance Table 3, Table 6, 60, Table 5 DaflUR Dicarboximide (vinclozolin) ultra 14, 20, 2 1 , 37 low resistance DaflS Dicarboximide (vinclozolin) 14, 20, 2 1 , 37, Table 2, 47, Table 3, sensitive Table 4, 60, Table 5 Daj2 - Dicarboximide (vinclozolin) 21 sensitive MATI MATt-1 Mating-type 1 (able t o mate with 18, 20, 21 , 33 Matl-2) MATI-2 Mating-type 2 (able to mate with 18, 20, 2 1 , 33' Mati-I) MATI-I/2 Mating-type 1 /2 (able to mate 1 8, 20, 21 , 33 with Matl- 1 and Matl-2) Sma - Small apothecia gene - wild-type 1 8, 21 cutA - Cutinase A 19 Bcpgal - A polygalacturonase expressed 1 9 early in pathogenesis. Self SellR Sodium selenate resistance class B Chapter 3-39, 60, Table 5, 88, Figure 37 Sel1S Sodium selenate sensitive Chapter 3-39, 60, Table 5, 88, Figure 37 nitl - Nitrogen utilisation gene - Chapter 3-39, 60, Table 5, 88 defective in nitrate reductase Pat1 Patl Pathogenicity related gene - non- 58, Table 4, Figure 25, Chapter 5-69 aggressive 1 36 Reference Faretta & Pollastro, 1 991 Faretra & Pollastro, 1 99 1 Beever & Parkes, 1 993 Faretta & Pollastro, 1991 Faretra & . Pollastro, 1 93 Faretra et ai, 1 988b Faretta & Pollastro, 1 992 Van K.an et aL, 1 997 Have etal, 1 997 Weeds et al, 1 997 Weeds et aL, 1 997 Weeds et aL, in prep Appendix 7 Pathogenicity related fungal attack chemicals 1 37 Appendix 7 Pathogenicity related fungal attack chemicals Table 12 Summary of references investigating the importance of B. cinerea attack chemicals to their aggressiveness on a particular host (correlation 'yes' indicates positive correlation unless otherwise stated) . Attack Reference No. Host Comment Aggressiveness chemical isolates correlation PG Zalewska-Sobczak et 2 Apple No al., 1 981 - . Grape Di Lenna et aI., 1981 3 Strawberry - No Bean Di Lenna & Fielding, 3 Apple Carrot 4 isoforms No 1983 Kovacs & Tuske, 27 Apple No 1 988 Paprika Bean - Have et aI., 1 997 2 Gene disruption Yes mutant PME Wasfy et aI., 1 978 3 Strawberry Yes (neg) Apricot Bean - Grape Di Lenna et aI., 1981 3 Strawberry - No Bean Lettuce Lorenz & Pommer, 1 0 Grape No 1987 Pepper - Kunz et aI., 1996 5 Apple Insertion No mutants PMG Wasfy et aI., 1 978 Strawberry Yes 3 Apricot Bean - Zalewska-Sobczak et 2 Apple No al., 1 981 - Lorenz & Pommer, 10 Grape Yes 1987 Pepper - Appendix 7 Pathogenicity related fungal attack chemicals 1 38 Attack Reference chemical PL Di Lenna et ai, 1 981 Movahedi & Heale, 1990 Protease Zalewska-Sobczak et ai, 1 981 Lorenz & Pommer, 1 987 Movahedi & Heale, 1 990 Laccase Kovacs & Tuske, 1 988 Faretra & Mayer, 1992 Sbaghi et ai, 1 996 ADS Edlich et ai 1 989 Weigend & Lyr, 1996 Tiedemann, 1 997 Cutinase Van Kan et ai, 1 997 Maltase Wasfy et ai, 1 978 Phenol oxidase Wasfy et ai, 1 978 Xylase Zalewska-Sobczak et ai, 1 981 Catalase Wasfy et ai, 1 978 f3-Glucosidase Sasaki & Nagayama, 1 994 and 1 996 PG = Polygalacturonase PL = Pectin-lyase AOS = Active oxygen species No. isolates 3 8 2 10 8 27 1 8 1 3 1 6 2 3 3 2 3 1 1 Host Comment Aggressiveness correlation GrapeStrawb erry Bean 2 isoforms Yes Lettuce Carrot Cabbage Strawberry Yes Raspberry - Grape Broad bean Apple - Yes Grape Yes Pepper - Carrot Cabbage Strawberry Yes Raspberry - Grape Broad bean Bean - �es Cream mutant Grape with low laccase No production Also ability to Grape degrade stilbene- Yes type phytoalexin VidaJaba - Yes VidaJaba - Yes Grape Sunflower Barley - Yes Broad bean Gerbera Gene disruption No Tomato Strawberry Yes - Apricot Bean Strawberry Yes - Apricot Bean (neg ) Apple - No Strawberry Yes Apricot Bean - Apples Grapes - Yes Lettuce PMG = Polymethylgalacturonase PME = Pectin-methyl-esterase Appendix 8 Polygalacturonase standard curves 1 39 Appendix 8 Polygalacturonase standard curves Preparation of PG standard curves follows Taylor & Secor [1 988] . A dilution series (10, 20: 50, 100 and 1000 fold) of standard PG solution (Aspergillus mdulans PG, 1440 units mg-I protein; 0.29 mg protein ml-I , Sigma) was included in each plate of B. cinerea filtrates assayed for PG activity (J3). Zone diameters for each series (one series from each of four plates) were plotted against the enzyme concentration expressed as a percentage of the PG standard. A best-fit line was plotted using Microsoft Excel 97 (Figure 40 p140) and the regression equation rearranged making enzyme concentration the subject as shown. Plate 1 Plate 2 Plate 3 Plate 4 [Enzyme] = antilog ((diam-17.092)/2.4639) [Enzyme] = antilog ((diam-16.41 1)/2.7102) [Enzyme] = antilog ((diam-1 6.442)/2.4822) [Enzyme] = antilog ((diam-16.264)/2.5827) ,- The equation was then used to convert zone diameters to a percentage of the standard, using the formula from the appropriate plate, and then to PG activity units using the formula: units = % (mg protein ml-I) (units .. ) from the PG standard. The converted data was analysed statistically (p37) . * 1 unit = the amount of enzyme required to catalyse the production of 1 flmole of reducing sugar per min. Appendix 8 Polygalacturonase standard curves 140 311 Plate 1 311 Plate 2 28 Y = 2.46391.0 (x) + 1 7.1192 28 Y = 2.7 1 1 121.0(x) + 1 6. 4 1 1 E R' = 0.9984 R' = 11.9995 g 26 26 � 24 24 .� 22 22 'U " c 211 � 20 li 1 8 1 8 " ::;: 1 6 1 6 I I I l Ol l 1 1 1 1 1 10 30 Plate 3 3il Plate 4 28 Y = 2.48221.0(x) + 1 6.442 28 Y = 2.58271.0 (x) + 1 6.264 E R' = 0.9989 R' = 0.9934 g 26 26 " ;;; 24 24 § 22 22 "" " c 0 20 20 II li 1 8 " 1 8 :;; 1 6 1 6 1 0 1 1 11 1 I I I 100 Enzyme series u ilu tion (°I.l of stanu anj) I ':ozymc series d ilu tioo (. '. o f st.od.rd) Figure 40 Polygalacturonase standard curves generated from zone diameters produced on substrate gel by a series of dilutions of the A . . (1�. PG standard. R2 values indicate the appropriateness of the fit (the closer to 1 the better the fit) . Appendix 9 Segregation of polygalacturonase isozymes 141 Appendix 9 Segregation of polygalacturonase isozymes The following poster was presented at the Australasian Plant Pathology Society 11 th Biennial Conference CAPPS) held in Perth in September 1997. Abstract SEGREGATION OF POLYGALACTURONASE ISOZYMES IN SEXUAL PROGENY OF BOTRYTIS CINEREA (BOTRYOTINIA FUCKELIANA) K.R. SharrockA, P.L. WeedsB and R. E. Beevel �e Horticulture and Food Research Institute of New Zealand, Private Bag 3 1 23, Hamiltoa, New Zealand BPlant Science Dept., Massey University, Palmerston North, New Zealand cManaaki Whenua - Landcare Research, Private Bag 92 170, Auckland, New Zealand INTRODUCTION Some strains of Botrytis cinerea can be readily crossed in the laboratory. We have compared polygalacturonase (PG) isozymes of a New Zealand and an Italian strain and found marked differences in certain areas of their PG isozyme profiles. Segregation of individual PG isozymes and linkage relationships with benzimidazole resistance and pathogenesis markers in single-ascospore progeny from the cross of these two strains are discussed. Little is known of the genetic basis for the wide range of isozymes of PG produced by many plant pathogenic fungi, including B. cinerea. It is possible that some of the diversity of PGs could be attributable to post­ translational modification, with a single gene thereby giving rise to several PG isozymes. In this case, such isozymes would segregate together in progeny of a sexual cross, whereas those, which are separate gene products, should show greater independence in segregation. To our knowledge, there have been no previous reports of the segregation of PG isozymes between progeny of a sexual cross in B. cinerea. MATERIALS AND METHODS B. cinerea strains SASS6 was a single-ascospore-derived reference strain supplied by F. Faretra (Bari, Italy). Mp97 was a non-aggressive mutant produced by u.v. mutagenesis from A4 (a single-ascospore strain from a cross of two NZ field isolates). SASS6 and Mp97 were crossed and 20 single spore progeny were selected for further study. Growth of the fungi in vitro and in vivo Liquid growth medium used for in vitro culture was modified Richard's solution with citrus pectin (O.S% w/v) as the carbon source. Flasks were inoculated with spores (2 x 104/rnJ final concentration) and filtrates were collected after 24 h growth on an orbital shaker at 20°C. French beans (Phaseolus vulgaris cv. Top Crop) were grown to the two true leaf stage. Plastic rings (3 mm diam.) placed on the adaxial leaf surfaces were loaded with 20 III containing 10,000 spores suspended in half normal strength Vogel's medium + sucrose (0.7S% w/v). After 48 h incubation under humid conditions at 23°C, the inoculum droplets were retrieved from above the developing lesions, and assessed for PG content. Separation and detection of PG isozymes Isoelectric focusing of culture filtrates and leaf infection droplets, with subsequent detection of PG isozymes, was performed as previously described. After focusing, an overlay gel containing buffered sodium polypectate (0.04% w/v) was placed in contact with the IEF gel for I S min and then incubated at 37°C for 4.S h (for culture filtrates) or I h (leaf infection fluids). The overlay was then stained for at least 2 h in 0.03% ruthenium red. Appendix 9 Segregation of polygalacturonase isozymes 142 RESULTS AND DISCUSSION PG isozyme profiles distinguished parent strains. A distinctive profile of PG isozymes was produced by each of the parent strains both in vitro and in vivo. In vitro the most obvious differences between the two were in the pI 8 vicinity, with Mp97 producing a prominent isozyme of pI 8.0 which was consistently absent from the SAS56 cultures. In vivo this distinction was not as clear-cut; the band at pI 8.0 was still prominent in Mp97, but SAS56 did produce a minor band at this pI. SAS56 in vivo produced a distinctive band at pI 5, which was not detected in the Mp97 inoculation droplet. Direct comparison of the range of isozymes produced in vivo was complicated by the large differences in growth and lesion development between the non-aggressive and aggressive strains, so in vitro profiles were used for analysis of the segregation of individual isozymes in the progeny. Segregation Patterns The pI 8 band which distinguished the parents in vitro segregated 1 : I in the progeny, indicating it is controlled by a single gene with a recombination pattern suggesting linkage to MbcI (benzimidazole resistance). Two other PG isozymes with pIs of ca. 8.4 and 8.8 were expressed in various relative intensities by the progeny. Almost half of the progeny appeared to lack the pI 8.4 isozyme that was expressed by both parents. As B. cinerea is haploid, this apparent loss of an isozyme in approximately half of the progeny is difficult to explain. The pI 8.8 isozyme was produced in both parents but was more intense in SAS56. The progeny matched one or other parental type in roughly equal proportions, except for one strain in which this particular band was extremely faint. More work is needed to determine if this is indicative of multiple isozymes of similar pI. No link between any PG isozyme and pathogenicity Progeny of the Mp97 x SAS56 cross were clearly distinguishable on bean leaves as either non-aggressive (Mp97 type) or invasively pathogenic (SAS56 type). This difference in pathogenicity could not be linked to any one PG isozyme produced either in vitro or in vivo. REFERENCES 1 . Weeds, P.L.; Beever, R.E.; Sharrock, K.R.; Long, P.G. A major gene controlling pathogenic aggressiveness in Botrytis cinerea (Botryotiniafuckeliana). In prep. 2. Sharrock K.R.; Labavitch I.M. ( 1994). Polygalacturonase inhibitors of Bartlett pear fruits: differential effects on Botrytis cinerea polygalacturonase isozymes, and influence on products of fungal hydrolysis of pear cell walls and on ethylene induction in cell culture. Physiological and Molecular Plant Pathology 45, 305-3 19. Segregation of polygalacturonase isozymes in sexual progeny of Botrytis cinerea (Botryotinia fuckeliana) Aims • To determine cilC nature of inheritance of polygalacruronase (PG) isozymes of BOlrytis cinerea. To d8 (.4.) IO""' -I2 (NA) I I -M (.4.) lZ - Mp97,CNAI lJ SAS564 (A) 1 4- SAS56c(.4.) 15 ..(l (NA) 10 - 21 (.4.) 1 1= -11 (NA) 15-4$( .4.) 19 M (A) 2O- Mp97b (NA) 1 1 · SAS(6b (A) J � 7 9 II lJ U 11 19 Figure I . /" vitro PC activit)' reveAled bY ;A polypccla.te ovotrla), .1(tcr Isoclectrlc fOCUSing of 24 h culture fihrat("5 from the part'1H $tr.lInS And .1 selection of progeny. A " J.ggressive; NA = non-.lggr('ssive. 1 - Blank lIloculation 2 '" SAS56 (A) .l " SAS56 (A) 4.: Mp97 (NA) 5 " Mp97 (NA) 6 - A4 (A) 7 -M (A) 8 � 37 (A) 9 : 47 (NA) 1 0- 42 (;o.lA) 11 =-40 (NA) 12 -= 3S (A) J J " 96 (A) 14 =- 3 (NA) 8 10 I:! IJ Figure 2. /n V;I'O PC activity rcvcald bY ;A polypcct